Estudio genético y molecular de mecanismos de resistencia a bilis en salmonella enterica

! ! ! ! ! ! ! Bile!resistance!mechanisms!in!Salmonella( enterica:!genetic!and!molecular!analysis! ! ! ! ! ! ! TESIS!DOCTORAL! VERÓNICA!URDANETA!PÁEZ! ! ! ! ! Departamento!de!Genética! ! Universidad!de!Sevilla! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Bile!resistance!mechanisms!in!Salmonella( enterica:!genetic!and!molecular!analysis! Memoria!elaborada!por!Verónica!Carolina!Urdaneta!Páez!!!!!!!!!!!!!!!!!!!!!!!! para!optar!al!grado!de!Doctor!en!Biología! ! ! Tesis!realizada!bajo!la!dirección!del!Dr.!Josep!Casadesús!Pursals! en!el!Departamento!de!Genética!de!la!Facultad!de!Biología!de!la! Universidad!de!Sevilla! ! ! !!!!!!!!!!!!!!! ! Josep!Casadesús!Pursals!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Verónica!Urdaneta!Páez!!!!!!!! ! Sevilla,!19!de!julio!de!2016! ! ! ! ! ! ! ! INDEX& RESUMEN' 1 ! INTRODUCTION' 7 ! The&genus&Salmonella& 9! Evolution!of!Salmonella(pathogenicity!10! Salmonella!infection!12! Salmonella(passage&through&the&host:µ8environment&conditions&and&stress&responses&14! Salmonella&and&bile&16! Bile!16! Bile!as!an!antimicrobial!agent!19! [!Disruption!of!bacterial!cell!membranes!20! [!Induction!of!macromolecular!instability:!secondary!structure!formation!in!RNA!and!DNA! damage!21! [!Misfolding!and/or!denaturation!of!proteins!21! Bile!resistance!mechanisms!in(Salmonella(enterica!21! [!Bacterial!cell!envelope!22! [!Efflux!pumps!23! [!DNA!repair!mechanisms!25! [!Salmonella!enterica!adaptation!to!bile!27! OBJECTIVES'29 ! MATERIALS'AND'METHODS' 33 ! Bacterial&strains&35! Bacteriophages&36! Culture&media,&solutions&and&growth&conditions&36! Bacterial!media!and!growth!conditions!36! Culture!media!36! Growth!conditions!38! Solutions!38! Bacterial&transduction&38! P22!lysates!38! Transduction!in!liquid!medium!39! Detection!of!lysogenic!transductants!40! P22!sensitivity!assay!40! ! DNA&manipulation&and&transfer&40! Plasmids!40! Extraction!of!plasmid!DNA!41! Extraction!of!genomic!DNA!41! Digestion,!modification!and!ligation!of!DNA!fragments!42! Agarose!DNA!gel!electrophoresis!42! Isolation!of!DNA!fragments!from!agarose!gels!43! Bacterial&transformation&43! High!efficiency!E.(coli!transformation!43! Bacterial!electroporation!44! Genetic&screens&44! T[POP!transposon!genetic!screen!44! pBR328[based!plasmid!library!genetic!screen!45! Construction&of&bacterial&strains&46! Oligonucleotides!46! Polymerase!chain!reaction!(PCR)!50! Semi8random,&two8step&PCR&(ST8PCR)&51! Chromosomal&gene&disruption&using&PCR&products&52! Preparation!of!DNA!for!substitution!53! Cell!transformation!53! Excision!of!the!resistance!marker!54! Strain!construction!by!transductional!transfer!of!genetic!markers!54! Adaptation&assays&in&bile&gradient&plates&54! Determination&of&minimal&inhibitory&concentrations&of&sodium&deoxycholate&55! In(vivo(assays:&Isolation&of&bile&hyper8resistant&candidates&from&mice&gallbladders&55! DNA&sequencing&56! DNA&sequence&analysis&56! RNA&manipulation&57! RNA!extraction!based!in!TRIsure™!method!57! RNA!phenol!extraction!58! Quantitative!RT[PCR!(qRT[PCR)!58! Quantification!of!qRT[PCR!results!59! Flow&cytometry&59! Statistical&analysis&60! ! RESULTS'61 ! CHAPTER'1:'EXPLORATION'OF'CELLULAR'FUNCTIONS'ESSENTIAL'FOR'BILE'ADAPTATION' AND'RESISTANCE'IN'SALMONELLA(ENTERICA'63 ! Surveys&of&bile&adaptation&in&bile8sensitive&mutants&65! Suppression&of&acrA,&acrB&and/or&tolC&bile&sensitive&phenotypes:&genetic&screen&with&a&pBR328& plasmid&genomic&library&69! T8POP&transposon&genetic&screen:&looking&for&bile&resistant&phenotypes&72! CHAPTER'2:'ESSENTIAL'ROLE'OF'ACRAB=MEDIATED'EFLLUX'IN'SALMONELLA(ENTERICA( ADAPTATION'TO'BILE'75 ! Surveys&of&adaptation&to&bile&in&mutants&lacking&efflux&systems&77! Lack&of&AcrAB&suppresses&bile&hyperresistance&79! Activation&of&acrAB&expression&by&sodium&deoxycholate&80! Single&cell&analysis&of&AcrAB8mediated&efflux&84! CHAPTER'3:'A'STUDY'OF'MECHANISMS'OF'BILE'RESISTANCE'IN(VIVO'89 ! Characterization&of&S.&enterica&populations&present&in&the&gallbladder&of&BALB/c&mice&(typhoid&fever& animal&model)&91! Characterization&of&bile8resistant&mutant&derivatives&isolated&from&the&gallbladder&of&BALB/c&mice&94! DISCUSSION'99 ! CONCLUSIONS'113 ! REFERENCES' 117 ! ' ! ! 1 ! & & & & & RESUMEN& ! ! ! 8 Introduction! 9 The&genus&Salmonella& & The!genus!Salmonella(belongs!to!the!Enterobacteriaceae!family!and!includes!facultative! anaerobic,!non[spore!forming,!rod[shaped!Gram[negative!bacteria.!Most!Salmonellae! are! motile! and! are! able! to! infect! a! wide! variety! of! animal! hosts! including! reptiles,! mammals,!birds!and!amphibians.!Salmonella(is!a!close!relative!of!Escherichia,!Shigella,! and!Citrobacter.(! The!genus!Salmonella( is!currently!divided!into!two!species,!Salmonella( enterica( and! Salmonella(bongori((Tindall,!Grimont,!Garrity,!&!Euzéby,!2005).!Salmonella(enterica(is! subdivided!within!six!subspecies!2:!enterica((I),!salamae((II),!arizonae((IIIa),!diarizonae( (IIIb),! houtenae( (IV)! and! indica( (VI).! Historically,! Salmonella( enterica( subsp.! V! was! bongori,!which!is!now!considered!a!different!species.! Salmonella( subspecies! are! classified! into! serovars! based! on! the! White[Kauffman! classification! scheme! 3,! which! relies! on! antisera! that! recognize! two! highly! variable! surface!antigens,!O!(lipopolysaccharide!O[antigen)!and!H!(flagellar!proteins)!2,4.!There! are!more!than!2,500!Salmonella(serovars,!most!of!which!belong!to!the!subsp.!enterica( 5.!Serovars!of!this!subspecies!regularly!colonize!warm[blooded!vertebrates!6,!and!they! account!for!99%!of!human!infections!by!Salmonella.!On!the!other!hand,!serovars!of! Salmonella(bongori(and!the!rest!of!Salmonella(enterica(subspecies!are!usually!associated! to!cold[blooded!vertebrates!or!to!the!environment!(Bäumler,!Tsolis,!Ficht,!&!Adams,! 1998).!! Serovars!belonging!to!subsp.!enterica(differ!in!their!host!specificity!and!in!the!types!of! diseases!they!produce.!Some!serovars!are!host[restricted,!while!others!can!infect!a!wide! variety!of!animal!hosts!8.!The!diseases!caused!by!subsp.!enterica(serovars!vary!from!self[ limiting!gastroenteritis!to!life[threatening!systemic!infection,!and!the!outcome!of!the! disease!depends!on!the!specific!serovar[host!combination.!For!example,!the!specialist! human[restricted!serovar!Typhi!produces!typhoid!fever,!whereas!the!generalist!serovar! Typhimurium!produces!mild!gastroenteritis!in!humans!but!causes!a!systemic!infection! similar!to!human!typhoid!fever!when!infecting!mice!9.!For!this!reason,!the!interaction! between! serovar! Typhimurium! and! mice! has! been! extensively! used! as! a! model! for! Introduction! 10 typhoid!fever!in!humans!10,!and!most!studies!in!Salmonella(have!employed!this!serovar.! In!this!work,!we!have!used!the!mouse[virulent!strain!Salmonella(enterica(subsp.!enterica( serovar!Typhimurium!SL1344,!from!the!B.A.D.!Stocker!collection.!! According!to!reports!from!the!World!Health!Organization!(2013),!salmonellosis!is!one!of! the!most!common!and!widely!distributed!foodborne!diseases,!with!tens!of!millions!of! human!cases!(from!which!more!than!a!hundred!thousand!result!in!death)!occurring! worldwide!every!year.!In!addition,!since!the!beginning!of!the!1990s,!Salmonella!strains! that!are!resistant!to!a!range!of!antimicrobials!have!emerged,!and!are!now!a!serious! public!health!concern.!Furthermore,!Salmonella!is!a!ubiquitous!and!resilient!bacterium! that!can!survive!several!weeks!in!a!dry!environment!and!several!months!in!water!(WHO,! Fact!sheet!N°139,!2013).! ! Evolution&of&Salmonella(pathogenicity(& The!genera!Salmonella(and!Escherichia(diverged!about!120[160!million!years!ago!11.! Almost!25%!of!the!Salmonella(genome!consists!of!genetic!material!that!is!absent!in! Escherichia( coli( 6,12.! The! evolution! of! Salmonella( pathogenicity! is! related! to! the! acquisition!of!virulence!factors:!the!Salmonella(pathogenicity!islands!(SPIs)!13,14,!which! are!arranged!as!clusters!of!virulence!genes!in!the!chromosome.!More!than!10!SPIs!have! been! described! 15,! including! some! which! are! serotype[specific.! The! fact! the! these! regions!are!absent!in!the!chromosome!of!other!Enterobacteriaceae!and!have!different! G+C!contents!than!the!average!of!the!Salmonella(chromosome!suggests!that!they!have! been! acquired! by! horizontal! gene! transfer! (Figure& I1)! 12,14. Introduction! 11 ! Figure&I1.&Phylogeny&of&the&genus&Salmonella.(The!acquisition!of!SPI[1,!SPI[2,!and!the! ability!to!infect!warm[blooded!vertebrates!is!indicated.!Modified!from!Ellermeier!et!al.,! 2006!16.! ! The!best!characterized!SPIs!are!Salmonella(pathogenicity!island!1!(SPI[1)!and!Salmonella( pathogenicity!island!2!(SPI[2).!SPI[1!was!acquired!first!by!the!common!ancestor!of!the! two!Salmonella(species,!and!is!involved!in!the!invasion!of!intestinal!epithelial!cells!in!the! animal!host!17.!SPI[1!acquisition!likely!allowed!Salmonella(to!become!an!intracellular! pathogen! associated! with! cold[blooded! vertebrates! 16.! SPI[2! allows! Salmonella( to! survive!in!macrophages!and!to!colonize!deeper!tissues!18,!and!its!acquisition!marked!the! split!of!the!two!Salmonella(species!16.!Hence,!only!members!of!Salmonella(enterica(have! the!ability!to!reach!deep!tissues!and!organs!to!produce!systemic!infections.!! The! ancestor! of! subsp.! enterica( acquired! the! capacity! to! infect! warm[blooded! vertebrates,!and!different!lineages!subsequently!evolved!to!colonize!a!variety!of!hosts.! Even!though!the!mechanisms!of!host!specificity!are!not!fully!understood,!the!presence! of!a!virulence!plasmid!in!some!serovars!of!subsp.!enterica(has!suggested!the!potential! involvement! of! plasmid! functions! 7.! Another! factor! that! may! be! involved! in! host! specificity!is!the!presence!of!different!sets!of!fimbrial!operons!in!different!serovars!7,19.!! ( ( Introduction! 12 Salmonella&infection& The! Salmonella( infection! process! usually! begins! with! the! ingestion! of! contaminated! water!or!food.!Figure!I2!depicts!the!biology!of!the!infection!by!Salmonella(in!humans.! The!bacteria!are!capable!to!survive!acid!pH!in!the!stomach!and!to!evade!the!defense! systems! that! it! encounters! in! the! small! intestine! until! it! reaches! the! intestinal! epithelium.!Invasion!of!this!epithelium!occurs!generally!trough!M!cells,!allowing!the! bacteria!to!reach!lymphocytes!B!and!T!which!are!bellow!Peyer!patches!20.!Translocation! across!the!intestinal!epithelium!is!mediated!by!the!virulence[associated!type!3!secretion! system!encoded!by!Salmonella!pathogenicity!island!1!(SPI[1)!21.! ! Once!the!epithelium!is!crossed,!S.(enterica(can!produce!three!main!types!of!infection:! gastroenteritis,!systemic!infection!and!asymptomatic!chronic!carriage.!! ! In!gastroenteritis,!non[typhoid!strains!produce!an!localized!inflammatory!response!that! induces!infiltration!of!polymorphonuclear!leukocytes!(PMN)!into!the!intestine!lumen,! and!diarrhea!by!secretion!of!fluids!and!electrolytes!in!the!small!and!large!intestines! (Figure!I2)!20,22.(! ( Systemic!infection!occurs!when!bacterial!serotypes!invade!intestinal!macrophages!and! disseminate!inside!the!organism!trough!the!lymphatic!system,!permitting!colonization! of!internal!organs!(liver,!spleen,!bone!marrow,!and!gallbladder)!(Figure!I2).!Within!the! macrophages,!S.(enterica!cells!activate!virulence!mechanisms!to!survive!and!replicate!in! the!intracellular!environment.!In!this!phase,!the!type!3!secretion!system!encoded!by! Salmonella(pathogenicity!island!2!(SPI[2)!plays!a!crucial!role!20,23.!! ! Following! systemic! infection,! a! fraction! of! individuals! can! become! chronic,! asymptomatic!Salmonella(carriers,!and!consequenctly!reservoirs!for!future!infections.! The! mesenteric! lymph! nodes,! the! liver! and! the! gallbladder! have! been! proposed! as! potential! Salmonella!reservoirs! 24.! Particularly,! colonization! of! the! gallbladder! by( Salmonella!in!asymptomatic!Salmonella!carriers!permits!constant!shedding!of!bacteria! into!the!medium!since!Salmonella!cells!are!released!into!the!small!intestine!each!time! Introduction! 13 the!gallbladder!contracts.!From!the!small!intestine,!the!bacteria!travel!downwards!in!the! gut,!ultimately!being!released!inside!the!host!feces!20,25.!!! ! ! Figure&I2.&S.(enterica(infection&pathogenesis.&(A)!In!humans,!typhoid!fever!is!caused!by! ingestion!of!food!or!water!contaminated!with!Salmonella(enterica(serovar!Typhi:!(1)!the! surviving!bacteria!go!through!the!acid!pH!in!the!stomach,!invade!the!intestine!epithelial! cells!and!migrate!to!the!lamina!propia.!(2)!In!the!intestine!mucosa,!Salmonella(enterica( serovar!Typhi!is!phagocytized!by!macrophages!and!survives!within!these!cells!due!to! virulence!factors,!which!interfere!with!the!host!cells!functions.!After!the!invasion,!the! bacteria! express! other! factors! that! inhibit! detection! by! the! host’s! innate! immune! system.!This!allows!the!systemic!dissemination!of!the!bacteria,!colonizing!macrophages! in!the!liver,!spleen!and!bone!marrow.!(3)!From!the!liver,!the!bacteria!can!reach!the! gallbladder,!this!infection!can!give!rise!to!a!state!of!Salmonella(asymptomatic!carrier.!(4)! The!Salmonella(enterica(serovar!Typhi!carriers!are!continuously!secreting!bacteria!from! the!gallbladder!to!the!small!intestine!with!the!bile,!(5)!excreting!viable!bacteria!in!their! feces,! (6)! consequently! infecting! other! hosts.! Image! adapted! from! Tischler! and! Mckinney,!2010!25.!(B)!Spleen!of!a!non[infected!129sv!mouse!(left)!and!the!spleen!of!a! 129sv!mouse!infected!with!Salmonella(enterica!serovar!Typhimurium!SL1344!60!days! post[infection!(right).!Image!adapted!from!Monack!et!al.,!2004!26.!(C)!Hystologic!sections! of!BALB/C!mice!ileum!dyed!with!hematoxilin!and!eosin!(scale:!100!µm).!The!upper!image! corresponds!to!a!nonn[infected!control,!the!loweer!image!shows!the!ileum!of!a!mouse! 5!days!post[infection!of!a!lethal!dose!of!S.(Enterica(serovar!typhimurium!14028!10. ! ! In! humans,! the! development! of! the! S.( enterica( chronic! carrier! state! is! frequently! associated!with!the!presence!of!gallbladder!abnormalities,!for!instance,!gallstones!27.! Salmonella(is!able!to!form!biofilms!in!gallstones,!a!matrix!that!provides!high!resistance! Epithelial)cell T)cell Macrophage Systemic)infection:)dissemination)to) the)lymph) nodes,)liver)and)spleen Bcell M)cell Gastroenteritis Introduction! 14 to! antimicrobial! agents! 28:! typically,! antibiotic! therapy! is! ineffective! in! carriers! of! S.( enterica!serovar!Typhi!who!suffer!of!gallstone!formation!29.!( ( Salmonella(passage&through&the&host:µ8environment&conditions&and& stress&responses&& As! described! above,! the! cycle! of! infection! of! S.( enterica( involves! a! variety! of! environments,!each!with!different!conditions!that!are!far!from!optimal!for!bacterial! growth!and!proliferation.!Hence,!as!a!foodborne!pathogen,!S.(enterica!must!resist!or! evade! multiple! levels! of! defense! to! survive! within! the! host.! For! this! purpose,! it! is! essential!that!the!bacterial!cells!are!able!to!sense!and!respond!rapidly!and!appropriately! to!the!vast!array!of!stresses!they!may!encounter.! After! entering! the! host! orally,! S.( enteric( encounters! the! acidic! pH! of! the! stomach.! Immediately! after,! survivors! reaching! the! intestine! must! face! reduced! oxygen! concentration,!bile!salts,!antimicrobial!peptides,!weak!acids!from!metabolic!products!of! resident! microbial! flora,! increased! osmolarity,! and! competition! with! resident! microorganisms!for!nutrients!and!space!30.!Furthermore,!serovars!able!to!adapt!and! survive! these! conditions! must! endure,! during! epithelium! colonization,! nutrient! depletion! within! the! M! cells.! Subsequent! release! into! the! intestinal! submucosa! is! followed!by!phagocytosis!by!resident!macrophages!allowing!the!bacteria!to!escape!host! humoral!defenses.!Within!the!macrophage,!the!salmonellae!reside!within!phagosomes! or!possibly!phagolysosomes,!which!present!many!dangers!to!the!invading!bacteria!(e.g.,! acidification,!nutrient!limitation,!generation!of!reactive!oxygen!and!nitrogen!species,! and!exposure!to!various!antimicrobial!peptides!(e.g.,!defensins).!Infected!macrophages! act!as!vehicles!for!dissemination!of!the!bacteria!throughout!the!host,!causing!a!systemic! infection!31,32.! A!relevant!consequence!of!systemic!infection!is!that!S.(enterica!reaches!and!colonizes! the!gallbladder,!a!highly!adverse!environment!for!bacterial!survival:!bile,!a!detergent[ like!compound!with!high!antimicrobial!activity,!is!concentrated!in!this!organ.!Further! ahead,!we!will!focus!on!the!strategies!that!S.(enterica(employs!to!survive!in!presence!of! this!substance.!! Introduction! 15 Table! I1! simplifies! different! environmental! stresses! Salmonella( endures! during! the! stages!of!host!infection!and!intestine!colonization,!and!the!primary!stress!responses!that! Salmonella(activates!to!survive.!! ! Table&I1.&Different&stresses&experienced&by&Salmonella&when&colonizing&a&susceptible& host,&and&main&genetic&loci&that&contribute&to&the&response&of&the&bacteria&to&tolerate& this&stresses.!Compiled!from!Rychlik!and!Barrow,!2005;!Alvarez[Ordonoez!et!al.,!2016! and!Spector!and!Kenyon,!2011!30,33,34. Environment& Stress&factor& Regulons&induced& Result& Before! entering! the! host! Cold!! Low! nutrients! csp(( rpoS,( rpoE,( cyaA,( crp.( General!stress!resistance.!! Activation! of! the! starvation[ stress!!response!(SSR)! Stomach! Extreme!acid! pH! rpoS,( fur,( phoPQ,( ompR,( adiA,( cadCBA.( General! stress! resistance.! Induction! of! acid! tolerance! proteins.! Decrease! of! intracellular! pH.! Activation! of! the! acid! tolerance! response! (ATR)! and! acid! resistance! mechanisms!(AR).!! Duodenum! Bile! phoPQ,( oxyR,( soxRS,( rec,( wecD,( wecA,(acrAB,(tolC( Membrane! modifications,! invasion! suppressed,! SOS! response,! DNA! repair,! efflux! activation,!adaptation.! Ileum! Decreased! O2!supply! ! Commensal! bacteria:! Short! chain! fatty! acids! (SCFAs),! Bacteriocins,! Competitive! flora!quorum! sensing! frn,(arcAB( ((((((((((((((((((((((((( (((((((((((((((((((((((((( rpoS,(sdiA,(luxS( ( Switch!from!aerobiosis!to! anaerobiosis,!defense!against! reactive!oxygen!and!nitrogen! intermediates.!! Acid! induced! resistance! to! SCFAs.! Sensing! of! AI[1.! production! of! AI[2.! Starvation! stress!response.!! Introduction! 16 Epithelium! Cationic! antimicrobial! peptides! (CAMPs)! phoPQ,(rpoE(( ( LPS!modifications,!resistance!to! macrophage!CAMPs.! After! leaving! the!host! Cold!shock!! Low! nutrients! Aerobiosis! csp,(rpoS( rpoS,( rpoE,( cyaA,( crp( arcAB,( fnr,( oxyR,( soxRS( General!stress!response! Activation! of! the! starvation[ stress!response!(SSR)! Switch! from! anaerobiosis! to! aerobiosis,! Protection! against! oxidative!stress! ! These!stress!responses!generate!a!resistant!state!that!allows!survival!and!persistence!in! the!different!and!changing!host!environments.!Thus,!these!stress!responses!and!the! survival! strategies! associated! with! them! can! have! a! profound! impact! on! the! epidemiology!and!pathogenesis!of!these!bacteria.!We!will!focus!on!the!stress!generated! by!bile!and!the!strategies!that!Salmonella(employs!to!endure!with!such!conditions.!! Salmonella&and&bile& Bile& Bile!is!a!thick,!bitter,!yellow[green!fluid!synthesized!in!the!liver!by!the!hepatocytes.!In! humans,!its!daily!production!is!approximately!0.5!to!1!liter,!and!its!pH!is!between!6.5! and!8! 35.! From! the! hepatocytes,! bile! is! transported! throughout! the! bile! duct! to! the! gallbladder,!where!bile!thickening!takes!place.!! Bile!serves!as!a!digestive!and!as!a!secretory!compound.!Although!bile!does!not!contain! digestive!enzymes,!it!plays!an!essential!role!in!lipid!digestion!and!absorption:!bile!is! released! into! the! duodenum! after! food! intake,! where! bile! acids,! which! are! potent! digestive!surfactants,!promote!lipid! absorption! 33.! This!occurs!as!acidic!and!partially! digested!fats!stimulate! the! secretion!of!the! enteric! hormone!cholecystokinin,!which! induces!contraction!of!the!gallbladder!and!the!discharge!of!bile.!In!addition,!bile!acts!as! an! excretory! agent! of! many! substances! and! metabolites! that! cannot! be! eliminated! Introduction! 17 efficiently!in!urine!because!they!are!insoluble!or!protein!bound!like!cholesterol,!bilirubin,! heavy!metals,!lipophilic!steroids!and!drug!metabolites!35–39.!! Bile!is!composed!by!both!organic!and!inorganic!compounds.!97%!of!bile!is!water,!but!it! also!has!bile!salts,!inorganic!ions!(Na+,!Cl[,!HCO3[),!cholesterol,!phospholipids!and!bile! pigments!(biliverdin).!This!fluid!is!rich!in!lipids!and!poor!in!proteins.!Aside!from!liver! isoenzymes!of!alkaline!phosphatase,!it!does!not!contain!enzymes.!Cholesterol!solubility! is!improved!by!phospholipids!and!bile!salts!through!the!formation!of!micelles!33,35,36.!! Bile! salts! are! produced! in! the! liver! from! cholesterol,! specifically! in! the! pericentral! hepatocytes.! Bile! salts! serve! as! a! cholesterol! degradation! products,! and! their! daily! production!is!approximately!350!mg.!Bile!salts!have!predominantly!24!carbon!atoms!and! a!complete!steroid!core!40.!Seventeen!different!enzymes!convert!cholesterol!into!bile! acids,!which!are!transformed!into!bile!salts!and!the!association!of!Na+!or!K+!ions!(this! also!takes!place!in!the!liver).!There!are!four!types!of!bile!salts:!primary!and!secondary,! conjugated!and!nonconjugated!35,41.!Conjugation!involves!the!formation!of!an!amide! bond!with!either!taurine!or!glycine.! Primary!bile!salts!are!the!immediate!products!of!the!conversion!of!cholesterol!into!bile! acids! and! their! structure! varies! widely! between! vertebrate! species.! For! instance,! in! humans!and!rats,!the!primary!bile!salts!are!cholate!and!chenodeoxycholate;!while!in! mice!they!are!cholate!and!muricholate!41.!! Secondary! bile! salts! are! produced! from! primary! bile! salts! by! intestinal! bacteria! by! removal!of!the!hydroxyl!group!at!C[7.!These!secondary!salts!are!absorbed!from!the!colon! and! recirculate! with! the! primary! bile! salts.! In! humans,! secondary! bile! salts! are! deoxycholate!(DOC,!from!cholate)!and!litocholate!(from!chenodeoxycholate)!36,42.! Although!other!bile!acids!are!present!in!human!bile!in!trace!proportions!43,!deoxycholate! and!litocholate!compose!>95%!of!all!biliary!bile!acids!38.!The!chemical!diversity!of!the! bile!acid!pool!is!further!expanded!by!enzymatic!actions!of!anaerobic!bacteria!in!the!gut,! which!convert!primary!bile!acids!into!dozens!of!secondary!and!tertiary!bile!acids!44.!The! variety!of!bile!acids!in!the!enterohepatic!circulation!ensures!complete!solubilization!of! hydrophobic!nutrients!in!the!small!intestine!41.! Introduction! 24 ! Figure& I5.& Multidrug& efflux& systems& in& Salmonella& enterica.! Diagrammatic! representation!of!the!structure!of!efflux!systems!and!their!location!on!the!membrane.! Antibiotic!substrates!are!also!listed!for!each!efflux!system.!Reproduced!from!Horiyama! et!al.,!2010!75.& ! AcrAB[TolC!is!the!best!characterized!efflux!system!in!enteric!bacteria!76–79.!This!efflux! pump!comprises!the!outer[membrane!channel!TolC,!the!secondary!transporter!AcrB! located!in! the! inner!membrane,!and!the! periplasmic! AcrA,!which!bridges! these! two! integral!membrane!proteins.!The!AcrAB–TolC!efflux!pump!is!able!to!transport!vectorially! Introduction! 25 a!diverse!array!of!compounds!with!little!chemical!similarity!80.!This!system!is!necessary! for!the!intrinsic!resistance!of!E.(coli!to!solvents,!detergents,!dyes,!lipophilic!antibiotics,! and!bile!salts.!In!S.(enterica,!deletion!of!the!acrA,(acrB!or!tolC!genes!confers!increased! susceptibility!to!antimicrobial!agents!and!chemical!compounds,!including!bile,!81.!Figure! I6!depicts!the!AcrAB[TolC!efflux!system!structure!and!location!within!the!bacterial!cell! membrane.! ! ! Figure&I6.&Schematic&drawing&of&the&tripartite&RND&multidrug&efflux&system&AcrAB8TolC& in&E.(coli.(Suggestions!on!the!stoichiometry!of!the!adaptor!AcrA!to!inner!membrane!RND! component!AcrB!(or!to!outer!membrane!channel!TolC)!vary!between!1!and!4.!AcrB!is!a! proton!force[dependent!drug!efflux!transporter,!and!AcrA!is!thought!to!aid!in!direct! cytoplasm!to!environment!efflux!by!bridging!the!inner!and!outer!membranes.!An!outer! membrane!protein,!TolC,!contributes!to!the!function!of!the!Acr!system.!Reproduced! from!Pos,!2009!83.!! ! The!genes!encoding!the!AcrAB[TolC!multidrug!efflux!system!are!under!the!control!of!a! transcriptional!regulator!known!as!RamA.!In!turn,!the!ramA( gene!is!transcriptionally! activated!by!bile,!mainly!by!inhibiting!transcriptional!repression!exerted!by!RamR!79,84.!! & [!DNA(repair(mechanisms( & DNA!adenine!methylase!(dam)!mutants!of!S.(enterica!are!bile[sensitive!85,!and!a!study! Introduction! 26 on!the!causes!of!this!phenotype!unveiled!the!involvement!of!Dam[directed!mismatch! repair! 63.! Mutations! in! any! of! the! mismatch! repair! genes! mutHLS!suppressed! bile! sensitivity! in! dam! mutants,! providing! evidence! that! bile! sensitivity! was! caused! by! MutHLS!activity.!An!analogy!between!these!observations!and!previous!studies!in!E.(coli! supported! the! view! that! DNA! strand! breakage! by! the! MutHLS! system! renders! dam! mutants!sensitive!to!bile!salts!(39).!Salmonella!mutHLS!mutants!are!not!sensitive!to!bile! (39),!indicating!that!bile[induced!DNA!damage!can!be!repaired!by!mechanisms!other! than!Dam[dependent!mismatch!repair.!In!dam!mutants,!however,!lack!of!DNA!strand! discrimination!causes!extensive!DNA!strand!breakage!when!the!MutHLS!systems!deals! with!bile[induced!lesions!63.! ! Surveys!of!bile!sensitivity!among!S.!enterica!DNA!repair!mutants!has!revealed!that!base! excision! repair! (BER),! SOS[associated! DNA! repair! and! recombinational! repair! by! the! RecBCD!enzyme!are!required!to!cope!with!bile[induced!DNA!damage!(40).!In!contrast,! nucleotide! excision! repair! (NER)! is! dispensable.! The! observation! that! bile! resistance! requires!BER!but!not!NER!suggests!that!bile[induced!DNA!lesions!are!unlikely!to!be!bulky,! thus!providing!indirect!support!for!the!oxidative!damage!hypothesis!63,64.!! ! Several! lines! of! evidence! suggest! that! bile! salts! may! impair! DNA! replication! in! Salmonella:!(i)(lexA(ind)!and!dinB!mutations!confer!bile!sensitivity,!suggesting!that!SOS[ associated!translesion!DNA!synthesis!may!be!required!to!overcome!bile[induced!DNA! damage!(40);!(ii)!recB,(recC,!and!recA(recD!mutants!are!also!bile!sensitive,!indicating!that! survival!to!bile!requires!RecB[dependent!homologous!recombination!64.!Primary!lesions! (e.!g.,!oxidized!cytosine!moieties)!can!be!expected!to!trigger!base!excision!repair,!and! the!activity!of!BER!exonucleases!will!produce!DNA!strand!breaks!as!an!intermediate!step! in!the!DNA!repair!process.!Furthermore,!as!indicated!above,!DNA!strand!breaks!can!also! be!formed!as!a!consequence!of!MutHLS!activity.!These!DNA!strand!breaks!may!impair! progression!of!DNA!replication!forks,!inducing!the!SOS!response;!as!a!consequence,! DinB[mediated!translesion!synthesis!may!occur.!It!is!also!conceivable!that!bile[induced! lesions!could!directly!block!DNA!replication,!thus!inducing!the!SOS!response!in!a!direct! fashion.!In!such!a!scenario,!the!need!of!homologous!recombination!mediated!by!the! RecBCD!enzyme!might!reflect!the!occurrence!of!stalled!DNA!replication!forks!64.! Introduction! 27 [!Salmonella(enterica(adaptation(to(bile( & Salmonella(enterica(can!be!adapted!to!growth!in!extremely!high!bile!concentrations!if! previously! exposed! to! sub[lethal! doses! 86.! This! increase! in! bile! resistance! levels! is! transitory:!removal!of!bile! from! the! medium!will!cause!that!the!levels! of! resistance! return!to!its!original!levels.!This!phenomenon!occurs!preferentially!if!the!bacteria!is! exposed! to! near! lethal! bile! concentrations! during! logarithmic! growth! phase! 47.! A! transcriptomic!analysis!performed!by!Prouty!et.(al((2004)!in!the!presence!of!a!sublethal! concentration! of! DOC! (3%)! identified! at! least! 230! Salmonella! genes! with! altered! expression!(about!3%!of!the!Salmonella!genome).!Of!these!genes,!101!were!activated! by!bile!and!129!were!repressed!by!bile!87.!In!turn,!a!study!carried!out!in!our!laboratory! in!the!presence!of!a!higher!concentration!of!DOC!(5%)!revealed!also!multiple!changes!in! gene!expression!88.!The!latter!study!proposed!that!activation!of!the!RpoS[dependent! general!stress!response!plays!a!crucial!role!in!the!adaptation!of!Salmonella!to!bile,!a!view! supported!by!the!observation!that!rpoS!mutants!are!bile[sensitive!88.!Adaptation!to!bile! involves!a!response!by!the!bacterial!population,!and!might!occur!during!the!infection! process,!specifically!along!the!passage!of!bacteria!through!the!host!intestine,!where!bile! is! released! in! low! concentrations.! This! exposure! might! allow! the! cell! to! endure! the! increasing!bile!concentrations!found!in!the!hepatobiliary!tract,!and!ultimately!in!the! gallbladder.!In!turn,!a!study!carried!out!in!our!laboratory!in!the!presence!of!a!higher! concentration!of!DOC!revealed!also!multiple!changes!in!gene!expression!88.!! ! ! 28 ! & ! 29 & & & & & & & & & & OBJECTIVES& & ! 30 & Objectives! 31 Previous!studies!of!our!laboratory!had!identified!cellular!functions!that!contribute!to! bile!resistance!in!Salmonella(enterica.(Such!functions!include!components!of!the!cellular! envelope,!stress!responses,!and!DNA!repair!networks.(These!studies!had!also!provided! evidence! that! the! phenomenon! known! as! "adaptation! to! bile"! (the! increase! of! bile! resistance!upon!exposure!to!sublethal!concentrations)!might!involve!multiple!changes! in!gene!expression.!This!Thesis!intends!to!understand!the!molecular!basis!of!adaptation! of! Salmonella( enterica! to! bile,! using! a! combination! of! classical! genetics,! molecular! biology,!single!cell!analysis,!and!infection!of!mice.!The!specific!objectives!are!as!follows:! ! Objective& 1.& Identification! and! characterization! of! Salmonella( enterica( functions! involved!in!adaptation!to!bile.! & Objective&2.&Investigation!of!the!roles!of!S.(enterica!efflux!pumps!in!bile!resistance!and! adaptation.! & Objective&3.&Characterization!of!S.(enterica!bile[resistant!isolates!from!the!gallbladder! of!BALB/c!mice.! & Objective&4.!Characterization!of!bile[resistant!mutants!of!S.(enterica(isolated!from!the! gallbladder!of!BALB/c!mice.! ! 32 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 33 & & & & & & & & & & & MATERIALS&AND&METHODS& & & Materials!and!methods! 40 This!method!does!not!yield!independent!transductants,!but!incubations!shorter!than!30! minutes!do!not!permit!transductants!to!divide,!so!the!proportion!of!twins!is!minimal.! On!the!other!hand,!it!has!the!advantages!of!being!fast!and!repetitive.!Using!dilutions!of! the!transduction!mixture,!a!number!of!multiplicities!of!infection!can!be!tested!for!the! same!transduction.!! Detection&of&lysogenic&transductants&& Transductans!harboring!a!selective!marker!could!have!been!infected!by!P22!phage!and! become!pseudolysogenic!(the!int!mutation!avoids!integration,!and!delays!the!formation! of!true!lysogens).!As!time!goes!on,!pseudolysogens!become!resistant!or!immune!to!new! P22!infections!and!cannot!be!lysed!or!transduced!again.!Pseudolysogeny!should!thus!be! avoided.! For! this! purpose,! transductant! colonies! were! isolated! in! EBU! plates! (with! antibiotics!if!necessary).!In!these!plates,!pseudolysogens!are!dark!colored!and!P22[free! colonies!are!light!colored.!This!color!difference!is!due!to!cell!lysis!in!the!pseudolysogenic! colony,! which! causes! acidification! of! the! medium! and! turning! of! the! pH! indicator,! darkening!the!agar.!A!transductant!was!considered!P22[free!when!streaking!did!not!give! rise!to!any!dark!colony.!! P22&sensitivity&assay&& In!EBU!plates,!isolates!that!forms!light!color!colonies!could!be!lysogens!that!do!not! undergo!visible!lysis.!These!isolates!are!P22[resistant!and!can!be!mistaken!by!real!P22[ free!isolates.!To!avoid!this!situation,!an!assay!to!detect!P22[sensitive!strains!is!advisable.! A!streak!with!a!P22!H5!lysate!is!done!on!an!LB!or!EBU!plate,!and!air[dried.!The!test!strain! is!then!streaked!in!a!perpendicular!way!to!the!H5!streak.!P22[sensitive!strains!grow!until! they!reach!the!H5!streak,!while!P22[resistant!strains!grow!over!the!streak.! ! DNA&manipulation&and&transfer&& Plasmids&& Plasmids!used!in!this!study!are!listed!in!Table!M3.!! ! Materials!and!methods! 41 Table&M3.&List&of&plasmids&used&in&this&thesis.&& Plasmid& Description& Reference& pCP20! bla!cat!cI857!λPR!flp!pSC101!oriTS,!Apr,!Cmr! Cherepanov!and! Wackernagel,!1995!91! pKD3! bla!FRT!cat!FRT!PS1!PS2!oriR6K,!Apr!,!Cmr! Datsenko!and!Wanner,! 2000!92! pKD4! bla!FRT!aph!FRT!PS1!PS2!oriR6K,!Apr!,!Kmr! Datsenko!and!Wanner,! 2000!92! pKD13! bla!FRT!aph!FRT!PS1!PS4!oriR6K,!Apr!,!Kmr! Datsenko!and!Wanner,! 2000!92! pKD46! bla!PBAD!gam!bet!exo!pSC101!oriTS,!Apr! Datsenko!and!Wanner,! 2000!92! pBR328! Cloning!vector!Ampr,!Kmr,!Cmr! Bolivar!and!Backman,! 1979!93! Extraction&of&plasmid&DNA&& For!the!extraction!of!plasmid!DNA!the!commercial!system!GenElute™!Plasmid!Miniprep! Kit,!provided!by!Sigma[Aldrich!Co!(St.!Louis,!Missouri,!USA),!was!used.!! Extraction&of&genomic&DNA&& For! the! extraction! of! genomic! DNA,! 5! ml! of! cells! grown! in! exponential! phase! were! collected!and!re[suspended!in!0.4!ml!of!lysis!buffer,!4!μl!of!RNAse!(10!mg/ml)!were! added! and! the! mixture! was! incubated! at! 37°!C! for! 30! minutes.! After! that,! 20! μl! of! proteinase!K!(20!mg/ml)!was!added!and!the!sample!was!incubated!for!2!h!at!65°!C.! Finally,!3!or!4!extractions!were!performed!with!phenol:chloroform[isoamyl!alcohol!in!a! 2:1!proportion.!Optionally,!one!last!extraction!with!chloroform:isoamyl!alcohol!(24:1)! can!be!performed.!DNA!was!precipitated!at![20°!C!by!adding!1/10!volume!of!sodium! acetate!3!M!and!2.5!volumes!of!ethanol.!After!precipitation,!genomic!DNA!was!washed! with!70!%!ethanol!and!re[suspended!in!20!μl!of!TER!buffer.!! •!Buffer!lysis!! Tris[HCl! ! ! 50!mM!pH!8!! EDTA!!!!!10!mM! NaCl!! ! ! ! 100!mM! SDS!!!!!0.2!%! Materials!and!methods! 42 ! •!TER:!! Tris[HCl!! ! ! 10!mM!pH!7.5! EDTA!! ! ! ! 1!mM!pH!8! RNAse!!!!20!μg/ml! Digestion,&modification&and&ligation&of&DNA&fragments&& Restriction! endonucleases! were! supplied! by! Roche! Diagnotics! GmbH! (Indianapolis,! Indiana,!USA),!New!England!Biolabs!(Beverly,!Massachusetts,!USA)!and!Promega!Biotech! (Madison,! Wisconsin,! USA).! In! each! case,! enzymes! were! used! following! the! manufacturer’s!instructions.!! For!ligation!of!DNA!fragments,!1!U!of!T4!DNA!ligase!(1U/μl,!Roche!Diagnostics)!was!used! in!the!buffer!supplied!by!the!manufacturer.!Routinely,!the!mixture!was!incubated!at!16°! C!for!12!hours!at!least.!! Agarose&DNA&gel&electrophoresis&& Electrophoresis!in!an!agarose!gel!was!used!to!test!the!quality!of!DNA!extraction,!to! determine! DNA! fragments! after! plasmid! restriction,! to! estimate! the! efficiency! of! endonuclease! restriction,! to! confirm! PCR! amplification,! etc.! The! agarose! gel! was! submerged!in!TAE!1x!buffer.!! Low! Electro! Endosmosis! agarose! (Pronadisa,! Conda,! Spain)! was! employed.! Its! concentration!varied!between!0.8!and!1.5!%!depending!on!the!size!of!the!fragments!to! be!separated.!The!loading!buffer!used!was!a!solution!of!bromophenol!blue!(0.125%)!and! Ficoll!400!(12.5%).!! The!1!Kb!ladder!(GIBCO!BRL,!Life!Technologies,!New!York,!USA)!was!used!as!molecular! weight!marker.!Samples!were!mixed!with!1/10!of!loading!buffer.!Ethidium!bromide!(0.5! μg/ml! final! concentration)! was! added! to! the! gels! to! make! bands! visible.! Gels! were! illuminated!with!a!UV!transilluminator;!pictures!were!taken!with!a!Polaroid!ISO3000/36! snapshot!film.!! Materials!and!methods! 43 •!TAE! Tris[acetate!! ! ! 40!mM!! EDTA! ! ! ! 10!mM!pH!7.7!! Isolation&of&DNA&fragments&from&agarose&gels&& For!the!isolation!of!DNA!fragments!from!agarose!gels!was!used!the!commercial! system!Wizard®!SV!Gel!and!PCR!Clean[Up!System!supplied!by!Promega!Co.!! Bacterial&transformation&& High&efficiency&E.(coli&transformation&& Competent! cells! were! prepared! using! a! variation! of! the! Inoue! method! 94,! which! guarantees!high!transformation!efficiency!(between!5x107!and!5x108!transformants!per! μg!of!plasmid!DNA).!An!overnight!culture!of!E.(coli!DH5α!was!diluted!100[1000!times!in! 200!ml!of!SOB,!and!incubated!at!22°!C!and!200!rpm!until!the!OD600!reached!0.5.!The! culture!was!chilled!quickly!on!ice!and!kept!on!it!for!10!minutes.!Cells!were!harvested!by! centrifugation!at!2500!g!and!4°!C!for!10!min.!The!pellet!was!re[suspended!in!20!ml!of! cold!TB,!and!1.5!ml!of!DMSO!was!added.!After!a!10!min!incubation!on!ice,!aliquots!of!0.2! ml!or!0.5!ml!were!prepared,!frozen!in!liquid!nitrogen,!and!stored!at![80°!C.!! For!transformation,!an!aliquot!of!competent!cells!was!slowly!thawed!on!ice!and!was! mixed!with!the!plasmid.!The!mixture!was!incubated!on!ice!for!30!minutes,!subjected!to! heat!shock!(42°!C,!45!s),!and!cooled!on!ice!for!1!min.!One!ml!of!LB!was!then!added.!The! mixture!was!incubated!at!37°!C!for!1!h;!finally,!the!cells!were!concentrated!in!100!μl!and! spread!on!selective!media.!! •!TB!! PIPES! ! ! ! 10!mM! CaCl2!!!!!15!mM! KCl!!!!!250!mM! pH!6.7with!KOH! MnCl2! ! ! 55!mM! Materials!and!methods! 44 ! This!solution!was!sterilized!by!filtration.! Bacterial&electroporation&& An!overnight!culture,!was!diluted!1/100!in!LB!and,!depending!on!the!strain,!was!grown! at!37°!C!or!30°!C!until!a!0.6[0.8!OD600!was!reached.!The!culture!was!chilled!on!ice!and! kept!on!it!for!5!minutes.!25!ml!were!transferred!to!a!tube,!and!the!cells!were!harvested! by!centrifugation!at!4000!rpm!for!5!min!at!4°!C.!The!supernatant!was!discarded!and!the! bacterial!pellet!was!softly!re[suspended!in!1!ml!of!cold!ddH2O.!Afterwards,!24!ml!of!cold! water!were!added;!this!wash!was!repeated!a!second!time.!Finally,!cells!were!harvested! and!re[suspended!in!250!μl!of!water.!! Electroporation!was!performed!by!mixing!1!μl!of!plasmid!DNA!or!10!μl!of!PCR!product! with!40!μl!of!competent!cells.!The!mixture!was!transferred!to!a!cooled!cuvette!with!2! mm!of!distance!between!the!plates.!The!cuvette!was!subjected!to!an!electric!discharge! in!the!electroporator!(2.5!KV,!200!Ω!and!25!μF).!The!electroporator!employed!was!a!BTX! Electrocell!Manipulator!600!(Harvard!Apparatus,!Holliston,!Massachusetts,!USA).!After! the!discharge,!1!ml!of!LB!was!added!to!the!cells,!which!were!then!transferred!to!a!10!ml! plastic!tube!and!incubated!at!37°!C!with!shaking!(200!rpm)!for!1!h.!Finally,!cells!were! concentrated!in!100!μl!and!spread!on!selective!media.!! Genetic&screens& T8POP&transposon&genetic&screen& T[POP! transposon! mutagenesis! was! done! using! the! strain! wt! strain! SL1344! as! the! recipient,!the!transducing!phage!P22!was!used!as!the!vector!of!choice!to!insert!the! transposon!into!cells,!the!aim!of!this!procedure!was!finding!bile!resistant!phenotypes!by! insertion!the!T[POP,!either!by!gene!inactivation!caused!by!its!disruption!or!by!increased! expression!of!the!surroundings!of!the!T[POP!insertion!site!95,96.Nine!pools!of!T[POP3! insertions,! which! were! previously! prepared,! were! used! to! generate! mutagenesis! by! chromosomal!homologous!recombination!within!the!recipient!strain.!! Materials!and!methods! 45 Briefly,!to!obtain!these!pools!a!lysate!carrying!a!T[POP3!(TetR)!insertion!in!an!F’!plasmid! was!prepared.!This!lysate!was!used!to!transduce!a!strain!(S.(enterica(wild!type!strain)! carrying!the!pNK2880!plasmid!97.!This!plasmid!produces!ATS!Tn10!transposase,!allowing! T[POP3! transposition! into! the! recipient! chromosome.! As! a! result,! all! tetracycline[ resistant!mutants!isolated!will!inherit!T[POP3!by!transposition!96.!ATS!transposase!is!less! specific!than!the!wild!type!transposase!of!Tn10.!During!transduction,!the!transposon! enters!the!receptor!strain!inserted!in!a!F’!fragment,!and!cannot!recombine!with!the!host! chromosome!because!there!is!no!homology.!! To! obtain! an! independent! collection! of! insertions! of! the! T[POP3! transposon,! the! following!steps!were!followed:!(i)!10!μl!of!a!T[POP3!(TetR)!lysate!were!transduced!to!100! μl!of!the!strain!harboring!pNK2880;!(ii)!transductions!were!incubated!at!37°!C!for!30! minutes;!(iii)! transductions! were! spread!in!LB! plates! and!incubated!for! 4! hours;!(iv)! replicates!were!made!in!LB!with!Tet!and!EGTA!10!mM!(compound!that!chelates!Ca2+!ions! and! avoids! the! P22! capsid! assembly,! by! this! way,! reinfections! are! avoided! and! the! probability! of! appearance! of! pseudolysogenics! decreases)! and! incubated! at! 37°!C! overnight;!(v)!transductant!colonies!were!recovered!with!1!ml!of!LB!with!EGTA.!Colonies! of!various!plates!can!be!mixed;!usually,!collections!are!made!with!2000[5000!colonies;! (vi)!1!ml!of!cells!were!frozen!with!75!μl!of!DMSO!at![20°!C;!(vii)!collection!tubes!were! thawed!in!ice!and!diluted!1:25!in!5!ml!of!LB!with!Tet!and!EGTA;!(viii)!cells!were!incubated! for!1.5!hours!at!37°!C,!and!centrifuged!at!13000!rpm!for!3!minutes.!The!pellets!were! washed!3[4!times!with!LB!to!eliminate!EGTA;!(ix)!cells!were!re[!suspended!in!1!ml!of!LB;! and!(x)!a!lysate!was!prepared!as!described!previously.! These!P22!phage!lysate!pools,!with!independent!T[POP3!chromosomal!insertions,!can! be!used!in!subsequent!crosses!involving!cells!that!do!not!express!transposase,!therefore! the!insertions!will!be!inherited!by!homologous!recombination.!! pBR3288based&plasmid&library&genetic&screen& A! S.( enterica( genomic! library! cloned! into! pBR328! plasmid! was! used! to! screen! bile! sensitive!phenotype!suppressors!on!the!acrA::Km(strain!background!(SV7248).!! This! library! was! previously! constructed! by! partial! digestion! of! genomic! DNA! from! Materials!and!methods! 46 Salmonella(enterica(Serovar!Typhimurium!SL1344!with!Sau3A.!DNA!fragments!7[11!kb! long!were!ligated!to!the!pBR328!vector,!which!was!previously!digested!with!BamHI!and! dephophorylated.!Salmonella(strain!TR5878!was!transformed!with!the!ligation!products,! and!ampicillin[resistant!clones!were!selected!on!LB!Ap!plates.!Pools!of!approximately! 1000!independent!transformants!were!collected!and!lysed!with!phage!P22!HT!105/1! int201.!As!a!quality!control,!the!ability!of!the!library!pools!to!complement!null!mutations! in! araA( (required! for! growth! with! L[arabinose! as! the! sole! carbon! source)! or! xylA( (required!for!growth!with!D[xylose!as!the!sole!carbon!source)!was!tested.!Lysates!that! permitted!successful!complementation!were!stored!and!used!for!plasmid!delivery!to! recipient!strains!in!subsequent!genetic!screens.! The!genetic!screen!was!done!by!mixing!100!µl!of!a!saturated!acrA::Km(culture!with!10! µl! of! each! P22! lysate! of! the! pBR328! plasmid! library! pools.! After! 45! minutes,! the! transductions! were! plated! in! LB! Cm! 0.5%! DOC! agar! plates! and! incubated! at! 37°! C! overnight.!There!were!9!different!pools,!and!10!transductions!were!made!out!of!each!of! the!pools!(90!transductions!total).!!As!a!control,!5!additional!transductions!using!as!the! recipient!the!wt!SL1344!strain!were!made!from!aleatory!pBR328!pools,!they!were!also! plated!in!LB!Cm!0.5%!DOC!agar!plates!and!incubated!at!37°!C!overnight.! The!following!day,!the!acrA(background!colonies!obtained!were!and!picked!in!LB!Cm! agar!plates!containing!increasing!DOC!concentrations!(from!1!to!7%).!The!candidates! that!showed!an!increased!level!of!DOC!resistance!were!cultured!in!LB!Cm!liquid!medium,! Minipreps!were!done!to!recover!the!pBR328!plasmid!derivatives!and!sent!out!for!DNA! sequencing!of!the!inserts.!! Construction&of&bacterial&strains&& Oligonucleotides& Oligonucleotides!used!in!amplification!and!sequencing!were!provided!by!Invitrogen!Life! Technologies!and!are!listed!in!Table!M4.!! ! ! Materials!and!methods! 47 Table&M4.&Oligonucleotides&used&in&this&study.& Oligonucleotide& name& Sequence&5’83’& MR[MLE[P4[ FOR! TGTATCACGAACCTGATAAATGGAGAAATTATGAAAA TTAATTCCGGGGATCCGTCGACC! MR[MLE[P1[ REV! GCATAAACCATAATCAGTATCACCAGATGCTATTTAAT CGTGTAGGCTGGAGCTGCTTC! MR[MLE[E1! GTTCAAGTCTTGGCGGATTC! MR[MLE[E2! CAATACCTCCCGTGTATACC! rtn[P4[FOR! AATGATTTGAAAGTAAGAAAGCTATTTCTGATGTTGTT TAATTCCGGGGATCCGTCGACC! rtn[P1[REV! TGAATTTTTTTCCGTAGCGAGTTCCTGCATTATTCAGC CGTGTAGGCTGGAGCTGCTTC! rtn[E1! CTAACGATAGTATCCTGCACC! rtn[E2! CAGATATCGTTATCGACTCTCCGG! sptP[P4[FOR! AGGAAGCGCTCAAAAACATACTGCAGGAATATGCTAA AGTATTCCGGGGATCCGTCGACC! sptP[P1[REV! GATAGTTCTAAAAGTAAGCTATGTTTTTATCAGCTTGC CGTGTAGGCTGGAGCTGCTTC! sptP[E1! TCTATCTGGCGGCAGATTATGG! sptP[E2! TTGGCGCTTATAATGCCG! udg[P4[FOR! TCATAATTAAGTTAATTCTGAGAGCGAATAATGAAAA TCAATTCCGGGGATCCGTCGACC! udg[P1[REV! TAAATCATCGGGACTCATCAGCCCCGATTGCCTTATTA AGTGTAGGCTGGAGCTGCTTC! udg[E1! GAATTGTTTTCGTCTGCGTTGCAC! udg[E2! CAATCTGTTCCGGATCGTTC! ulaA[P4[FOR! TAAACAATAATTATTGCTGGCGAGGTGGATATGTTTA TCCATTCCGGGGATCCGTCGACC! ulaA[P1[REV! AATATCGCGCGCCAGTTGGGTGATTTCGGTTACGTTC ATGTGTAGGCTGGAGCTGCTTC! ulaA[E1! AGGTGGAACACTCCGATCTTTC! ulaA[E2! ATGGCCTTTCGACAGTACGAAG! yfbQ[P4[FOR! GTGGCAATAACAGCTAAAAGGTCAAGATTCATGTCCC CCAATTCCGGGGATCCGTCGACC! yfbQ[P1[REV! CAGGCTACCGAAGTAGCCTGAATTTAAGATTATTGAT GGGTGTAGGCTGGAGCTGCTTC! yfbQ[E1! AACTCACCGTCGTGGGAGTAC! yfbQ[E2! TGCAGACTATGTTCGGAGAC! Materials!and!methods! 48 yohI[P4[FOR! AGCGCCTCTTTTTTTGCTGTGGATACCGATATGCGTGT TTATTCCGGGGATCCGTCGACC! yohI[P1[REV! CCCGGCACACGTTCTGGACGCTGTAGAGATTAGATTT TAGTGTAGGCTGGAGCTGCTTC! yohI[E1! TGTGGAAAGGGAAATCGC! yohI[E2! GCAGCGATAGTATCTG! acrA[E1! ACA!TCC!AGG!ATG!TGT!TGT!CG! acrA[E2! CAC!TGT!TGG!AGG!ACA!TAT!AC! acrA[P1[FOR! GAC!CAA!TTT!GAA!ATC!GGA!CAC!TCG!AGG!TTT!ACA!TAT!GAA!CTG! TAG!GCT!GGA!GCT!GCT!TCG!! acrA[P2[REV! AGA!AAT!TAG!GCA!TGT!CTT!AAC!GGC!TCC!TGT!TTA!AGT!TAA!GCA! TAT!GAA!TAT!CCT!CCT!TAG! acrB[E2! GAC!AGG!AGA!AAA!TAG!CCA!GG! acrB[E1! GTT!GGC!GCT!GAT!AAC!AAA!GTG! acrB[P1[FOR! GTC!TTA!ACT!TAA!ACA!GGA!GCC!GTT!AAG!ACA!TGC!CTA!ATT!TCT! GTA!GGC!TGG!AGC!TGC!TTC!G! acrB[P2[REV! CTT!GCG!CGG!CCT!TAT!CAA!CAG!TGA!GCA!AAT!CAG!CGA!TGT!TCC! ATA!TGA!ATA!TCC!TCC!TTA!G! acrD[E1! CCAACAAGGAAGAGAGTCAG! acrD[E2! TTGAACGTGAACTGGGGAAC! acrF[P4[FOR! CTGCAGCGAAAACGGCGCAATAAGGTAACCGTACATGGCAATTCCGG GGATCCGTCGACC! acrF[P1[REV! AGGCGTCCGAAGACGCCTCTGTTTACCGGTTAATCATGATTGTAGGCT GGAGCTGCTTCG! acrF[E1! GCTTCCCGGTATGTTTGTTC! acrF[E2! GTATTGGTATGAACCAGCCG! emrA[E1! CTG!GTC!ATC!ACT!TAG!CAC!AG!! emrA[E2! GAC!TTC!GCC!AAA!ACG!TTT!CG!! emrA[P1[FOR! CAG!CCG!ATG!TGA!ATA!ATA!AGA!TCG!TGG!AGA!ACA!ACA!TGA! GTG!TAG!GCT!GGA!GCT!GCT!TCG!! emrA[P2[REV! GCG!GTT!TTT!GCT!GTT!GCA!TCA!CAC!GCA!CCT!CAG!GAT!TAA!CCA! TAT!GAA!TAT!CCT!CCT!TAG! macA[E1! GTA!ATC!TGA!AGT!GTA!CCA!GG! macA[E2! GAC!AAC!AGA!TGG!TAG!CGC!TG!! macA[P1[FOR! TTA!CTT!TCT!GGT!!TAA!AAT!TTT!GCC!GTC!AGG!GTT!TCT!ATG!CTG! TAG!GCT!GGA!GCT!GCT!TCG!! macA[P2[REV! TAC!GAC!TCA!CAT!TGC!ACA!GTT!CAA!GCA!ATG!CCG!TCA!TGG!CCA! TAT!GAA!TAT!CCT!CCT!TAG! mdfA[E1! GTA!ACA!GGG!AGG!AAA!AGC!AG!! mdfA[E2! CGG!CAA!CGA!TAT!TGA!GAT!GC! Materials!and!methods! 49 mdfA[P1[FOR! CAC!TCT!GCG!CGG!TTT!TTA!TTG!GCG!AAG!AGA!TGG!TAT!GCA!GTG! TAG!GCT!GGA!GCT!GCT!TCG!! mdfA[P2[REV! GAT!AAC!AGA!AAG!CTT!ATC!AGG!CGT!AGC!CGC!TCT!CTC!AGA!CCA! TAT!GAA!TAT!CCT!CCT!TAG! mdsA[E1! TAT!TCA!GGC!TGC!ACG!GTT!C! mdsA[E2! GAC!CAT!CAA!TGG!CCA!TCT!G!! mdsA[P1[FOR! GGA!AAC!GAC!ATG!CGT!AGA!ACA!TTC!AAA!ATT!ATG!TTG!ATA!GTG! TAG!GCT!GGA!GCT!GCT!TCG!! mdsA[P2[REV! GAT!GGG!GCG!TGC!AAT!GAA!AAA!GTG!GGT!GAA!TTT!CAT!TGG! CGC!CAT!ATG!AAT!ATC!CTC!CTT!AG! mdtA[E1! GAA!ATC!GGT!GGC!TTA!CCT!TC! mdtA[E2! GAC!ATC!TGC!TTC!AGT!CCT!GA!! mdtA[P1[FOR! CGC!AAA!ACG!TTT!CAG!GAT!GAG!AAA!CTT!ATA!CCG!ATG!AAA!GTG! TAG!GCT!GGA!GCT!GCT!TCG! mdtA[P2[REV! GGC!CGC!CCG!TGC!TGC!CCG!GAG!GTA!ATA!CCT!GCA!TCA!GGC! GCA!TAT!GAA!TAT!CCT!CCT!TAG! mdtK[E1! CCAGCATGTAGTCAGGTAAC! mdtK[E2! TATGCTTCTTCAGGCCACTG! mdtK[P1[FOR! TACGTGCAGAAGTATACCAGTGAAGCGCGTCAGTTATTAGTGTAGGC TGGAGCTGCTTCG! mdtK[P2[REV! CCATGCGACGGCTATCGTCGTATTATCGCGCAGCGCGCTGCATATGA ATATCCTCCTTAG! RT[acrA[FOR! TTTGCGCGCCATCTTCCC! RT[acrA[REV! GACGTGCGCGAACGAAC! acrB[RT[PCR! FOR! CGCTGGGCGTATCTATTAGC! acrB[RT[PCR! REV! CGCGATCGATAAAGTCGTTT! RT[acrD[FOR! GCTGCACAAAGGCGAACA! RT[acrD[REV! TACGATTGAAGGTACGGTTAAACCA! RT[acrF[FOR! TGTCGCCTCTAACATTAAGGATTCT! RT[acrF[REV! CGTACTGTGCGCCAAATAGCT! RT[emrA[FOR! TCGGTCTGGATATGGGAACAG! RT[emrA[REV! AGTTACCCGTCGCATTTTGC! RT[macA[FOR! GGTATCGCAACAGGATCTGGAT! RT[macA[REV! GCCAATACGCGCCTGTTTA! RT[mdfA[FOR! CCGCGATTCAGGAGTCCTT! RT[mdfA[REV! CGCGATTAACGCCACGTT! RT[mdsA[FOR! CCGTGCTGATTGACGATCT! RT[mdsA[REV! CTCCACCTGATTCTCCTTGC! Materials!and!methods! 56 the!candidates!were!selected,!gDNA!was!isolated!as!described!and!the!samples!were! sent!for!whole!genome!sequencing.!As!a!control,!our!laboratory!wt!strain!genome!was! also!sequenced.! DNA&sequencing&& Plasmid!DNA!and!chromosomal!DNA!obtained!by!PCR!were!sequenced!in!the!sequencing! service!of!Stab!Vida!(Oeiras,!Portugal).!! ! Whole!bacterial!genome!sequencing!was!done!by!the!Biology!Sequencing!Service!in!the! University!of!Seville,!(Centros!de!Investigación,!Tecnología!e!Innovación!CITIUS,!Edificio! Celestino!Mutis,!Seville,!Spain).!As!a!control,!the!genome!of!the!laboratory!stock!of!S.( enterica(SL1344!was!also!sequenced.!Briefly,!draft!genome!sequences!were!obtained! using!Roche!454!FLX+!technology!on!a!GS!FLX!titanium!system!(Roche!Diagnostic,!GmbH)! 100.! Samples! were! prepared! according! to! the! GS! Rapid! Library! Preparation! Method! Manual.! Emulsion! PCR! and! 454! pyrosequencing! were! performed! following! the! manufacturer’s!instructions!101.!! ! DNA&sequence&analysis&& Bioinformatics! analysis! of! DNA! sequences! was! performed! using! the! algorithms! of! molecular! biology! of! the! National! Center! for! Biotechnology! Information! (NCBI)! at! www.ncbi.nlm.nih.gov.!! ! Whole! genome! sequences! were! analyzed! in! collaboration! with! the! Institute! of! Integrative! Biology,! Department! of! Functional! and! Comparative! Genomics! in! the! University! of! Liverpool.! The! Burrow[Wheeler! Alignment! tool! (BWA)!102! was! the! algorithm!of!choice!for!such!purpose.!The!first!step!was!the!alignment!of!the!454!GS! FLX+!/!XL+!sequence!reads!to!the!reference!genome,!in!our!case!the!S.(enterica(SL1344! genome!sequence!(ftp://ftp.sanger.ac.uk/pub/pathogens/Salmonella/STmSL1344.dbs),! to! identify! DNA! sequence! differences.! The! alignment! tool! BWA! consists! of! three! algorithms:!BWA[backtrack,!BWA[SW!and!BWA[MEM.!The!first!algorithm!is!designed!for! Materials!and!methods! 57 Illumina!sequence!reads!up!to!100bp,!while!the!rest!two!for!longer!sequences!ranged! from!70bp!to!1Mbp!102,103l.!In!our!case,!the!BWA[MEM!algorithms!was!employed,!which! is!recommended!for!high[quality!queries!as!it!is!faster!and!more!accurate.!BWA[MEM! also!has!better!performance!than!BWA[backtrack!for!70[100bp!Illumina!reads!(Li!and! Durbin,! unpublished).! The! genome! sequence! of! the! laboratory! stock! of! S.( enterica( SL1344!was!also!analyzed!and!!as!a!control.!! ! RNA&manipulation&& RNA&extraction&based&in&TRIsure™&method& To!prepare!cells!for!RNA!extraction,!5!ml!of!fresh!LB!±!5%!DOC!was!inoculated!with!100μL! from!an!overnight!bacterial!culture,!and!incubated!with!shaking!at!37°C.!Three!biological! replicates!were!performed!for!each!strain.!For!exponential!phase!cultures!(O.D.600!≈!0.6),! a!2!ml!aliquote!from!each!culture!was!saved!1.5!and!3h!later,!depending!if!the!culture! was!in!LB!or!in!LB!+!5%!DOC,!respectively;!for!stationary!phase!cultures!(O.D.600!≈!2),!a!2! mL! aliquote! of! o/n! cultures! was! saved! (>12h).! The! samples! were! centrifuged! at! 13000rpm,!4°!C!for!2!minutes!(Centrifuge!5415!R,!Eppendorf).!The!pellet!was!washed! three! times! with! NaCl! 0.9%! and! resuspended! in! 100! µl! of! a! solution! of! lysozyme! (3mg/ml;!Sigma!Chemical!Co.).!Cell!lysis!was!facilitated!by!a!freeze[thaw!cycle.!After! lysis,!RNA!was!extracted!using!1!ml!of!TRIsure!reagent!(Bioline,!Taunton,!Massachusetts,! USA)!was!added,!and!the!preparation!was!incubated!for!5!min!at!room!temperature.! Samples!were!centrifuged!at!4°!C!at!13000!rpm!for!10!min.!The!supernatants!were! recovered!and!poured!out!in!clean!tubes!(eliminating!the!genomic!DNA!in!this!step).!200! μl!of!chloroform!were!added,!and!the!samples!were!vortexed!for!15!s!and!centrifuged! for!15!min!at!4°!C!and!13000!rpm.!The!supernatants!were!carefully!recovered,!avoiding! recovering!the!interphase,!and!transferred!to!clean!tubes.!500!μl!of!isopropanol!were! added.!Samples!were!mixed!by!inversion!2[3!times!and!they!were!incubated!at!room! temperature!for!10!min,!followed!by!a!10!min!centrifugation.!The!supernatants!were! discarded! and! the! pellets! were! washed! with! cold! 70%! ethanol.! The! samples! were! centrifuged!at!4°!C!and!13000!rpm!for!5!min.!The!supernatants!were!discarded!and! pellets!were!air[dried!and!then!kept!at![20°!C!until!use.!The!pellets!were!re[suspended! Materials!and!methods! 58 in!30!μl!of!RNAse[free!H2O!(autoclaved!ddH2O!is!enough).!To!obtain!a!homogeneous! mixture,! the! samples! were! incubated! at! 65°!C! for! a! few! minutes.! The! quantity! and! quality!of!the!RNA!was!determined!using!a! ND[1000!spectrophotometer!(NanoDrop! Technologies).!!! RNA&phenol&extraction&& Whenever! RNA! samples! were! not! clean! enough,! they! were! subjected! to! phenol! treatment.!First,!ddH2O!was!added!until!a!final!volume!of!150!μl.!The!same!volume!of! phenol!was!then!added!and!the!preparation!was!mixed!by!vortexing.!The!samples!were! centrifuged!at!4°!C!and!13000!rpm!for!5!min.!The!aqueous!layer!was!recovered!in!a!clean! tube.!The!same!volume!of!chloroform:!isoamyl!acid!(24:1)!was!then!added.!The!samples! were!mixed!by!vortexing!and!centrifuged!at!4°!C!at!13000!rpm!for!5!min.!The!aqueous! layer!was!recovered!and!2.5!volumes!of!ethanol!and!1:10!volumes!of!sodium!acetate!3! M!pH!5.2!were!added.!The!samples!were!precipitated!at![20°!C!for!at!least!30!min.!After! precipitation,!the!samples!were!centrifuged!at!4°!C!and!13000!rpm!for!30!min,!and! washed!with!ethanol!70!%.!! Quantitative&RT8PCR&(qRT8PCR)&& Retrotranscription!was!performed!using!QuantiTect®&Reverse!Transcription!Kit!(Qiagen,! Venlo,! The! Netherlands)! following! manufacturer’s! instructions.! Quantitative! RT[PCR! reactions!were!performed!in!a!LightCycler!480!II!apparatus!(Roche).!Each!reaction!was! carried! out! in! a! total! volume! of! 10! μl! on! a! 480[well! optical! reaction! plate! (Roche)! containing!5!μl!SYBR,!0.5!μl!DYE!II!(Takara,!Japan),!4.6!μl!cDNA!(1/10!dilution)!and!two! gene[!specific!primers!at!a!final!concentration!of!0.2!mM.!Real[time!cycling!conditions! were!as!follows:!(i)!95°C!for!10!min!and!(ii)!40!cycles!at!95°C!for!15s,!60°C!for!1min.!A! non!RT!control!was!included!for!each!primer!set.!Triplicates!were!run!for!each!reaction,! and! the! Ct! value! is! averaged! from! them.! The! expression! of! the! target! genes! was! normalized!to!the!expression!of!a!constitutive!gene!used!as!internal!control.!Absence!of! primer!dimers!was! corroborated!by!running!a!dissociation!curve!at!the!end!of!each! experiment! to! determine! the! melting! temperature! of! the! amplicon.! Melting! curve! analysis! verified! that! each! reaction! contained! a! single! PCR! product.! Gene[specific! Materials!and!methods! 59 primers!were!designed!with!Primer3Plus!software!(http://www.bioinformatics.nl/cgi[ bin/primer3plus/primer3plus.cgi/)!and!are!listed!in!Table!M4.! Quantification&of&qRT8PCR&results&& For!quantification,!the!efficiency!of!each!primer!pair!was!determined!to!be!between! 90%[110%,! following! the! instructions! for! efficiency! determination! described! in! the! “Guide! to! Performing! Relative! Quantification! of! Gene! Expression! Using! Real[Time! Quantitative!PCR”!(Applied!Biosystems).!These!efficiencies!indicate!that!the!amount!of! DNA!is!doubled!in!each!PCR!cycle,!and!allows!for!direct!comparison!between!different! genes.!Relative!RNA!levels!were!determined!using!the!∆∆Ct!method!as!described!in!the! above!mentioned!guide.!Briefly,!each!gene!Ct!value!is!normalized!to!the!Ct!value!for!the! internal!control!(hdfR),!which!gives!the!∆Ct!value.!This!value!is!then!related!to!a!given! gene!in!the!reference!strain!(SL1344,!in!this!case)!giving!us!the!∆∆Ct!value.!Since!the! amount!of!DNA!doubles!in!each!PCR!cycle,!the!relative!amount!of!input!cDNA!can!be! determined!by!using!the!formula!2[∆∆Ct.!Each!∆∆Ct!determination!was!performed!at!least! in!three!different!RNA!samples!(three!biological!replicates),!and!the!results!presented! here!are!a!representative!example!of!such!determinations.! &Flow&cytometry&& Flow!cytometry!was!used!to!monitor!the!efflux!pump!AcrAB!activity!over!time.!Data! acquisition! and! analysis! were! performed! using! a! Cytomics! FC500[MPL! cytometer! (Beckman! Coulter).! Data! were! collected! for! 100000! events! per! sample,! and! were! analyzed!with!CXP!and!FlowJo!8.7!Software.! ! To!study!AcrAB!activity,!accumulation!and!efflux!of!EtBr!was!monitored!on!a!real[time! basis!104,105..(EtBr!was!excited!at!488!nm,!and!fluorescence!was!detected!using!a!585[nm! filter.! To! study(AcrAB! activity! with! DOC,! exponential! cultures! of! strains! SV7636! (ΔacrDmdtK(mdfA(emrA(acrF(mdtA,(macA::Km,(mdsA::Cm)!and!SL1344!were!diluted!with! and!without!5%!DOC!and!incubated!for!2!hours!(for!exponential!phase)!and!o/n!(for! stationary!phase).!EtBr!was!then!added!(1!μg/mL)!and!the!cultures!were!incubated!for! an!additional!60!minutes!at!37°C!in!the!dark!to!permit!EtBr!accumulation.!Finally,!0.5!mL! Materials!and!methods! 60 of!each!culture!was!taken,!the!bacterial!suspension!was!centrifuged!for!3!min!at!13000! rpm!and!the!pellet!was!resuspended!in!PBS!for!EtBr!fluorescence!measurement.!To! study!AcrAB!activity!during!adaptation!to!DOC,!the!same!strains!were!cultured!in!20!mL! LB!up!to!an!OD≈0.35,!when!DOC!was!added!to!a!final!concentration!of!5%.!1mL!samples! were!taken!every!hour,!treated!with!EtBr!as!described!previously,!and!analyzed!in!the! cytometer!for!8!hours.!! ! Statistical&analysis&& To! calculate! averages! and! standard! deviations! of! the! experiments! performed,! “Microsoft!Excel”!was!employed.!In!addition,!the!Student’s!t!test!was!used!to!determine! if!the!differences!in!our!experiments!were!statistically!significant.! ! ! 61 & & & & & & & & & & & RESULTS& & & ! 62 & ! 63 & & & & & & & & & & & CHAPTER&1:&EXPLORATION&OF&CELLULAR& FUNCTIONS&ESSENTIAL&FOR&BILE&ADAPTATION& AND&RESISTANCE&IN&Salmonella(enterica( ! 64 Chapter!1! 65 Surveys&of&bile&adaptation&in&bile8sensitive&mutants& ! The!goal!of!this!assay!was!to!ascertain!if!different!bile[sensitive!mutants!were!able!to! adapt!or!not!to!survive!bile!after!previous!incubation!with!sub[lethal!concentrations!of! sodium!deoxycholate!(DOC).!The!aim!was!to!try!to!find!if!the!adaptation!process!was!a! synergic!process!depending!on!several!gene!functions!or!if!it!was!mostly!dependent!on! a!specific!one.!The!mutants! used!carried!loss[of[function!mutations!in!the!following! genes:!! W!dam,! encoding! a! DNA! adenine! methylase! involved! in! genome! defense,! DNA! replication!and!repair,!nucleoid!segregation,!regulation!of!gene!expression,!control! of!transposition,! and!host[pathogen! interactions!106–108.!A! dam( mutation!causes! pleiotropic!defects!but!is!not!lethal!109.! W!damX,(a!gene!whose!protein!localizes!in!the!septal!ring!in!E.coli.!Loss!of!DamX!results! in!a!variety!of!division!phenotypes,!suggesting!a!role!in!cytokinesis!110.!In!Salmonella( enterica,! DamX! is! located! in! the! inner! membrane! and! its! loss! causes! severe! sensitivity!to!bile!111.! W!phoP,!a!gene!that!encodes!one!of!the!components!of!the!PhoPQ!sensor!system,! which! comprises! a! membrane! bound! sensor! kinase! PhoQ! (which! has! a! binding! domain!and!a!kinase/phosphatase!domain)!and!a!response!regulator!PhoP!in!the! bacterial! cytoplasm! 112.! PhoPQ! is! essential! for! the! virulence! of! Gram[negative! bacterial!pathogens!in!humans!and!mice!113.!According!to!van!Velkingburg!et.(al.( (1999),!the!PhoPQ!system!is!required!for!enhanced!resistance!to!bile!in!Salmonella! 86.! W!rpoS,( the!gene! encoding! sigma!factor! RpoS,! which! is! required!for! expression! of! genes!involved!in!a!variety!of!environmental!stresses!(starvation,!oxidation,!low!pH)! 114.!In!addition,!RpoS!is!required!for!Salmonella!virulence!in!mice!115.!This!gene!is!up[ regulated!by!5%!sodium!deoxycholate!and!rpoS(mutants!are!bile[sensitive!88.! W!wecD,!a!gene!involved!in!the!synthesis!of!enterobacterial!common!antigen!(ECA).! ECA!plays!a!role!in!Salmonella(virulence,!particularly!upon!oral!infection,!and!may! protect!the!pathogen!from!bile!salts!73.!! Chapter!1! 72 The!results!from!this!genetic!screen!were!interesting!enough!to!focus!the!following! stages!of!our!research!project!into!the!description!of!the!roles!that!efflux!systems!might! have!in!the!process!of!adaptation!and!resistance!to!bile!in!Salmonella(enterica.( & T8POP&transposon&genetic&screen:&looking&for&bile&resistant&phenotypes.& ( A!genetic!screen!based!on!the!T[POP3!transposon!95!was!used!to!search!for!genes!that! could!alter!bile!resistance!levels!either!by!mutation!(gene!disruption)!or!by!increased! expression!above!normal!levels.!For!this!purpose,!the!wild! type! strain! (SL1344)! was! subjected! to! mutagenesis! using! a! TPOP3! library! from! the! laboratory! collection.! Mutagenesis! involved! transduction! with! P22! HT! phage.! Isolates! carrying! T[POP3! insertions!were!selected!on!LB!agat!+!Tc!+!7%!sodium!deoxycholate.! ! One!hundred!and!twenty!colonies!were!obtained;!these!were!individually!analyzed!by! determination!of!minimal!inhibitory!concentrations!of!sodium!deoxycholate!(DOC)!in! 96[well!plates.!The!MICs!were!done!in!two!conditions,!with!and!without!tetracycline.! This! is! done! because! gene! disruption! due! to! T[POP3! insertion! is! insensitive! to! the! presence!of!tetracycline,!while!increased!expression!of!the!genome!regions!surrounding! T[POP3!is!tetracycline[dependent!95.!Tetracycline!induces!the!expression!of!the!outward! pointing!tetRA(promoters!present!in!the!transposon,!thus!allowing!the!transcription!of! adjacent!genes!95,96.! ! The! sodium! deoxycholate! MICs! with! and! without! Tc! revealed! that,! out! of! the! 120! candidates,!10!showed!a!higher!DOC!resistance!level.!However,!none!of!the!candidates! showed! enhanced! DOC! resistance! level! in! presence! of! Tc.! This! indicates! that! the! candidates! found! presented! a! higher! DOC! resistance! level! due! to! gene! inactivation! rather!than!to!increased!gene!expression.! ! To!identify!the!regions!where!the!T[POP3!element!had!inserted,!the!semi[random!two[! step! polymerase! chain! reaction! (ST[PCR)! method! was! employed.! The! list! of! genes! identified!are!shown!in!table!C1.4.!! Chapter!1! 73 ! Table&C1.4.&Identification&of&the&T8POP3&transposon&insertion®ions&on&the&different& bile&resistant&candidates&found&on&the&screen.&For!each!candidate!the!genomic!region! of!the!transposon!(position!in!the!chromosome)!and!the!genes!present!in!such!region! are!denoted.!! Candidate& T8POP3&insertion&position& Gene& A684A& 135302! LysR[type!transcription!regulator!(leuO) B6816A& 2455187! hypothetical! membrane! protein! SL1344_2311!(ulaA)! 4.7& 2156785! UDP[glucose!6[dehydrogenase!(udg)! 11.7B& 2060185! bacteriophage!terminase!(large!subunit)! 6.1.1& 3045128! Type!III!secretion!system!effector!protein! (sptP)! 6.2.1& 2268871! conserved!hypothetical!protein!(yohI)! 8.1.1A& 2060845! predicted!bacteriophage!protein! 8.2.4& 2373343! hypothetical! MR[MLE[family! protein! (SL1344_2242)! 8.2.6A& 2440118! hypothetical!aminotransferase!(yfbQ)! 8.2.7A& 388445! hypothetical!rtn!protein!(SL1344_0338) ! Since!the!phenotypes!observed!were!due!to!gene!inactivation!rather!than!increased! expression! of! genes! flanking! the! T[POP3! insertion! site,! deletion! mutants! were! constructed!with!the!lambda!Red!gene!disruption!method!92.!Two!candidates!(8.1.1A! and! 11.7B)! were! left! out! as! their! genes! did! not! belong! to! the! Salmonella( enterica( genome!but!they!were!bacteriophage!protein!genes.!! Determination!of!the!minimal!inhibitory!concentration!of!sodium!deoxycholate!for!each! of!the!mutants!showed!no!differential!bile!resistance!level!when!compared!with!the!wild! type!strain!(SL1344)!(Figure!C1.2).!! Chapter!1! 74 ! Figure&C1.2.&Minimal&inhibitory&concentration&(MICs)&of&sodium&deoxycholate&(DOC)& for&Salmonella(enterica(strains&mutants&for&the&genes&identifies&in&the&T8POP3&genetic& screen.&MIC!values!were!compared!with!the!wt!strain!(SL1344!his+)..! ! The!results!on!this!screen!did!not!provide!interesting!nor!conclusive!results!on!the!role! these!genes!could!play!in!bile!resistance!or!adaptation!in!Salmonella.!Therefore,!the! results!of!this!screen!were!not!further!analyzed.!! ! 12 3 4 5 6 7 8 9 10 11 0 SL#his⁺ DOC.%  MR*MLE 12 3 4 5 6 7 8 9 10 11 0  sptP  rtn  udg  ulaA  yfbQ  yohI  leuO DOC.% ! 75 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! CHAPTER&2:&ESSENTIAL&ROLE&OF& AcrAB8MEDIATED&EFLLUX&IN&Salmonella( enterica(ADAPTATION&TO&BILE& ! 76 Chapter!2! 77 Surveys&of&adaptation&to&bile&in&mutants&lacking&efflux&systems& ! The!results!obtained!for!adaptation!in!bile!sensitive!mutants!showed!that!only!efflux! pumps!related!mutants!(acrA,!acrB(and!tolC)(were!not!able!to!adapt!and!had!a!very!high! susceptibility!to!bile.!These!lead!us!to!study!the!role!in!bile!resistance!and!adaptation!of! the!other!eight!efflux!systems!reported!in!S.enterica(75,81.!Therefore,!single!efflux!pump! mutants!were!constructed!(Table!M1),!and!minimal!inhibitory!concentrations!(MICs)!of!! sodium! deoxycholate! (DOC)! were! determined.! The! results! confirmed,! that! not! only! AcrAB!is!essential!for!bile!resistance!122,!but!also!that!the!other!efflux!pumps!have!a! minimal!contribution!in!such!resistance,!having!MICs!of!DOC!values!very!similar!to!that! of! the! wt! strain! (Figure! C2.1).! These! mutants! were! able! to! adapt! when! cultured! overnight!with!5%!DOC.!! ! !!!!!! ! Figure&C2.1.&Minimal&inhibitory&concentration&(MICs)&of&sodium&deoxycholate&(DOC)& for&Salmonella(enterica(strains&lacking&efflux&systems.&MIC!values!were!compared!with! the!wt!strain!(SL1344!his+),!a!bile!resistant!mutant!(BR6,!a!previously!described!yrbK( mutant!88)!and!a!bile!sensitive!strain!(∆dam,(Dam!methylase!mutant).!! ! The!preliminary!results!on!single!efflux!pump!mutants!led!us!to!hypothesize!that,!even! though!bile!resistance!in!Salmonella!was!achieved!mainly!by!the!active!efflux!of!the! AcrAB!system,!it!could!be!possible!that!the!other!efflux!pumps!played!an!additional! secondary!role!in!bile!resistance.!To!study!this!possibility,!mutants!that!lacked!from!two! to!eight!efflux!systems!were!designed!and!constructed!by!sequential!replacement!of! SL#1344#his⁺%(wt) acrA::Km acrD::Km acrF::Km emrA::Km macA::Km mdfA::Km mdtA::Km mdtK::Km BR6 ∆dam MIC#of#DOC#(%) 12 3 4 5 6 7 8 9 10 11 0 mdsA::Cm Chapter!2! 78 each!efflux!pump!gene!with!an!antibiotic!resistance!cassete!92.!As!a!result,!eight!new! mutants!were!obtained! (Table! M1):!all!of! them! having!the!acrAB( genes!untouched.! These!new!strains!were!assayed!by!determining!its!minimal!inhibitory!concentration! (MICs)!in!ox!bile!gradient!plates!(0[30%)!to!study!their!resistance!level!and!if!they!were! able!to!adapt!as!well!(Figure!C2.2).!! ! ! Figure&C2.2.&Adaptation& assays&in& 0830%&ox& bile& gradient&plates& of&mutants&lacking& efflux&systems.&Plates!on!the!left!side!column!(A)!show!the!bile!resistance!levels!of!each! strain!cultured!overnight!in!LB!broth.!On!the!right!side!column!(B)!the!plates!of!the!same! strains!cultured!overnight!in!LB!with!sublethal!concentrations!of!sodium!deoxycholate! Bile% 0%% 30%% Bile% 0%% 30%% LB%Cultures%o/n% LB%+%DOC%Cultures%o/n% Δ"acrD"mdtK" mdfA"emrA" acrF"mdtA," macA::Km," mdsA::Cm% acrD::Km" acrB::Km" acrA::Km" SL1344" (WT)% 5%%DOC% 5%%DOC% 5%%DOC% 1%%DOC% 1%%DOC% o/n$LB$cultures 0% 30% Bile o/n$LB$+$DOC$ cultures 0% 30% Bile SL1344$ (wt) Δ"acrD mdtK mdfA emrA acrF mdtA," macA::Km,"mdsA::Cm AB acrD::Km acrA::Km acrB::Km 0.5%$DOC 0.5%$DOC 5%$DOC 5%$DOC 5%$DOC Bacterial$ lawn Bacterial$ lawn Bacterial$ lawn Chapter!2! 79 (DOC).!In!the!wt,!SV7636!(Δ(acrD(mdtK(mdfA(emrA(acrF(mdtA,(macA::Km,(mdsA::Cm),! and!the!acrD::Km!strain,!a!bacterial!lawn!appeared.!The!percentage!of!DOC!used!for! adaptation!is!in!the!right!side!of!the!right!column.!The!strain!used!as!control!was!SL1344.! & The!results!showed!that!it!was!evident!that!these!efflux!pumps!did!not!have!an!additive! effect!in!bile!resistance,!all!of!them!showed!a!very!similar!level!of!resistance!as!that!seen! in!the!wild!type!strain!(SL1344!his+).!Moreover,!even!the!mutant!that!lacked!all!of!the! efflux!pump!systems!but!AcrAB!(SV7636:!Δ(acrD(mdtK(mdfA(emrA(acrF(mdtA,(macA::Km,( mdsA::Cm)!showed!the!same!bile!resistance!level!than!the!wt!strain!(Figure!C2.2!A).!! ! Once!the!levels!of!resistance!of!these!mutants!were!determined,!exponential!phase! cultures! of! these! strains! were! incubated! overnight! with! sublethal! concentrations! of! sodium!deoxycholate!(5%!DOC!for!most!strains!and!0.5%!for!acrA(or(acrB(mutants).!!The! following!day,!dilutions! of!these!cultures! were! plated! in!ox!bile! gradient! plates!and! incubated!overnight!at!37°C.!Results!demonstrated!the!ability!of!these!mutants,!with! multiple!efflux!systems!impaired,!to!adapt!to!bile!(Figure!C2.2!B).! ! Finally,!it!is!important!to!note!that!two!efflux!pumps!require!the!subunit!AcrA!as!part!of! its!system:!AcrAB!and!AcrAD!75,81.!Nevertheless,!an!acrD!mutation!does!not!affect!in!bile! resistance!nor!adaptation!(Figure!C2.2).!These!results!corroborate!the!importance!and! specificity!of!AcrAB[mediated!efflux!in!bile!resistance!and!adaptation.!!! ! Lack&of&AcrAB&suppresses&bile&hyperresistance& ( Previous!studies!made!in!our!laboratory!had!described!mutants!that!display!bile!hyper[ resistance!phenotypes.!The!findings!about!the!importance!of!the!AcrAB!efflux!system!in! bile! resistance! and! adaptation! raised! the! question! of! whether! this! cellular! function! might!be!likewise!involved!in!bile!hyperesistance.!The!idea!of!the!experiment!was!to!test! if!lack!of!the!AcrAB!efflux!system!affected!the!levels!of!bile!resistance!in!such!mutants.! For! this! purpose,! an! acrA::Km! allele! was! transduced! with! P22! HT! into! three! hyperesistant!strains:!!(i)!BR6,!a!mutant!carrying!a!deletion!in!the!yrbK!gene,!involved!in! Chapter!2! 80 lipopolysaccharide!transport!88;!(ii)!an!asmA!mutant!lacking!the!outer!membrane!protein! AsmA!123:!(iii)!a!prc(mutant!lacking!Prc!periplasmic!protease!124.! ! Exponential!cultures!of!acrA(yrbK,!acrA(asmA,!and!acrA(prc!were!diluted!10!fold!and! aliquots!of!100!µl!were!plated!on!0[20%!ox!bile!extract!gradient!plates.!The!plates!were! incubated!overnight!at!37°C!and!growth!was!monitored!afterwards.! ( ( Figure&C2.3.&Suppression&of&bile&hyperesistance&by&lack&of&AcrAB.&Assays!were!carried! out!on!0[20%!ox!bile!extract!gradient!plates.!! ! The! results! displayed! on! figure! C2.3! show! that! lack! of! AcrAB! suppresses! the! bile! hyperesistant!phenotypes!of!yrbK,!asmA,!and!prc(mutants,!as!shown!by!disappearance! of!the!bacterial!lawn.!These!findings!further!outline!the!importance!of!the!AcrAB!efflux! pump!to!maintain!homeostasis!in!the!bacterial!cell:!even!though!the!alterations!that! render!yrbK,!asmA,!and!prc(mutants!bile[resistant!seem!to!be!unrelated!to!AcrAB,!lack! of!the!AcrAB!efflux!pump!made!them!bile[sensitive.! ! Activation&of&acrAB&expression&by&sodium&deoxycholate& The!relevance!of!AcrAB[mediated!efflux!in!both!bile!resistance!and!bile!adaptation!led!us! to!study!the!expression!pattern!of!the!acrA(and!acrB!genes!during!the!bile!adaptation! process.!For!this!purpose,!transcription!analysis!was!performed!in!the!presence!of!sub[ SL1344&(WT)& acrA::Km( BR6& BR6,&acrA::Km& asmA::Km( ΔasmA,(acrA::Km( prc::Km( Δprc,(acrA::Km( Bile& 0%& 20%& Bile& 0%& 20%& Bile& 0%& 20%& Bile& 0%& 20%& Chapter!2! 81 lethal!concentrations!of!sodium!deoxycholate!(DOC).!The!acrA!and!acrB!mRNA!levels!were! determined!by!qRT[PCR!in!exponential!and!stationary!cultures!grown!with!5%!DOC.!The! strains!under!study!were!SV7636!(ΔacrD(ΔmdtK(ΔmdfA(ΔemrA(ΔacrF(ΔmdtA(ΔmacA::Km,( ΔmdsA::Cm)!and!the!wild!type!strain!(SL1344).!In!the!wild!type,!the!expression!levels!of! efflux!pumps!other!than!AcrAB!were!also!determined!(Table!C2.1).!! ! Table&C2.1.& Relative& expression&values& of& different&efflux& pump& genes&obtained& from& transcriptomic& analysis& (qRT8PCR)& performed& in& the& wt& (SL1344)& and& SV7636& (Δ( acrD( mdtK(mdfA(emrA(acrF(mdtA,(macA::Km,(mdsA::Cm)&strains&grown&in&LB&and&LB&5%&DOC& and& in& both& exponential& and& stationary& phase.& The! values! are! expressed! as! the! fold! change!in!expression!when!compared!with!the!wild!type!strain!grown!on!LB.!Data!are! normalized!to!the!RNA!levels!obtained!in!the!wild!type!strain!cultured!in!LB!(value!for!the! wild!type!=!1).!Data!are!from!3!independent!experiments.& Wild!type!strain!(SL1344)& & Relative!expression!values!(fold!change)!in!LB!5%!DOC! Gene& Exponential!phase! Stationary!phase! acrA( 14! 4.5! acrB( 10! 2! acrD( 2.5! 2! acrF( 1! 4! emrA( 2.5! 0.9! macA( 2! 1! mdfA( 2! 7! mdsA( 2! 3! mdtA( 2.5! 2! mdtK( 3! 3! SV7636!strain!(Δ(acrD(mdtK(mdfA(emrA(acrF(mdtA,(macA::Km,(mdsA::Cm)! ( Relative!expression!values!(fold!change)!in!LB!5%!DOC! Gene( Exponential!phase! Stationary!phase! acrA( 12! 5.5! acrB( 12! 3! & Most!efflux!pumps!appear!to!be!up[regulated!in!exponential!phase!in!the!presence!of!DOC! (Table!C2.1).!Notably,!the!acrA(and!acrB!genes!showed!a!gene!expression!increase!of!10[ fold!or!more.!The!results!are!similar!in!the!8!efflux!pump!mutant!strain!SV7636!(Δ(acrD( mdtK( mdfA( emrA( acrF( mdtA,( macA::Km,( mdsA::Cm),! with! a! 12[fold! increase! in! gene! expression!for!both!genes.!Table!C2.1!includes!also!additional!efflux!pump!genes,!whose! expression!was!analyzed!in!the!wild!type:!acrF,!reported!to!export!bile!75,81!showed!no! Chapter!2! 88 ! Figure&C2.7.&EtBr&fluorescence&means&over&time.!Values!were!obtained!from!cytometry! assays!with!the!eight!efflux!pump!mutant!SV7636!(ΔacrD(ΔmdtK(ΔmdfA(ΔemrA(ΔacrF( ΔmdtA,( macA::Km,( mdsA::Cm)! and! the! wild! type! strain! SL1344.! A! representative! experiment!is!shown.! ! Figure! C2.7! confirms! that! during! exponential! growth! efflux! is! active! in! a! sodium! deoxycholate[independent!manner,!as!indicated!by!the!low!ethidium!bromide!levels! detected!in!all!cultures.!After!6[7!hours,!as!the!samples!began!to!enter!stationary!phase,! cultures!of!both!strains!in!LB!started!to!accumulate!EtBr!at!a!higher!rate!than!the!cultures! in!LB!5%!DOC.!By!hour!eigth!of!incubation,!cultures!of!SL1344!and!SV7636!in!LB!showed! EtBr!accumulation!around!300!and!450!fluorescence!units,!respectively.!On!the!contrary,! when!both!strains!grew!in!the!presence!of!5%!DOC,!moderate!accumulation!of!EtBr!was! detected! (150! units! for! the! SV7636! strain! and! around! 100! for! the! wild! type).! The! differences! between! LB! and! LB! +! DOC! increased! further! after! 24! h,! suggesting! that! cultures! in! LB! decrease! their! efflux! activity! upon! entrance! into! stationary! phase.! In! contrast,!cultures!grown!with!5%!DOC!seem!to!keep!their!efflux!active!irrespectively!of! the!growth!phase.!! & 0 100 200 300 400 500 600 700 800 0 5 10 15 20 25 30 Fluorescence*mean Time EtBr*Fluorescence*mean SL1344,LB SL1344,5%,DOC SV7636,LB SV7636,5%,DOC ! 89 & & & & & & & & & & CHAPTER&3:&A&STUDY&OF& MECHANISMS&OF&BILE&&&&&&&&&&&&&&&&&& RESISTANCE&IN(VIVO( ! 90 ( Chapter!3! 91 Characterization&of&S.&enterica&populations&present&in&the&gallbladder&of& BALB/c&mice&(typhoid&fever&animal&model).& ( BALB/c! mice! are! particularly! susceptible! to! the! S.( enterica( serovar! Typhimurium! infection!because!they!are!homozygous!for!a!mutation!in!the!Bcg(locus,!also!known!as! Ity(or!Lsh.!This!gene!codes!for!the!Nramp1!(natural[resistance[associated!macrophage! protein! 1)! protein,! which! controls! the! innate! resistance! or! susceptibility! of! mice! to! infection! with! a! group! of! unrelated! intracellular! microorganisms! that! include! Salmonella,( Leishmania( and! Mycobacterium( 128,129.! To! analyze! the! S.( enterica( populations!that!colonize!the!gallbladder!upon!systemic!infection,!19!BALB/c!mice!were! orally!infected!with!108!colony!forming!units!(c.f.u.)!from!overnight!anaerobic!cultures! of! the! wild! type! strain! (SL1344).! Five! days! later,! the! mice! showed! the! previously! described!clinical!disease!symptoms.!At!this!stage!the!mice!were!euthanized!and!the! gallbladders!were!recovered.!! ! The!gallbladders!were!homogenized,!and!serial!dilutions!(10[1,!10[2,!10[3,!and!10[4)!were! prepared! with! NaCl! 0.9%.! To! select! Salmonella! isolates! and! avoid! growth! of! other! intestinal!bacteria,!the!dilutions!were!plated!on!LB!agar!+!streptomycin!200!µg/l,!and! incubated!overnight!at!37°C!(SL1344!is!resistant!to!low!concentrations!of!streptomycin).! The!following!day,!the!colonies!were!counted.!Table!C3.1!shows!the!number!of!colony[ forming[units!(c.f.u.)!present!in!the!gallbladder!of!each!mouse!(applying!dilution!factors! to!the!calculation!when!necessary).! ! ! ! ! ! ! ! ! ! Chapter!3! 92 Table&C3.1.&Estimated&number&of&c.f.u.&recovered&from&the&gallbladder&of&BALB/c&mice.& Mouse& Number&of&c.f.u.&recovered& from&the&gallbladder& 1& 63! 2& 548! 3& 105! 4& 475! 5& 6694! 6& 51! 7& 31204! 8& 37! 9& 1042! 10& 222! 11& 2858! 12& 203400! 13& 182! 14& 422! 15& 202500! 16& 1599000! 17& 1018! 18& 2783! 19& 11203! Although! BALB/c! mice! are! genetically! identical! and! the! experiment! consisted! in! inoculating!all!the!mice!with!the!same!strain!and!with!identical!numbers!of!cells,!the! numbers!of! c.f.u.! present! in! the! gallbladder! extracts! show! high!variability! from! one! mouse!to!another.!This!variation!could!have!several!causes.!For!instance,!because!the! inoculation! was! done! orally,! the! number! of! bacteria! that! ultimately! reached! the! digestive! system! might! be! different! in! each! mouse.! Non[genetic! variability! in! each! mouse!might!alter!the!ability!to!respond!to!the!infection.!Another!source!of!variation! could! be! stochastic! variation! in! the! number! of! S.( enterica( cells! able! to! invade! and! replicate!within!the!different!gallbladder!microenvironments.! Certain! plates! used! for! the! isolation! of! gallbladder! Salmonellae! contained! isolated! colonies!while!others!contained!a!bacterial!lawn.!Plates!containing!isolated!colonies! were!replicated!with!sterile!velvets!to!LB!agar!containing!high!concentrations!of!ox!bile! (from!12%!to!20%).!This!experiment!permitted!to!distinguish!bile[resistant!mutants!from! Chapter!3! 93 isolates!sensitive!to!bile,!which!were!by!far!the!most!abundant!type!(Figure!C3.1).!The! four!lawns!obtained!were!also!replicated,!and!provided!1,!32,!49,!and!122!mutants;!a! ratio!between!bile[adapted!and!bile[resistant!isolates!could!not!be!calculated!in!those! gallbladders.! ! Figure&C3.1.&Characterization&of&S.(enterica(BALB/c&mice&gallbladder&populations:&ratio& between&bile&adapted&and&bile&resistant&mutant&isolates&recovered&from&each&BALB/c& mouse&gallbladder.&The!ratio!is!represented!in!percentage!values.!On!the!right!side!of! the! chart,! the! actual! numbers! of! bile[adapted! isolates! vs! bile[resistant! mutants! are! shown.!! ! The!main!conclusion!from!these!experiments!was!that!colonization!of!the!gallbladder!by! Salmonella!is!made!possible!mainly!by!adaptation!(in!other!words,!without!mutation).! However,!bile[resistant!mutants!also!appear!at!low!frequencies.!These!observations!are! in!agreement!with!previous!studies!of!bile!resistance!in!vitro!which!also!suggested!that! non[mutational!adaptation!to!bile!was!more!frequent!than!mutation!88.!An!additional! observation!is!that!the!numbers!of!bile[resistant!mutants!show!significant!differences! from!one!gallbladder!to!another,!and!that!correlations!do!not!seem!to!exist!between! the!number!of!mutants!and!the!total!number!of!c.f.u.!This!observation!suggests!that! Salmonella!may!grow!in!the!gallbladder!for!a!significant!number!of!generations!in!the! gallbladder,! and! that! Luria! &! Delbrück! fluctuation! may! occur.! Unfortunately,! this! Chapter!3! 94 hypothesis!cannot!be!subjected!to!statistical!analysis!due!to!the!lack!of!an!outgroup!that! might!serve!as!control.! ! Characterization&of&bile8resistant&mutant&derivatives&isolated&from&the& gallbladder&of&BALB/c&mice.& Ten!bile[resistant!mutants!of!independent!origin!(each!isolated!from!a!different!mouse! gallbladder)!where!chosen!for!full!genome!sequencing.!The!mutants!were!propagated! from!colonies!grown!in!the!original!plates!inoculated!with!gallbladder!homogenates.! Therefore,!the!mutants!had!not!arisen!after!recovery!from!the!gallbladder.!Genome! sequencing!employed!Roche!454!FLX+!technology!on!a!GS!FLX!titanium!system.!Sample! preparation!included!gDNA!isolation!by!phenolic!extraction!and!ethanol!precipitation.! Emulsion!PCR!and!454!pyrosequencing!were!performed!following!the!manufacturer’s! instructions!and!1,031,375!reads!with!an!average!read!length!of!810!basepairs!were! obtained,!totaling!1,000Mb.!! Genome!sequencing!was!followed!by!alignment!with!the!S.(enterica(SL1344!genome! sequence! (ftp://ftp.sanger.ac.uk/pub/pathogens/Salmonella/STmSL1344.dbs)! to! identify! DNA! sequence! differences.! The! alignment! tool! used! was! BWA! (Burrows[ Wheeler!Alignment),!which!consists!of!three!algorithms.!In!our!case,!the!BWA[MEM! algorithm!was!employed!(Li!and!Durbin,!unpublished).!The!genome!of!the!laboratory! stock!of!S.(enterica(SL1344!was!also!sequenced!as!a!control.!Table!C3.2!describes!the! mutations!found!in!the!mutants!isolated!from!murine!gallbladders.!!! ( ( ( ( ( ( ( ( ( Chapter!3! 95 Table&C3.2.&Mutations&present&in&the&genome&of&bile&resistant&derivatives&of&S.(enterica( SL1344&isolated&from&BALB/c&mice&gallbladder.( ( Chapter!3! 96 ( The!following!observations!may!be!relevant:!! a.!Mutant! 1! shows! a! nucleotide! substitution! in! rlpB,! a! gene! that! encodes! a! lipoprotein! B! precursor.! A! similar! point! mutation! (transversion)! has! been! previously!reported!in!a!study!that!isolated!spontaneous!bile!resistant!mutants! from!agar!plates!containing!high!(lethal)!ox!bile!concentrations!88.!Even!though! this!protein!has!not!been!widely!studied!in!S.(enterica,!its!E.(coli(homolog!has! been!described!to!form!a!protein!complex!with!the!product!of!the!imp(gene! (Imp/RlpB)! that! is! responsible! for! LPS! transport! to! the! outer! membrane.! In! addition,!in!E.(coli,(rlpB(appears!to!be!essential!for!cell!viability!130.!Interestingly,! this!gene!has!been!reported!to!be!induced!by!bile!131.! b.!Mutant!2!shows!a!point!mutation!(transition)!in!yejM,!a!gene!that!remains!poorly! known!in!S.(enterica(and!may!encode!a!hypothetical!sulphatase.!Transcription!of! yejM!is! induced! by! bile! 131.! Its! homolog! in! E.( coli! is! classified! as! an! inner! membrane! protein! and! its! deletion! is! lethal.! YejM! might! be! involved! in! cell! permeability!and!lipid!A!production!132,133.!! c.!Mutant!3!harbors!a!transversion!in!the!dipZ(gene,!which!encodes!the!DsdB!thiol[ disulfide! interchange! protein.! In! E.( coli,! studies! have! shown! that! DsdB! is! a! cytoplasmic!membrane!protein!(more!specifically,!a!periplasmic!protein!disulfide! bond! isomerase)! that! transfers! reducing! power! from! the! cytoplasm! to! the! periplasm!to!aid!in!the!formation!of!disulphide!bonds!and!c[type!citochromes! 134,135.!In!addition,!136!have!suggested!that!this!gene!might!have!a!role!in!copper! tolerance.!! d.!Mutants!4!and!7!harbor!mutations!that!involve!cell!division!factors!(ftsQ(and!ftsK)! and!show!increased!expression!in!the!presence!of!bile!131.!In!the!case!of!mutant! 4! there! is! a! transversion! that! changes! the! ftsQ( gene! start! codon! from! ATG! (methionine)!to!TTG!(leucine),!which!is!the!second!most!used!start!codon!in!E.( coli(137.!Further!analysis!will!be!required!to!ascertain!whether!translation!of!the! mutant!mRNA!actually!uses!the!new!triplet!as!start!codon.!Mutant!7!presents!a! deletion!of!36!nt!that!results!in!the!loss!of!12!amino!acids.!Several!studies!made! in!E.(coli!regarding!these!division!factors!have!demonstrated!that!FtsQ!and!FtsK! both!belong!to!a!group!of!9!essential!proteins!that!participate!during! Gram[ Chapter!3! 97 negative!bacteria!in!septum!formation.!The!remaining!proteins!are!FtsA,!FtsI,! FtsL,!FtsN,!FtsW,!FtsZ,!and!ZipA!and!all!are!localized!in!the!cell!septum!138,139.!A! previous!study!from!our!laboratory!identified!another!cell!division!factor!whose! synthesis! is! induced! by! bile.! This! gene! is! is! currently! annotated! as! yiiU! in! S.! enterica,!and!is!an!homolog!of!E.!coli!zapB!88.! e.!Four!independent!mutants!(5,!7,!9!and!10)!show!mutations!in!intergenic!regions.! Interestingly,!in!candidates!5!and!10,!the!genomic!regions!where!the!mutations! are!present!have!been!reported!to!be!highly!induced!by!bile!131,!particularly,!the! small!RNAs!STnc400!and!GlmZ,!respectively.!! f.!Mutant! 6! shows! a! synonymous! nucleotide! substitution! in! the! DNA! adenine! methylase!gene!dam.!This!result!is!curious!as!dam(mutations!causes!pleiotropic! defects!which!include!increased!bile!sensitivity!63,85,140,141.!! g.!Mutant!8!harbors!a!mutation!in!a!gene!of!known!function!which!is!induced!by! bile! 131,! yhbG.! This! gene! has! not! been! described! in! S.( enterica( but! its! E.( coli( homolog!appears!to!be!essential,!and!might!have!a!role!in!cell!envelope!integrity! 142.!This!gene!is!predicted!to!be!an!ABC!transporter!143,144.!!! ! Discussion! 104 antibacterial!activities!(Prieto!et!al.!2006).! !( A!search!for!multicopy!suppressors!of!the!bile[sensitive!phenotype!(in!the!absence!of! AcrAB[TolC!strenghtened!our!view!that!efflux!is!essential!for!adaptation:!most!of!the! pBR328!derivatives!that!restored!bile!resistance!and!permitted!bile!adaptation!harbored! efflux!pump!components!such!as!AcrF,!AcrE!and!AcrB!(Table!C1.3).!! ! The!acrE(and!acrF(genes!encode!the!RND!efflux!system!AcrEF,!and!AcrF!is!homologous! to!AcrB!in!S.(enterica,!with!around!81%!of!amino!acid!identity!and!similar!substrate! specificity!81.!It!has!been!reported!that!AcrEF!might!be!required!for!drug!resistance!when! AcrAB!is!impaired!in!S.(enterica(152.!Evidence!of!this!kind!was!found!by!Olliver!et!al.!(2005)! in!the!case!of!antibiotic!resistance:!an!otherwise!sensitive!acrB!mutant!was!found!to! have!elevated!fluoroquinolone!tolerance!due!to!increased!expression!of!acrEF,!in!turn! caused!by!an!insertion!sequence!integrated!upstream!of!the!acrEF(operon!153.!A!similar! case!was!observed!by!Horiyama!et.!al!(2010):!the!enhanced!expression!of!acrEF!in!an! acrB( background! increased! resistance! levels! to! a! large! number! of! antibacterial! compounds!including!sodium!deoxycholate!75.!An!analogous!situation!might!occur!in!our! case:!increased!expression!of!acrEF!could!rescue!the!bile[sensitive!phenotype!of!the! acrA!mutant.! ! Because! both! the! adaptation! survey! performed! with! bile[sensitive! mutants! and! the! multicopy!suppressor!screen!showed!the!relevance!of!active!efflux!for!adaptation!to! bile,!(Table!C1.3),!subsequent!experiments!were!focused!on!the!role!played!by!efflux! pumps!in!adaptation!to!bile.!The!involvement!of!the!remaining!efflux!systems!reported! to!exist!in!S.(enterica(81(was!thus!investigated.!Figure!D2!shows!the!distribution!of!the! corresponding!loci!in!the!genome!S.( enterica( serovar!Typhimurium,!along!with!their! transcriptional!orientation!and!identity!percentage!with!their!E.(coli(homologs.!!! Discussion! 105 ! Figure&D2.&Drug&efflux&genes&encoded&on&the&S.(enterica&serovar&Typhimurium&genome.& Positions!of!genes!encoding!putative!drug!transporters,!outer!membrane!proteins!and! membrane! fusion! proteins! are! indicated! in! the! chromosome! of! S.( enterica( serovar! Typhimurium!strain!LT2!6.!Arrows!indicate!the!direction!of!transcription.!Amino!acid! identity!between!homologous!proteins!in!S.(enterica(and!E.(coli!are!indicated!as!numbers! under!the!genes.!Reproduced!from!81.! ( Gene$encoding$outer$membrane$protein Gene$encoding$membrane$fusion$protein Gene$encoding$RND6type$efflux$protein Gene$encoding$MFS6type$efflux$protein Gene$encoding$MATE6type$efflux$protein Gene$encoding$ABC6type$efflux$protein (kb) (kb) (kb) (kb) (kb) (kb) (kb) (kb) (kb) (kb) (kb) 3349 3350 tolC 89.7% 529 530 531 532 533 acrB 94.6% acrA 91.7% acrD 94.3% acrE 87.5% acrF 88.7% 2594 2595 2596 2597 3561 3562 3563 3564 3565 2214 2215 2216 2217 2218 2219 2220 2221 2222 mdtA 82.7% mdtB 91.6% mdtC 91.7% 393 394 395 396 397 398 mdsC (STM0350) Nonexistent$ in$ E.coli2 51.5%$to$OprM in$ Pseudomonas2 aeruginosa mdsB (STM0351) Nonexistent$ in$ E.coli2 62.3%$to$MexF in$ Pseudomonas2 aeruginosa mdsA (STM0352) Nonexistent$ in$ E.coli2 43.8%$to$MexE in$ Pseudomonas2 aeruginosa 2962 2963 2964 938 939 940 emrA 89.7% emrB 95.7% mdfA 90.6% 1502 1503 mdtK 92.3% 1019 1020 1021 macA 83.2% macB 83.3% Discussion! 106 Using!the!available!information!concerning!the!efflux!systems!present!in!S.(enterica,( mutants! lacking! at! least! one! of! the! genes! of! each! efflux! system! were! constructed.! Minimal! inhibitory! concentrations! (MICs)! of! sodium! deoxycholate! (DOC)! were! then! determined.!Remarkably,!all!mutants!but!acrAB(showed!DOC!resistance!levels!similar!to! that!of!the!wild!type!(Figure!C2.1).!This!was!a!surprise!as!several!of!these!efflux!pumps! (including!AcrAB)!had!been!reported!to!export!bile!and!bile!salts!(AcrAD,!AcrEF,!mdtABC,! EmrAB)!75,81,154.!These!results!could!be!explained!in!two!different!ways:! ! 1.!Deletion!of!these!genes!might!be!unnoticeable!by!the!presence!of!a!functional! AcrAB!efflux!system.!It!is!known!that!AcrAB!has!an!extremely!wide!specificity,! exporting! multiple! types! of! antibacterial! agents! (with! the! exception! of! aminoglycosides),!detergents,!dyes,!free!fatty!acids,!and!simple!solvents!154–156.! Particularly,! reconstitution! assays! of! the! purified! AcrAB! ! efflux! system! into! proteoliposomes!concluded!that,!after!testing!different!ligands,!the!properties! of!AcrB!were!optimized!for!the!exclusion!of!bile!salts!78.! 2.!The! deleted! genes! might! not! be! expressed! under! the! conditions! of! the! experiment.! ! To! probe! the! first! possibility,! a! set! of! strains! lacking! multiple! efflux! pumps! was! constructed.!These!strains!were!tested!for!bile!resistance!and!adaptation!ability!(Figure! C2.2).!The!strains!had!from!2!to!8!efflux!systems!impaired,!but!AcrAB!remained!intact.! The!results!on!bile!resistance!levels!were!conclusive:!even!the!strains!that!lack!every! efflux! system! but! AcrAB! had! a! bile! resistance! level! similar! to! that! of! the! wild! type.! Additionally,!all!these!mutants!were!able!to!adapt!to!bile!after!incubation!with!sublethal! levels!of!DOC.!These!results!underline!again!the!relevance!of!AcrAB[mediated!bile!efflux! in!the!adaptation!process.!! ! The!need!of!AcrAB!in!the!transient!increase!of!bile!resistance!levels!(adaptation)!raised! the!possibility!that!mutation!of!this!efflux!system!might!impair!bile!resistance!and!bile! adaptation!in!bile[resistant!mutants.!The!mutants!chosen!to!address!this!question!were! (yrbK,!asmA::Km!and!prc::Km),!all!carrying!mutations!that!affected!the!structure!of!the! cellular! envelope.! The! results! of! these! experiments! showed! that! an! acrA! mutation! Discussion! 107 generated!a!bile[sensitive!phenotype!in!the!three!backgrounds!under!study.!This!can!be! explained! by! admitting! that! some! efflux! systems,! including! AcrAB,! may! act! synergistically! with! the! permeability! barrier! 148.! In! other! words,! the! mechanisms! of! Gram[negative!bacteria!to!survive!when!exposed!to!toxic!compounds!are!mainly!two! (permeability!barriers!and!active!pumping!out),!and!they!must!function!together!147,157.! This!means!that!the!permeability!barriers!are!insufficient!to!prevent!influx!of!obnoxious! compounds,!and!their!action!simply!slows!down!uptake.!The!efflux!pumps,!which!form! multisubunit!complexes!by!association!with!outer!membrane!channels!(like!TolC)!and! accessory!proteins!(AcrA)!are!thus!necessary!extrude!toxic!compounds!directly!into!the! medium,!bypassing!the!outer!membrane!barrier!148.!Even!if!this!complex!mechanism!has! been!so!extensively!described!78,147,157–160,!it!still!comes!as!a!surprise!that!mutants!that! show!an!exceptionally!high!resistance!to!bile!rely!so!greatly!in!the!bile!extrusion!role!of! AcrAB.! This! result! only! confirms! the! weight! the! efflux! system! has! in! conferring! the! bacteria!the!ability!to!survive!in!the!presence!of!bile!and!to!adapt!to!grow!in!the!presence! of!increasing!concentrations.!!! ! To!monitor!the!expression!patterns!of!each!S.(enterica( efflux!system!during!the!bile! adaptation!process,!qRT[PCR!experiments!were!carried!out!under!different!conditions:! with!and!without!sublethal!concentrations!of!sodium!deoxycholate,!and!in!exponential! and!stationary!growth!phase.!The!strains!assayed!were!the!wild!type!and!the!mutant! lacking!eight!efflux!systems!(SV7636),!and!the!results!are!shown!in!Table!C2.1!and!Figure! C2.4.!Relevant!results!were!as!follows:! ! 1.!In!exponential!phase,!transcription!of!both!acrA(and!acrB(genes!is!highly!activated!by! sodium!deoxycholate;!on!the!contrary,!the!expression!level!of!other!genes,!including! those!which!encode!components!of!efflux!systems!reported!to!export!bile!salts,!are! unaffected!or!poorly!induced!by!the!presence!of!DOC! ! 2.!In!stationary!phase,!the!level!of!expression!of!acrAB!decreases!drastically!while!most! of!the!other!genes!do!not!show!remarkable!changes,!except!acrF(and!mdfA.!! ! Discussion! 108 These! observations! give! further! support! to! the! view! that! AcrAB! is! fundamental! for! adaptation.!It!seems!that!the!key!event!for!adaptation!occurs!during!the!first!encounter! of!the!bacteria!with!bile,!which!leads!to!increased!expression!of!genes!that!encode!the! efflux!pump.!As!time!passes!by!and!adaptation!takes!place,!activation!of!gene!expression! decreases.!In!stationary!phase,!it!is!curious!to!observe!that!the!mdfA(gene!is!so!highly! induced,!even!if!the!efflux!system!is!not!reported!to!have!bile!salts!as!a!substrate!75,81.! On! the! other! hand,! the! increased! expression! of! acrF! could! make! sense! as! AcrEF! is! somewhat!similar!to!AcrAB,!both!in!amino!acid!identity!and!in!substrate!specificity!81.! Activation!of!acrEF!expression!might!indicate!that!in!this!growth!phase,!once!the!culture! is!adapted,!this!efflux!pump!might!contribute!to!bile!salt!export!along!with!AcrAB.!The! mutant!lacking!all!efflux!systems!but!AcrAB!shows!a!similar!pattern!of!gene!expression! to!that!seen!in!the!wild!type!for!both!acrA(and!acrB!genes.!Since!the!mutant!is!able!to! adapt!and!grow,!this!result!and!the!observations!made!in!stationary!cultures!of!the!wild! type!strain!suggest!that!the!role!of!the!two!differentially!expressed!efflux!systems!(acrEF( and!mdfA)!in!stationary!phase!do!not!play!a!relevant!role!in!bile!salt!export.! ! While!the!leading!function!of!the!efflux!system!AcrAB!in!the!adaptation!process!is!out! of! question,! it! was! curious! to! observe! how,! in! spite! of! such! an! importance,! the! expression!of!both!genes!greatly!decreased!in!stationary!phase,!when!the!cultures!are! already!adapted!(Table!C2.1,!Figure!C2.4).!To!obtain!more!information!about!how!the! AcrAB!efflux!system!contributes!to!bile!adaptation,!AcrAB!activity!was!monitored!by! flow!cytometry!using!ethidium! bromide! (an!AcrAB!substrate)!as!a! reporter.! For! this! purpose,!ethidium!bromide!accumulation!was!tested!in!the!wild!type!and!in!the!mutant! lacking! all! efflux! systems! but! AcrAB! (SV7636)! with! and! without! 5%! DOC.! Ethidium! bromide!accumulation!within!individual!cells!changed!with!time,!especially!when!in!the! presence!of!DOC.!The!results!shown!in!Figure!C2.5!provide!several!conclusions:!! ! 1.! In! the! absence! of! sodium! deoxycholate! both! strains! showed! a! similar! pattern! of! eithidium!bromide!accumulation.! ! 2.!Addition!of!DOC!caused!a!decrease!of!EtBr!accumulation!within!the!cells!of!both! strains,!suggesting!DOC[dependent!activation!of!the!efflux!systems.! Discussion! 109 ! 3.!Even!if!DOC!remained!present!in!the!medium,!gradual!accumulation!of!EtBr!occurred.! This!trend!was!faster!for!the!wild!type!than!for!the!SV7636!strain.!During!this!shift,!two! subpopulations! appeared:! one! that! accumulated! EtBr! and! one! that! did! not.! Since! adapted! cultures! seem! to! accumulate! more! EtBr! than! not! adapted! cultures,! we! hypothesize!that!these!two!subpopulations!might!correspond!to!bile[adapted!bacteria! (right!peak)!and!bacteria!that!are!still!undergoing!adaptation!(left!peak).! ! 4.!The!bile!adaptation!process!takes!less!than!8!hours!after!the!addition!of!the!bile!salt! sodium!deoxycholate!and!ends!with!an!increased!intracellular!EtBr!level,!but!such!levels! never!reach!those!from!cultures!never!exposed!to!DOC.!These!observations!may!have! two! alternative! explanations,! which! are! not! mutually! exclusive.! One! is! that,! once! cultures! are! adapted,! the! intracellular! concentration! of! bile! salts! increases! as! the! bacterium! has! already! activated! mechanisms! that! permit! to! endure! the! damaging! effects! of! bile! within! the! cells:! activation! of! the! RpoS[dependent! general! stress! response,!activation!of!the!SOS!system,!DNA!repair!mechanisms,!etc.!This!hypothesis!is! supported!by!the!fact!that!these!responses!are!elicited!indeed!by!the!presence!of!bile! (Cano!et!al.,!2002;!Hernandez!et!al.,!2012;!Merritt!&!Donaldson,!2009;!Prieto!et!al.,! 2006).!An!alternative!possibility!is!that!bile[adapted!cultures!may!have!remodeled!their! cellular!envelope!to!significantly!decrease!influx!of!bile!salts,!thus!permitting!a!decrease! in!AcrAB!activity.!Is!has!been!previously!shown!that!changes!in!the!cellular!envelope! (LPS,!charge,!hydrophobicity,!etc.)!alter!bile!resistance!levels!in!several!bacterial!species! 57–60.( Figure(C2.6(shows(an(overlap(between(the(EtBr(fluorescence(histograms(of(the(wild(type( and(the!mutant!lacking!eight!efflux!systems!(SV7636),!both!in!the!presence!and!in!the! absence! of! DOC.( In( cultures( grown( without( DOC,( accumulation( of( EtBr( is( higher,( suggesting(a(decrease(in(AcrAB(activity(when(DOC(is(not(present.(The(cultures(with(DOC( show(less(EtBr(accumulation(after(24(h,(indicating(that(the(efflux(system(remains(active( in( a( DOCWdependent( manner.( Figure( C2.7( shows( a( comparison( over( time( of( the( EtBr( fluorescence(mean(in(each(strain(and(treatment.(In(this(scatter(chart,(activation(of(the( AcrAB( efflux( system( can( be( indirectly( monitored( by( EtBr( accumulation.( For( both( Discussion! 110 treatments,(the(efflux(pump(remained(active(in(exponential(phase,(indicating(that(AcrAB( activity(at(this(stage(is(independent(of(the(presence(of(DOC.(On(the(other(hand,(as(the( culture(grows(and(adaptation(occurs,(the(chart(shows(how(the(efflux(system(remains( more(active(in(the(presence(of(DOC.(Also,(in(LB(the(mutant(lacking(most(efflux(systems( (SV7636)(accumulates(more(EtBr(than(the(wild(type,(suggesting(that(the(wild(type(strain( exports(EtBr(more(efficiently(than(the(SV7636(strain.(This(may(be(easily(understood(if( other(efflux(systems(aside(from(AcrAB(export(DOC(75,154.((( ! Taking!into!consideration!the!experimental!data!discussed!above!as!well!as!information! from! the! literature,! we! propose! that! AcrAB! is! the! only! cell! function! essential! for! adaptation,!and!that!activation!of!acrAB(expression!may!be!crucial!at!early!stages!of! adaptation.! As! soon! as! a! critical! amount! of! AcrAB! is! present! in! the! cell,! however,! sustained!AcrAB!activity!may!be!sufficient!to!confer!bile!resistance.!! ! Because!animal!models!are!essential!to!understand!pathogenesis!161!a!goal!of!this!Thesis! was!to!test!adaptation!to!bile!in!an!animal!model.!BALB/c!mice!may!be!an!imperfect! model!of! typhoid!fever! but! the! relevance! of!this! infection!can! make!in( vivo! studies! worthwhile,!no!matter!if!imperfect!or!reductionist.!On!the!other!hand,!bile!resistance! may!be!a!crucial!trait!in!typhoid!fever!as!the!chronic!carrier!state!may!be!a!key!feature! for! continued! maintenance! of! the! pathogen! within! human! populations! 162.! Chronic! carriers!intermittently!shed!the!bacteria!for!a!prolonged,!ill[defined!period!of!time!in!the! local!environment!and!thus!may!spread!the!disease!in!the!community!and!maintain!a! reservoir!of!infection!163.! ! Our!study!of!bile!adaptation!in(vivo!involved!the!analysis!of!populations!of!S.(enterica! isolated!from!BALB/c!mice!gallbladders!5!days!after!oral!infection.!The!analysis!consisted! on! counting! the! number! of! colony! forming! units! recovered! from! each! individual! gallbladder!as!well!as!determining!their!bile!resistance!levels!and!the!stability!of!their! resistance.! The! number! of! c.f.u.! present! in! each! gallbladder! was! extremely! variable! (Table!C3.1).!More!remarkably,!bile!resistant!was!transient!in!over!80%!of!the!isolates.! Because!these!isolates!had!been!able!to!survive!in!the!gallbladder,!simple!statistical! analysis!seems!to!support!the!conclusion!that!bile!adaptation!is!a!common!mechanism! Discussion! 111 employed! by! S.( enterica! to! endure! the! bactericidal! action! of! bile! upon! gallbladder! colonization.!! ! Bile[resistant!mutants!were!also!isolated!from!the!gallbladder!of!BALB/c!mice,!and!full! genome!sequencing! identified!the! mutations!listed! in!Table! C3.2.!Certain! mutations! mapped!in!genes!related!to!the!bacterial!cell!envelope:!rlpB,( yejM,( dipZ,( yhbG.( This! finding!is!in!agreement!with!considerations!made!above!and!also!with!literature!data! that! underline! the! relevance! of! the! envelope! as! a! barrier! 33,38,45,47,123,164.! Curiously! enough,!one!of!the!mutations,!in!the!gene!rlpB,!involved!in!LPS!transport!and!induced! by!bile!131,!had!been!previously!described!by!in!a!study!that!selected!bile!resistant[ mutants!!in( vitro( 88.!LPS!seemed!to! be! altered! in! another!mutant!isolated!from!the! murine!gallbladder,!which!carried!a!mutation!in!the!poorly!known!yejM!gene,!which!is! also!induced!by!bile!131.!In!E.(coli,(yejM!may!be!related!to!cell!permeability!and!lipid!A! production!132,133.!Lipid!A!is!one!of!the!three!structural!regions!of!LPS,!and!it!is!a!highly! conserved!hydrophobic!molecule!that!serves!as!an!anchor!to!the!membrane!for!the!rest! of!the!LPS!70.!! ! Other!mutations!that!may!membrane!alterations!were!detected!in!bile[resistant!isolates! from! the! gall! bladder.! One! mapped! in! the! gene! encoding! in! DsdB,! a! cytoplasmic! membrane!protein!that!could!play!a!role!in!copper!tolerance!136.!Another!mutant!carried! a!mutation!in!yhbG,!a!gene!induced!by!bile!131!which!is!predicted!to!encode!an!ABC! transporter! 143,144.! In! E.( coli,! this! gene! is! essential! and! plays! a! role! in! cell! envelope! integrity!142.! ! Mutations!in!the!division!factor!genes!ftsQ(and!ftsK!were!also!found!in!the!gallblader! isolates.!!Interestingly,!transcription!of!these!genes!is!activated!by!bile!131.!In!E.(coli,!FtsQ! and!FtsK!belong!to!a!set!of!nine!proteins!that!participate!in!!septum!formation!138,139.! Interestingly,!previous!studies!from!our!laboratory!identified!another!cell!division!factor! (ZapB)!that!is!induced!by!bile!salts!88.! ( Altogether,!the!investigations!presented!in!this!study!provide!a!mechanistic!model!to! understand!adaptation!to!bile,!a!phenomenon!that!may!be!fundamental!during!systemic! Discussion! 112 infection!by!Salmonella(enterica,!and!perhaps!during!chronic!infection!as!well.! ! ! 113 ! & & & & & & & & & & CONCLUSIONS& & References! 120 Nat.(Rev.(Microbiol.!6,!53–66!(2008).! 21.! Zhou,!D.!&!Galán,!J.!Salmonella!entry!into!host!cells :!the!work!in!concert!of!type! III!secreted!effector!proteins.!Microbes(Infect.!1293–1298!(2001).! 22.! Zhang,!S.!et(al.!MINIREVIEW!Molecular!Pathogenesis!of!Salmonella!enterica! Serotype!typhimurium[induced!diarrhea.!Infect.(Immun.!71,!1–12!(2003).! 23.! Bäumler,!A.!J.,!Winter,!S.!E.,!Thiennimitr,!P.!&!Casadesús,!J.!Minireview!Intestinal! and!chronic!infections :!Salmonella!lifestyles.!Environ.(Microbiol.(Rep.!3,!508– 517!(2011).! 24.! Watson,!K.!G.!&!Holden,!D.!W.!Microreview!Dynamics!of!growth!and! dissemination!of!Salmonella!in!vivo.!Cell.(Microbiol.!12,!1389–1397!(2010).! 25.! Tischler,!A.!D.!&!Mckinney,!J.!D.!Contrasting!persistence!strategies!in!Salmonella! and!Mycobacterium.!Curr.(Opin.(Microbiol.!4,!93–99!(2010).! 26.! Monack,!D.!M.,!Bouley,!D.!M.!&!Falkow,!S.!Salmonella!typhimurium!Persists! within!Macrophages!in!the!Mesenteric!Lymph!Nodes!of!Chronically!Infected! Nramp1!ϩ!/!ϩ!Mice!and!Can!Be!Reactivated!by!IFN!␥!Neutralization.!J.(Exp.(Med.! 199,!(2004).! 27.! Lai,!C.!W.,!Chan,!R.!C.,!Cheng,!A.!F.,!Sung,!J.!Y.!&!Leung,!J.!W.!Common!bile!duct! stones:!a!cause!of!chronic!salmonellosis.!Am.(J.(Gastroenterol.!87,!1198–1199! (1992).! 28.! Prouty,!A.!M.,!Schwesinger,!W.!H.!&!Gunn,!J.!S.!Biofilm!Formation!and! Interaction!with!the!Surfaces!of!Gallstones!by!Salmonella!spp!.!Infect.(Immun.! 70,!2640–2649!(2002).! 29.! Crawford,!R.!W.!et(al.!Gallstones!play!a!signi!fi!cant!role!in!Salmonella!spp!.! gallbladder!colonization!and!carriage.!Proc.(Natl.(Acad.(Sci.!107,!4353–4358! (2010).! 30.! Rychlik,!I.!&!Barrow,!P.!A.!Salmonella!stress!management!and!its!relevance!to! behaviour!during!intestinal!colonisation!and!infection.!FEMS(Microbiol.(Rev.!29,! 1021–1040!(2005).! 31.! Clements,!M.,!Eriksson,!S.,!Tezcan[merdol,!D.!&!Hinton,!J.!C.!D.!Virulence!gene! regulation!in!Salmonella!enterica.!178–185!(2001).! 32.! Finlay,!B.!B.!&!Falkow,!S.!MicroReview!Salmonella!as!an!intracellular!parasite.! Mol.(Microbiol.!3,!1833–1841!(1989).! 33.! Alvarez[Ordonez,!A.!et(al.!Salmonella!spp!.!survival!strategies!within!the!host! gastrointestinal!tract.!Food(Res.(Int.!3268–3281!(2016).! doi:10.1099/mic.0.050351[0! 34.! Spector,!M.!P.!&!Kenyon,!W.!J.!Resistance!and!survival!strategies!of!Salmonella! enterica!to!environmental!stresses.!FRIN!(2011).! doi:10.1016/j.foodres.2011.06.056! 35.! Fontana,!J.!et(al.!Functions!of!cells!and!human!body.!Multimedia!textbook.!at! <http://fblt.cz/en/>! 36.! Hofmann,!A.!F.!Bile!Acids :!The!Good!,!the!Bad!,!and!the!Ugly.!News(Physiol.(Sci.! (1999).! 37.! Hofmann,!A.!F.!Bile!Secretion!in!Mice!and!Men.!Hepatology!847–852!(2001).! doi:10.1053/jhep.2001.28191! 38.! Begley,!M.,!Gahan,!C.!G.!M.!&!Hill,!C.!The!interaction!between!bacteria!and!bile.! FEMS(Microbiol.(Rev.!29,!625–651!(2005).! 39.! Casadesús,!J.,!Hernández,!S.!B.,!Cota,!I.!&!Ramos[Morales,!F.!in!The(Lure(of( References! 121 Bacterial(Genetics!(eds.!Casadesús,!J.,!Hughes,!K.!T.!&!Maloy,!S.)!153–162! (American!Society!of!Microbiology,!2011).!doi:10.1128/9781555816810! 40.! Hofmann,!A.!F.!&!Hagey,!L.!R.!Bile!acids:!Chemistry,!pathochemistry,!biology,! pathobiology,!and!therapeutics.!Cell.(Mol.(Life(Sci.!65,!2461–2483!(2008).! 41.! Russell,!D.!W.!The!enzymes,!regulation,!and!genetics!of!bile!acid!synthesis.! Annu.(Rev.(Biochem.!72,!137–174!(2003).! 42.! Monte,!M.!J.,!Marin,!J.!J.!G.,!Antelo,!A.!&!Vazquez[Tato,!J.!Bile!acids:!Chemistry,! physiology,!and!pathophysiology.!World(J.(Gastroenterol.!15,!804–816!(2009).! 43.! Ridlon,!J.!M.,!Kang,!D.[J.!&!Hylemon,!P.!B.!Bile!salt!biotransformations!by!human! intestinal!bacteria.!J.(Lipid(Res.!47,!241–59!(2006).! 44.! Hylemon,!P.!B.!&!Harder,!J.!Biotransformation!of!monoterpenes,!bile!acids,!and! other!isoprenoids!in!anaerobic!ecosystems.!FEMS(Microbiol.(Rev.!22,!475–488! (1998).! 45.! Merritt,!M.!E.!&!Donaldson,!J.!R.!Effect!of!bile!salts!on!the!DNA!and!membrane! integrity!of!enteric!bacteria.!J.(Med.(Microbiol.!58,!1533–1541!(2009).! 46.! Hofmann,!A.!F.!&!Eckmann,!L.!How!bile!acids!confer!gut!mucosal!protection! against!bacteria.!Pnas!103,!4333–4334!(2006).! 47.! Gunn,!J.!S.!Mechanisms!of!bacterial!resistance!and!response!to!bile.!907–913! (2000).! 48.!Leverrier,!P.!et(al.!Susceptibility!and!Adaptive!Response!to!Bile!Salts!in! Propionibacterium!freudenreichii :!Physiological!and!Proteomic!Analysis! Susceptibility!and!Adaptive!Response!to!Bile!Salts!in!Propionibacterium! freudenreichii :!Physiological!and!Proteomic!Analysis.!Appl.(Environ.(Microbiol.! 69,!3809–3818!(2003).! 49.! Fujisawa,!T.!&!Mori,!M.!Influence!of!bile!salts!on!β[glucuronidase!activity!of! intestinal!bacteria.!Lett.(Appl.(Microbiol.!22,!271–274!(1996).! 50.! Noh,!D.!O.!&!Gilliland,!S.!E.!Influence!of!bile!on!cellular!integrity!and!beta[ galactosidase!activity!of!Lactobacillus!acidophilus.!J.(Dairy(Sci.!76,!1253–1259! (1993).! 51.! Coleman,!R.,!Lowe,!P.!J.!&!Billington,!D.!Membrane!lipid!composition!and! susceptibility!to!bile!salt!damage.!BBA(W(Biomembr.!599,!294–300!(1980).! 52.! Gómez!Zavaglia,!A.,!Kociubinski,!G.,!Pérez,!P.,!Disalvo,!E.!&!De!Antoni,!G.!Effect! of!bile!on!the!lipid!composition!and!surface!properties!of!bifidobacteria.!J.(Appl.( Microbiol.!93,!794–799!(2002).! 53.! Záratea,!G.,!Gonzaleza,!S.,!Chaia,!A.!P.!&!Oliver,!G.!Effect!of!bile!on!the!β![ galactosidase!activity!of!dairy!propionibacteria.!Lait!80,!267–276!(2000).! 54.! Hofmann,!M.!et(al.!LUV’s!lipid!composition!modulates!diffusion!of!bile!acids.! Chem.(Phys.(Lipids!110,!165–171!(2001).! 55.! Cabral,!D.!J.,!Small,!D.!M.,!Lilly,!H.!S.!&!Hamilton,!J.!A.!Transbilayer!movement!of! bile!acids!in!model!membranes.!Biochemistry!26,!1801–1804!(1987).! 56.! Schubert,!R.,!Jaroni,!H.,!Schoelmerich,!J.!&!Schmidt,!K.!H.!Studies!on!the! Mechanism!of!Bile!Salt[Induced!Liposomal!Membrane!Damage.!Digestion!28,! 181–190!(1983).! 57.! Chou,!C.!&!Cheng,!S.!Recovery!of!low[temperature!stressed!E!.!coli!O157 :!H7! and!its!susceptibility!to!crystal!violet!,!bile!salt!,!sodium!chloride!and!ethanol.!61,! 127–136!(2000).! 58.! Chou,!L.!S.!&!Weimer,!B.!Isolation!and!characterization!of!acid[!and!bile[tolerant! References! 122 isolates!from!strains!of!Lactobacillus!acidophilus.!J.(Dairy(Sci.!82,!23–31!(1999).! 59.! King,!T.,!Ferenci,!T.!&!Szabo,!E.!A.!The!effect!of!growth!atmosphere!on!the!ability! of!Listeria!monocytogenes!to!survive!exposure!to!acid,!proteolytic!enzymes!and! bile!salts.!Int.(J.(Food(Microbiol.!84,!133—143!(2003).! 60.! Fernández!Murga,!M.!L.,!Bernik,!D.,!Font!de!Valdez,!G.!&!Disalvo,!!a!E.! Permeability!and!stability!properties!of!membranes!formed!by!lipids!extracted! from!Lactobacillus!acidophilus!grown!at!different!temperatures.!Arch.(Biochem.( Biophys.!364,!115–21!(1999).! 61.! Bernstein,!C.,!Bernstein,!H.,!Payne,!C.!M.,!Beard,!S.!E.!&!Schneider,!J.!Bile!salt! activation!of!stress!response!promoters!in!Escherichia!coli.!Curr.(Microbiol.!39,! 68–72!(1999).! 62.! Chou,!J.!H.,!Greenberg,!J.!T.!&!Demple,!B.!Posttranscriptional!repression!of! Escherichia!coli!OmpF!protein!in!response!to!redox!stress:!Positive!control!of!the! micF!antisense!RNA!by!the!soxRS!locus.!J.(Bacteriol.!175,!1026–1031!(1993).! 63.! Prieto,!A.!I.,!Ramos[Morales,!F.!&!Casadesús,!J.!Bile[induced!DNA!damage!in! Salmonella!enterica.!Genetics!168,!1787–1794!(2004).! 64.! Prieto,!A.!I.,!Ramos[Morales,!F.!&!Casadesús,!J.!Repair!of!DNA!damage!induced! by!bile!salts!in!Salmonella!enterica.!Genetics!174,!575–584!(2006).! 65.! Gahan,!C.!G.!M.,!O’Mahony,!J.!&!Hill,!C.!Characterization!of!the!groESL!operon!in! Listeria!monocytogenes:!Utilization!of!two!reporter!systems!(gfp!and!hly)!for! evaluating!in!vivo!expression.!Infect.(Immun.!69,!3924–3932!(2001).! 66.! Rince,!A.,!Flahaut,!S.!&!Auffray,!Y.!Identification!of!general!stress!genes!in! Enterococcus!faecalis.!Int.(J.(Food(Microbiol.!55,!87–91!(2000).! 67.! Schmidt,!G.!&!Zink,!R.!Basic!features!of!the!stress!response!in!three!species!of! bifidobacteria:!B.!longum,!B.!adolescentis,!and!B.!breve.!Int.(J.(Food(Microbiol.! 55,!41–45!(2000).! 68.! T.!J.!Silhavy,!D.!Kahne,!!and!S.!W.!The!bacterial!cell!envelope.!Cold(Spring(Harb.( Perspect.(Biol.!2,!1–17!(2010).! 69.! Lerouge,!I.!&!Vanderleyden,!J.!O[antigen!structural!variation :!mechanisms!and! possible!roles!in!animal!/!plant!^!microbe!interactions.!FEMS(Microbiol.(Rev.!26,! (2001).! 70.! Raetz,!C.!R.!H.!&!Whitfield,!C.!L!<scp>IPOPOLYSACCHARIDE</scp>!E! <scp>NDOTOXINS</scp>.!Annu.(Rev.(Biochem.!71,!635–700!(2002).! 71.! Picken,!R.!&!Beacham,!I.!Bacteriophage[resistant!mutants!of!Escherichia!coli! K12.!Location!of!Receptors!within!the!Lipopolysaccharide.!J.(Gen.(Microbiol.! 305–318!(1977).! 72.! Crawford,!R.!W.!et(al.!Very!Long!O[antigen!Chains!Enhance!Fitness!during! Salmonella[induced!Colitis!by!Increasing!Bile!Resistance.!PLoS(Pathog.!8,!(2012).! 73.! Ramos[morales,!F.,!Prieto,!A.!I.,!Beuzon,!C.!R.,!Holden,!D.!W.!&!Casadesús,!J.! Role!for!Salmonella!enterica!Enterobacterial!Common!Antigen!in!Bile!Resistance! and!Virulence.!J.(Bacteriol.!185,!5328–5332!(2003).! 74.! Thanassi,!D.!G.,!Cheng,!L.!W.!&!Nikaido,!H.!Active!efflux!of!bile!salts!by! Escherichia!coli!.!Active!Efflux!of!Bile!Salts!by!Escherichia!coli.!179,!2512–2518! (1997).! 75.! Horiyama,!T.,!Yamaguchi,!A.!&!Nishino,!K.!TolC!dependency!of!multidrug!efflux! systems!in!Salmonella!enterica!serovar!Typhimurium.!J.(Antimicrob.(Chemother.! 65,!1372–1376!(2010).! References! 123 76.! Ma,!D.!et(al.!Genes!acrA!and!acrB!encode!a!stress[induced!efflux!system!of! Escherichia!coli.!Mol.(Microbiol.!16,!45–55!(1995).! 77.! Ma,!D.!et(al.!Molecular!Cloning!and!Characterization!of!acrA!and!acrE!Genes!of! Escherichia!coli.!175,!6299–6313!(1993).! 78.! Zgurskaya,!H.!I.!&!Nikaido,!H.!Bypassing!the!periplasm :!Reconstitution!of!the! AcrAB!multidrug!efflux!pump!of!Escherichia!coli.!Proc.(Natl.(Acad.(Sci.!96,!7190– 7195!(1999).! 79.! Nikaido,!E.,!Yamaguchi,!A.!&!Nishino,!K.!AcrAB!Multidrug!Efflux!Pump!Regulation! in!Salmonella!enterica!serovar!Typhimurium!by!RamA!in!Response!to! Environmental!Signals!*.!283,!24245–24253!(2008).! 80.! Murakami,!S.,!Nakashima,!R.,!Yamashita,!E.!&!Yamaguchi,!A.!Crystal!structure!of! bacterial!multidrug!efflux!transporter!AcrB.!Nature!419,!587–593!(2002).! 81.! Nishino,!K.,!Latifi,!T.!&!Groisman,!E.!A.!Virulence!and!drug!resistance!roles!of! multidrug!efflux!systems!of!Salmonella!enterica!serovar!Typhimurium.!Mol.( Microbiol.!59,!126–141!(2006).! 82.! Pos,!K.!M.!Trinity!revealed:!Stoichiometric!complex!assembly!of!a!bacterial! multidrug!efflux!pump.!Proc.(Natl.(Acad.(Sci.(!106&,!6893–6894!(2009).! 83.! Pos,!K.!M.!Biochimica!et!Biophysica!Acta!Drug!transport!mechanism!of!the!AcrB! ef!fl!ux!pump.!BBA(W(Proteins(Proteomics!1794,!782–793!(2009).! 84.! Baucheron,!S.!et(al.!Bile[mediated!activation!of!the!acrAB!and!tolC!multidrug! efflux!genes!occurs!mainly!through!transcriptional!derepression!of!ramA!in! Salmonella!enterica!serovar!Typhimurium.!1,!2400–2406!(2014).! 85.! Cano,!D.!A.,!Pucciarelli,!M.!G.,!Garc??a[Del!Portillo,!F.!&!Casades??s,!J.!Role!of! the!RecBCD!recombination!pathway!in!Salmonella!virulence.!J.(Bacteriol.!184,! 592–595!(2002).! 86.! van!Velkinburgh,!J.!C.!&!Gunn,!J.!S.!PhoP[PhoQ[Regulated!Loci!Are!Required!for! Enhanced!Bile!Resistance!in!Salmonella!spp!.!67,!1614–1622!(1999).! 87.! Prouty,!A.!M.!et(al.!Transcriptional!regulation!of!Salmonella!enterica!serovar! Typhimurium!genes!by!bile.!41,!177–185!(2004).! 88.! Hernandez,!S.!B.,!Cota,!I.,!Ducret,!A.,!Aussel,!L.!&!Casadesus,!J.!Adaptation!and! Preadaptation!of!Salmonella!enterica!to!Bile.!8,!(2012).! 89.! Schmieger,!H.!Phage!P22[mutants!with!increased!or!decreased!transduction! abilities.!MGG(Mol.(Gen.(Genet.!119,!75–88!(1972).! 90.! Smith,!H.!O.!&!Levine,!M.!Two!sequential!repressions!of!DNA!synthesis!in!the! stablishment!of!lysogeny!by!phage!P22!and!its!mutants.!Proc.(Natl.(Acad.(Sci.!52,! 356–363!(1964).! 91.! Cherepanov,!P.!P.!&!Wackernagel,!W.!Gene!disruption!in!Escherichia!coli:!TcR! and!KmR!cassettes!with!the!option!of!Flp[catalyzed!excision!of!the!antibiotic[ resistance!determinant.!Gene!158,!9–14!(1995).! 92.! Datsenko,!K.!A.!&!Wanner,!B.!L.!One[step!inactivation!of!chromosomal!genes!in! Escherichia!coli!K[12!using!PCR!products.!2000,!(2000).! 93.!Bolivar,!F.!&!Backman,!K.!Plasmids!of!Escherichia!coli!as!cloning!vectors.! Methods(Enzymol.!68,!245–267!(1979).! 94.! Inoue,!H.,!Nojima,!H.!&!Okayama,!H.!High!efficiency!transformation!of! Escherichia!coli!with!plasmids.!Gene!96,!23–28!(1990).! 95.! Rappleye,!C.!A.!&!Roth,!J.!R.!A!Tn!10!Derivative!(!T[POP!)!for!Isolation!of! Insertions!with!Conditional!(!Tetracycline[Dependent!)!Phenotypes.!179,!5827– References! 124 5834!(1997).! 96.! Lee,!C.,!Wozniak,!C.,!Karlinsey,!J.!E.!&!Hughes,!K.!T.!Genomic!Screening!for! Regulatory!Genes!Using!the!T[POP!Transposon.!Methods(in(Enzymology!421,! 159–167!(2007).! 97.! Kleckner,!N.,!Bender,!J.!&!Gottesman,!S.!Uses!of!transposons!with!emphasis!on! Tn10.!Methods(Enzymol.!204,!139–180!(1991).! 98.! Chun,!K.!T.,!Edenberg,!H.!J.,!Kelley,!M.!R.!&!Goebl,!M.!G.!Rapid!Amplification!of! Uncharacterized!Transposon[!tagged!DNA!Sequences!from!Genomic!DNA.!DNA( Seq.!240,!233–240!(1997).! 99.! Szybalski,!W.!&!Bryson,!V.!Genetic!Studies!on!Microbial!Cross!Resistance!To! Toxic!Agents.!J.(Bacteriol.!64,!489–499!(1952).! 100.! Rothberg,!J.!M.!&!Leamon,!J.!H.!The!development!and!impact!of!454!sequencing.! Nat.(Biotechnol.!26,!1117–1124!(2008).! 101.! Roche!Diagnostic.!Rapid!Library!Preparation!Method!Manual!GS!FLX+!Series![! XL+.!Roche(Diagnostic!(2011).! 102.! Li,!H.!&!Durbin,!R.!Fast!and!accurate!short!read!alignment!with!Burrows[ Wheeler!transform.!Bioinformatics!25,!1754–1760!(2009).! 103.! Li,!H.!&!Durbin,!R.!Fast!and!accurate!long[read!alignment!with!Burrows[Wheeler! transform.!Bioinformatics!26,!589–595!(2010).! 104.! Viveiros,!M.!et(al.!Demonstration!of!intrinsic!efflux!activity!of!Escherichia!coli!K[ 12!AG100!by!an!automated!ethidium!bromide!method.!Int.(J.(Antimicrob.( Agents!31,!458–462!(2008).! 105.! Martins,!M.!et(al.!An!instrument[free!method!for!the!demonstration!of!efflux! pump!activity!of!bacteria.!In(Vivo((Brooklyn).!20,!657–664!(2006).! 106.! Marinus,!M.!G.!&!Løbner[Olesen,!A.!DNA!Methylation.!EcoSal(Plus!6,!1–62! (2014).! 107.!Løbner[olesen,!A.,!Skovgaard,!O.!&!Marinus,!M.!G.!Dam!methylation :! coordinating!cellular!processes.!Curr.(Opin.(Microbiol.!154–160!(2005).! doi:10.1016/j.mib.2005.02.009! 108.! Low,!D.!A.,!Weyand,!N.!J.!&!Mahan,!M.!J.!Roles!of!DNA!Adenine!Methylation!in! Regulating!Bacterial!Gene!Expression!and!Virulence.!Infect.(Immun.!69,!7197– 7204!(2001).! 109.! Torreblanca,!J.!&!Casadesús,!J.!DNA!Adenine!Methylase!Mutants!of!Salmonella! Eylbhimurium!and!a!Novel!Dam[Regulated!Locus.!Genetics!26,!15–26!(1996).! 110.! Arends,!S.!J.!R.!et(al.!Discovery!and!Characterization!of!Three!New!Escherichia! coli!Septal!Ring!Proteins!That!Contain!a!SPOR!Domain :!DamX!,!DedD!,!and!RlpA! .!J.(Bacteriol.!192,!242–255!(2010).! 111.!Lopez[Garrido,!J.,!Cheng,!N.,!Garcia[Quintanilla,!F.,!García[del!Portillo,!F.!&! Casadesús,!J.!Identification!of!the!Salmonella!enterica!damX!Gene!Product!,!an! Inner!Membrane!Protein!Involved!in!Bile!Resistance!!†.!J.(Bacteriol.!192,!893– 895!(2010).! 112.! Hancock,!R.!E.!W.!&!McPhee,!J.!B.!Salmonella!’!s!Sensor!for!Host!Defense! Molecules.!Cell!320–323!(2005).!doi:10.1016/j.cell.2005.07.021! 113.! Gunn,!J.!S.!Bacterial!modification!of!LPS!and!resistance!to!antimicrobial! peptides.!J.(Endotoxin(Res.!7,!57–62!(2001).! 114.! Nickerson,!C.!A.!&!Curtiss!III,!R.!Role!of!Sigma!Factor!RpoS!in!Initial!Stages!of! Salmonella!typhimurium!Infection.!Infect.(Immun.!65,!1814–1823!(1997).! References! 125 115.!Chen,!C.!et(al.!Central!Regulatory!Role!for!the!RpoS!Sigma!Factor!in!Expression! of!Salmonella!dublin!Plasmid!Virulence!Genes.!J.(Bacteriol.!177,!5303–5309! (1995).! 116.! Glauner,!B.,!Holtje,!J.[V.!&!Schwarz,!U.!The!Composition!of!the!Murein!of! Escherichia!coli!*.!J.(Biol.(Chem.!263,!10088–10095!(1988).! 117.! Koronakis,!V.!TolC![!the!bacterial!exit!duct!for!proteins!and!drugs.!FEBS(Lett.!555,! 66–71!(2003).! 118.! Nishino,!K.,!Nikaido,!E.!&!Yamaguchi,!A.!Regulation!and!physiological!function!of! multidrug!ef!fl!ux!pumps!in!Escherichia!coli!and!Salmonella.!BBA(W(Proteins( Proteomics!1794,!834–843!(2009).! 119.! Broadbent,!S.!E.,!Balbontin,!R.,!Casadesus,!J.,!Marinus,!M.!G.!&!Woude,!M.!Van! Der.!YhdJ!,!a!Nonessential!CcrM[Like!DNA!Methyltransferase!of!Escherichia!coli! and!Salmonella!enterica!.!J.(Bacteriol.!189,!4325–4327!(2007).! 120.! Ramos,!J.!L.!et(al.!The!TetR!Family!of!Transcriptional!Repressors.!69,!326–356! (2005).! 121.! Frisch,!R.!L.!et(al.!Separate!DNA!Pol!II[!and!Pol!IV[Dependent!Pathways!of!Stress[ Induced!Mutation!during!Double[Strand[Break!Repair!in!Escherichia!coli!Are! Controlled!by!RpoS!.!J.(Bacteriol.!192,!4694–4700!(2010).! 122.! Prouty,!A.!M.,!Brodsky,!I.!E.,!Falkow,!S.!&!Gunn,!J.!S.!Bile[salt[mediated!induction! of!antimicrobial!and!bile!resistance!in!Salmonella!typhimurium.!Microbiology! 775–783!(2004).!doi:10.1099/mic.0.26769[0! 123.! Prieto,!A.!I.!et(al.!Roles!of!the!Outer!Membrane!Protein!AsmA!of!Salmonella! enterica!in!the!Control!of!marRAB!Expression!and!Invasion!of!Epithelial!Cells!.! 191,!3615–3622!(2009).! 124.! Hernández,!S.!B.,!Ayala,!J.!A.,!Rico[pérez,!G.,!García[del!Portillo,!F.!&!Casadesús,! J.!Increased!bile!resistance!in!Salmonella!enterica!mutants!lacking!Prc! periplasmic!protease.!87–92!(2013).!doi:10.2436/20.1501.01.183! 125.!Usui,!M.,!Asai,!T.!&!Sato,!S.!Low!expression!of!AcrB!in!the!deoxycholate[sensitive! strains!of!Salmonella!enterica!subspecies!enterica!serovar!Pullorum.!366–368! (2011).!doi:10.1111/j.1348[0421.2011.00321.x! 126.! Viveiros,!M.!et(al.!Demonstration!of!intrinsic!efflux!activity!of!Escherichia!coli!K[ 12!AG100!by!an!automated!ethidium!bromide!method!(PDF!Download! Available).pdf.!Int.(J.(Antimicrob.(Agents!458–462!(2008).! 127.! Sánchez[romero,!M.!A.!&!Casadesús,!J.!Contribution!of!phenotypic! heterogeneity!to!adaptive!antibiotic!resistance.!111,!355–360!(2014).! 128.! Canonne[Hergaux,!F.,!Gruenheid,!S.,!Govoni,!G.!&!Gros,!P.!The!Nramp1!protein! and!its!role!in!resistance!to!infection!and!macrophage!function.!Proc.(Assoc.(Am.( Physicians!111,!283–289!(1999).! 129.! Gruenheid,!S.,!Pinner,!E.,!Desjardins,!M.!&!Gros,!P.!Natural!resistance!to! infection!with!intracellular!pathogens:!the!Nramp1!protein!is!recruited!to!the! membrane!of!the!phagosome.!J.(Exp.(Med.!185,!717–730!(1997).! 130.!Wu,!T.!et(al.!Identification!of!a!protein!complex!that!assembles! lipopolysaccharide!in!the!outer!membrane!of!Escherichia!coli.!Proc.(Natl.(Acad.( Sci.(U.(S.(A.!103,!11754–11759!(2006).! 131.!Kr??ger,!C.!et(al.!An!infection[relevant!transcriptomic!compendium!for! salmonella!enterica!serovar!typhimurium.!Cell(Host(Microbe!14,!683–695!(2013).! 132.! Hirvas,!L.,!Nurminen,!M.,!Helander,!I.!M.,!Vuorio,!R.!&!Vaara,!M.!The!lipid!a! References! 126 biosynthesis!deficiency!of!the!Escherichia!coli!antibiotic[supersensitive!mutant! LH530!is!suppressed!by!a!novel!locus,!ORF195.!Microbiology!143,!73–81!(1997).! 133.! De!Lay,!N.!R.!&!Cronan,!J.!E.!Genetic!interaction!between!the!Escherichia!coli! AcpT!phosphopantetheinyl!transferase!and!the!YejM!inner!membrane!protein.! Genetics!178,!1327–1337!(2008).! 134.! Stewart,!E.!J.,!Katzen,!F.!&!Beckwith,!J.!Six!conserved!cysteines!of!the!membrane! protein!DsbD!are!required!for!the!transfer!of!electrons!from!the!cytoplasm!to! the!periplasm!of!Escherichia!coli.!EMBO(J.!18,!5963–5971!(1999).! 135.! Gordon,!E.!H.,!Page,!M.!D.,!Willis,!A.!C.!&!Ferguson,!S.!J.!Escherichia!coli!DipZ:! anatomy!of!a!transmembrane!protein!disulphide!reductase!in!which!three!pairs! of!cysteine!residues,!one!in!each!of!three!domains,!contribute!differentially!to! function.!Mol.(Microbiol.!35,!1360–1374!(2000).! 136.! Gupta,!S.!D.,!Wu,!H.!C.,!Rick,!P.!D.!&!Rick,!P.!D.!A!Salmonella!typhimurium! genetic!locus!which!confers!copper!tolerance!on!copper[sensitive!mutants!of! Escherichia!coli!A!Salmonella!typhimurium!Genetic!Locus!Which!Confers!Copper! Tolerance!on!Copper[Sensitive!Mutants!of!Escherichia!coli!†.!179,!4977–4984! (1997).! 137.! Blattner,!F.!R.!et(al.!The!Complete!Genome!Sequence!of!Escherichia!coli!K[12.! Science((80W.().!277,!1453–1462!(1997).! 138.! Chen,!J.!C.!&!Beckwith,!J.!FtsQ,!FtsL!and!Ftsl!require!FtsK,!but!not!FtsN,!for!co[ localization!with!FtsZ!during!Escherichia!coli!cell!division.!Mol.(Microbiol.!42,! 395–413!(2001).! 139.! Buddelmeijer,!N.!&!Beckwith,!J.!A!complex!of!the!Escherichia!coli!cell!division! proteins!FtsL,!FtsB!and!FtsQ!forms!independently!of!its!localization!to!the!septal! region.!Mol.(Microbiol.!52,!1315–1327!(2004).! 140.! Heithoff,!D.!M.!et(al.!Salmonella!DNA!adenine!methylase!mutants!confer!cross[ protective!immunity.!Infect.(Immun.!69,!6725–6730!(2001).! 141.! Giacomodonato,!M.!N.,!Sarnacki,!S.!H.,!Caccuri,!R.!L.,!Sordelli,!D.!O.!&!Cerquetti,! M.!C.!Host!response!to!a!dam!mutant!of!Salmonella!enterica!serovar!enteritidis! with!a!temperature[sensitive!phenotype.!Infect.(Immun.!72,!5498–5501!(2004).! 142.! Sperandeo,!P.,!Pozzi,!C.,!Dehò,!G.!&!Polissi,!A.!Non[essential!KDO!biosynthesis! and!new!essential!cell!envelope!biogenesis!genes!in!the!Escherichia!coli!yrbG!–! yhbG!locus.!157,!547–558!(2006).! 143.! Dassa,!E.!&!Bouige,!P.!The!ABC!of!ABCS:!a!phylogenetic!and!functional! classification!of!ABC!systems!in!living!organisms.!Res.(Microbiol.!152,!211–229! (2001).! 144.! Saurin,!W.,!Hofnung,!M.!&!Dassa,!E.!Getting!in!or!out:!early!segregation!between! importers!and!exporters!in!the!evolution!of!ATP[binding!cassette!(ABC)! transporters.!J.(Mol.(Evol.!48,!22–41!(1999).! 145.! Blair,!J.!M.!&!Piddock,!L.!J.!Structure,!function!and!inhibition!of!RND!efflux! pumps!in!Gram[negative!bacteria:!an!update.!Curr.(Opin.(Microbiol.!12,!512–519! (2009).! 146.! Paulsen,!I.!T.,!Park,!J.!H.,!Choi,!P.!S.!&!Jr.,!M.!H.!S.!A!family!of!Gram[negative! bacterial!outer!membrne!factors!that!function!in!the!export!of!protiens,! carbohydrates,!drugs!and!heavy!metals!from!Gram[negative!bacteria.!FEMS( Microbiol.(Lett!156,!1–8!(1997).! 147.! Nikaido,!H.!Multidrug!efflux!pumps!of!gram[negative!bacteria.!J.(Bacteriol.!178,! References! 127 5853–5859!(1996).! 148.! Nikaido,!H.!Preventing!drug!access!to!targets:!cell!surface!permeability!barriers! and!active!efflux!in!bacteria.!Semin.(Cell(Dev.(Biol.!12,!215–223!(2001).! 149.! Lacroix,!F.!J.!C.!et(al.!Salmonella!typhimurium!acrB[like!gene:!Identification!and! role!in!resistance!to!biliary!salts!and!detergents!and!in!murine!infection.!FEMS( Microbiol.(Lett.!135,!161–167!(1996).! 150.! Nikaido,!H.,!Basina,!M.,!Nguyen,!V.,!Emiko,!Y.!&!Rosenberg,!E.!Y.!Multidrug!Efflux! Pump!AcrAB!of!Salmonella!typhimurium!Excretes!Only!Those!β![Lactam! Antibiotics!Containing!Lipophilic!Side!Chains!Multidrug!Efflux!Pump!AcrAB!of! Salmonella!typhimurium!Excretes!Only!Those!␤![Lactam!Antibiotics!Containing! Lipophilic!Side!Ch.!180,!4686–4692!(1998).! 151.! Prouty,!A.!M.,!Van!Velkinburgh,!J.!C.!&!Gunn,!J.!S.!Salmonella!enterica!serovar! typhimurium!resistance!to!bile:!Identification!and!characterization!of!the!tolQRA! cluster.!J.(Bacteriol.!184,!1270–1276!(2002).! 152.! Nishino,!K.,!Hayashi[Nishino,!M.!&!Yamaguchi,!A.!H[NS!modulates!multidrug! resistance!of!Salmonella!enterica!serovar!typhimurium!by!repressing!multidrug! efflux!genes!acrEF.!Antimicrob.(Agents(Chemother.!53,!3541–3543!(2009).! 153.! Olliver,!A.,!Vallé,!M.,!Chaslus[Dancla,!E.!&!Cloeckaert,!A.!Overexpression!of!the! multidrug!efflux!operon!acrEF!by!insertional!activation!with!IS1!or!IS10!elements! in!Salmonella!enterica!serovar!typhimurium!DT204!acrB!mutants!selected!with! fluoroquinolones.!Antimicrob.(Agents(Chemother.!49,!289–301!(2005).! 154.! Li,!X.,!Plésiat,!P.!&!Nikaido,!H.!The!Challenge!of!Efflux[Mediated!Antibiotic! Resistance!in!Gram[Negative!Bacteria.!Clin.(Microbiol.(Rev.!28,!337–418!(2015).! 155.! Tsukagoshi,!N.!&!Aono,!R.!Entry!into!and!release!of!solvents!by!Escherichia!coli! in!an!organic[aqueous!two[liquid[phase!system!and!substrate!specificity!of!the! AcrAB[TolC!solvent[extruding!pump.!J.(Bacteriol.!182,!4803–4810!(2000).! 156.! Lennen,!R.!M.,!Politz,!M.!G.,!Kruziki,!M.!A.!&!Pfleger,!B.!F.!Identification!of! transport!proteins!involved!in!free!fatty!acid!efflux!in!Escherichia!coli.!J.( Bacteriol.!195,!135–144!(2013).! 157.! Nikaido,!H.!MINI[REVIEWS!The!role!of!outer!membrane!and!efflux!pumps!in!the! resistance!of!Can!we!improve!drug.!93–98!(1998).! 158.! Nikaido,!H.!Outer!membrane!barrier!as!a!mechanism!of!antimicrobial!resistance.! Antimicrob.(Agents(Chemother.!33,!1831–1836!(1989).! 159.! Wandersman,!C.!Secretion!across!the!bacterial!outer!membrane.!Trends(Genet.! 8,!317–322!(1992).! 160.! Zgurskaya,!H.!I.!&!Nikaido,!H.!Multidrug!resistance!mechanisms:!drug!efflux! across!two!membranes.!Mol(Microbiol!37,!219–225!(2000).! 161.! Garai,!P.,!Gnanadhas,!D.!P.!&!Chakravortty,!D.!Salmonella!enterica!serovars! Typhimurium!and!Typhi!as!model!organisms:!Revealing!paradigm!of!host[ pathogen!interactions.!Virulence!3,!377–388!(2012).! 162.!Gunn,!J.!S.!et(al.!Salmonella!chronic!carriage:!Epidemiology,!diagnosis,!and! gallbladder!persistence.!Trends(Microbiol.!22,!648–655!(2014).! 163.! Gonzalez[Escobedo,!G.,!Marshall,!J.!M.!&!Gunn,!J.!S.!Chronic!and!acute!infection! of!the!gall!bladder!by!Salmonella!Typhi:!understanding!the!carrier!state.!Nat(Rev( Micro!9,!9–14!(2011).! 164.! Raivio,!T.!L.!Envelope!stress!responses!and!Gram[negative!bacterial! pathogenesis.!Mol.(Microbiol.!56,!1119–1128!(2005).! References! 128 ! !