Molecular Mechanisms Used by Salmonella to Evade the Immune System

Molecular Mechanisms Used by Salmonella to Evade the Immune System Joaquín Bernal-Bayard1* and Francisco Ramos-Morales2 1Département de Microbiologie, Institut Pasteur, Paris, France. 2Departamento de Genética, Universidad de Sevilla, Seville, Spain. *Correspondence: [email protected] https://doi.org/10.21775/cimb.025.133 Abstract Human and animal pathogens are able to circumvent, at least temporarily, the sophisticated immune defences of their hosts. Several serovars of the Gram-negative bacterium Salmonella enterica have been used as models for the study of pathogen–host interactions. In this review we discuss the strategies used by Salmonella to evade or manipulate three levels of host immune defences: physical barriers, innate immunity and adaptive immunity. During its passage through the digestive system, Salmonella has to face the acidic pH of the stomach, bile and antimicrobial peptides in the intestine, as well as the competition with resident microbiota. After host cell invasion, Salmonella manipulates inflammatory pathways and the autophagy process. Finally, Salmonella evades the adaptive immune system by interacting with dendritic cells, and T and B lymphocytes. Mechanisms allowing the establishment of persistent infections are also discussed. Introduction Salmonella spp. are pathogenic Gram-negative, rod-shaped, motile bacteria belonging to the family Enterobacteriaceae. The genus Salmonella includes the species S. bongori and S. enterica. S. enterica is further divided into several subspecies and more than 2500 serovars (Tindall et al., 2005). Salmonella can cause gastroenteritis, typhoid fever, abortion, and bacteraemia, depending on the serovar and the host. For instance, S. enterica subspecies enterica serovar Typhi (S. Typhi) is responsible for typhoid fever in humans, whereas S. enterica subspecies enterica serovar Typhimurium (S. Typhimurium) causes gastrointestinal inflammation in humans and a systemic typhoid-like disease in mice (Garai et al., 2012). Salmonella is a facultative intracellular pathogen that usually resides in a modified phagolysosome known as Salmonella-containing vacuole (SCV) during host infection. However, recent data indicate that this facultative intracellular pathogen has a distinct bimodal lifestyle in epithelial Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 134 | cells, where, there are subpopulations of vacuolar and cytosolic Salmonella (Knodler et al., 2010). Target cells include M cells, gut epithelial cells, dendritic cells (DCs), macrophages, monocytes, neutrophils, B cells, and T cells. During its passage through the host, Salmonella must resist or evade multiple levels of immune defence. Many virulence genes contribute to the survival of these bacteria inside the host. Some of them are clustered in horizontally acquired genomic regions known as Salmonella pathogenicity islands (SPIs) (Gerlach and Hensel, 2007). The largest and best known are SPI1, which is present in all the members of the genus, and SPI2, which is present in S. enterica but not in S. bongori. These SPIs encode two distinct type III secretion systems, type III secretion system 1 (T3SS1) and type III secretion system 2 (T3SS2), respectively (Galán and Curtiss, 1989; Ochman et al., 1996; Shea et al., 1996). These systems are able to inject directly into the eukaryotic host cells a number of bacterial proteins known as effectors (Ramos-Morales, 2012a). Collectively, these virulence factors contribute to invade host cells, interfere with host cellular functions, subvert immunity, establish an intracellular niche, and promote pathogen proliferation. In this review we summarize the mechanisms of immunity that pathogens in general have to face and the specific strategies that S. enterica uses to deal with the different levels of defence of the host. Overview of mechanisms of immune defence Humans, like any other animal are confronted every day to a myriad of microorganisms. However, these potentially infectious agents do not cause disease regularly due to a series of defence mechanisms that have been shaped over millions of years of evolution. Three levels of immune defence can be recognized (Delves et al., 2011; Kenneth, 2011): (i) physical barriers, (ii) the innate immune system and (iii) the adaptive immune system. Physical barriers The best way to avoid infections is by keeping potentially infectious agents out of the body. Physical barriers are very effective for this purpose. They are represented by the skin and other epithelia covering the gastrointestinal, respiratory and urogenital tracts. These surfaces provide, not only mechanical, but also chemical and microbiological barriers. The primary mechanical protection common to all epithelia consists of epithelial cells linked by tight junctions. The internal epithelia, or mucosal epithelia, are covered with mucous secretions. Mucus blocks the adherence of bacteria to epithelial cells. Microorganisms trapped within it can be removed by the movement of cilia, coughing or sneezing. In the intestinal mucosa, mucus is secreted by specialized goblet cells and creates a physical barrier against microbial pathogens (Liévin-Le Moal and Servin, 2006). Other general mechanical factors that contribute to this first level of defence are the flow of air or other fluids, like the tears in the eyes. In addition to these physical obstacles, a chemical barrier to infections is provided by substances that kill microbes or that inhibit their growth. These chemical substances include a group of antibacterial enzymes present in body fluids, like lactoperoxidase in milk, lysozyme and secretory phospholipase A2 in tears, nasal secretions and saliva. Lysozyme breaks the peptidoglycan (PG) in the bacterial cell wall of Gram-positive and Gram-negative bacteria. Phospholipase A2 kills bacteria by hydrolysing phospholipids in the cell membrane. Antimicrobial peptides, like defensins, cathelicidins and histatins, represent another important Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 135 group of antimicrobial agents associated to epithelia. All of them are amphipathic peptides activated by proteolysis. Defensins and cathelicidins disrupt membranes and are toxic to many microorganisms, whereas histatins act against pathogenic fungi. Additional chemical barriers that are found by potential food pathogens include the acidity of the stomach pH and the bile in the intestine. Microbiota is the microbial community composed of bacteria, protozoa, archaea, viruses and fungi that reside in different body niches. This microbiota competes with pathogenic microorganisms for nutrients and attachment sites. Therefore, the normal microbiota that is present in the intestine provides a microbiological physical defence against potential microbial pathogens that, like Salmonella, usually invade through the gut. In addition, some microbiota-produced metabolites affect the growth and virulence of pathogens. These metabolites include short-chain fatty acids, succinate, mucin O-glycans, molecular hydrogen, secondary bile acids, and the quorum sensing autoinducer AI-2 (Vogt et al., 2015). Innate immune defence The second level of defence is the innate immune response. This response occurs immediately after entry of a foreign agent into the body, but its effectiveness does not improve upon secondary encounter with the same agent. Microorganisms are recognized and killed by two main classes of phagocytic cells: polymorphonuclear neutrophils and macrophages. These cells carry pattern recognition receptors (PRRs) on their cytoplasmic membranes that adhere to pathogen-associated molecular patterns (PAMPs) on the microbe surface. PRRs include Toll-like receptors (TLRs), C-type lectin receptors (CTLRs), NOD-like receptors (NLRs), RIG-like receptors (RLRs) and scavenger receptors. PAMPs that activate TLRs include lipopolysaccharide (LPS), PG, lipoproteins, flagellin and other pathogen-derived ligands. Binding of a TLR to its ligand leads to activation of nuclear factor κB (NF-κB) and several members of the interferon-regulated factor (IRF) family of transcription factors. CTLRs are very diverse transmembrane proteins. RLRs are found in the cytoplasm and activate NF-κB and IRF3/4 to induce antiviral type I interferons in response to doublestranded RNA. Scavenger receptors recognize modified low-density proteins, LPS and other ligands, and some of them can cooperate with TLRs (Murshid et al., 2016). NLRs are soluble cytosolic proteins that usually are composed of three domains: an N-terminal domain that recruits proteases or kinases, a central oligomerization domain and C-terminal leucine-rich repeats (LRRs) that recognize PAMPs. NLRs exist in an autoinhibited conformation that is released upon binding to a PAMP, allowing oligomerization and recruitment of different host ligands depending on the particular NLR (Kim et al., 2016). The NLRs NOD1 and NOD2 induce autophagy to remove pathogens by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry. Autophagy is a conserved intracellular degradation pathway that involves the formation of double-membrane vesicles, known as autophagosomes, which deliver cytosolic components to the lysosome for degradation. Microbe-induced autophagy, or xenophagy, is important to restrict the growth of a number of intracellular pathogens, including S. Typhimurium (Gomes and Dikic, 2014). NOD1 and NOD2 activate NF-κB and mitogen-activated protein kinase (MAPK) signalling pathways, whereas NLRP2 and NLRP4 are negative regulators of the NF-κB pathway. Some NLRs, as well as absent in melanoma 2-like receptors (ALRs) and pyrin, act as inflammasome sensors (Sharma and Kanneganti, 2016). The inflammasome is mainly activated in myeloid cells as an essential component of innate immunity that promotes caspase-1-induced conversion of Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 136 | procytokines into active IL-1β and IL-18. It can result in an inflammatory form of cell death known as pyroptosis. Inflammasomes are cytosolic protein complexes that usually contain a specific sensor, an adaptor molecule (ASC) and pro-caspase-1. Canonical inflammasomes contain NLRP1, NLRP3, NLRC4, AIM2 or pyrin as sensors. NLRP3 is also involved in a non-canonical inflammasome that activates pro-caspase-11 in response to Gram-negative bacteria. This inflammasome is primed by recognition of LPS through a TLR and type I interferon signalling (Rathinam and Fitzgerald, 2016). The general consequences of binding to PRRs include the activation of macrophages and neutrophils to increase their phagocytic activity, and the release of cytokines and chemokines that amplify the immune response. Once inside the phagocytic cell, the microorganism can be killed by reactive oxygen or nitrogen intermediates, by preformed antimicrobials like the defensins mentioned above or a neutral protease. This takes place with the assistance of other factors like low pH, lysozyme and lactoferrin. Other important cells in the innate immune system are natural killer cells, which identify host cells expressing abnormal patterns of proteins and induce apoptosis of these cells. In addition to the cellular components of innate immunity, the complement system and other soluble circulating defensive proteins integrate the humoral innate immunity. The complement system is a multicomponent enzyme cascade that facilitates phagocytosis and lysis of microorganisms. This system can be activated by the lectin, classical or alternative pathways. All of the aforementioned generate a C3 convertase that leaves C3b bound to the microbial surface and releases C3a. C3b acts as an opsonin that increases the ability of phagocytes to ingest bacteria. C3a, together with C5a, recruit phagocytic cells to the site of infection and promote inflammation. Complement activation also leads to the formation of a membrane-attack complex that causes cell lysis. In addition, complement activation together with products of activated mast cells promotes inflammation, a response that leads to local swelling, redness, pain and temperature elevation. Adaptive immune response Unlike innate responses, acquired or adaptive immunity is able to recognize specific antigens and develop a response against these pathogens. In contrast to innate immunity, adaptive immunity is different in each individual of a species and responds to specific antigenic challenges, displaying then a flexible spectrum of action, as it is able to recognize millions of different antigenic molecules. The two major varieties of lymphocytes, B-lymphocytes and T-lymphocytes, are essential to generate humoral and cellular immunity, respectively. B-lymphocytes are able to make antibodies, specific antigen-recognition molecules, known as immunoglobulins, which deal with extracellular infections. T-lymphocytes are involved in the control of intracellular infections. Their receptors recognize processed antigens in association with molecules of the major histocompatibility complex (MHC). T cells are classified in two categories: CD4 (helpers) and CD8 (cytotoxic) T cells, which are activated upon recognition of an antigen presented by major histocompatibility complex class II (MHC-II) and major histocompatibility complex class I (MHC-I), respectively by the TCR. While MHC-I is present on most cell surfaces, MHC-II is expressed on antigen presenting cells (APC). A connection between the innate and adaptive immune systems at the level of cellular immunity is provided by DCs, which are relevant APCs. Stimulation of DCs by PAMPs triggers their maturation. Mature DCs present antigens to T cells via MHC molecules and Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 137 also provide co-stimulatory signals via B7 family ligands, promoting the activation of naive T cells. The interconnection between innate and adaptive immunity is also important for humoral immunity since the classical pathway to activate the complement requires immunoglobulin M (IgM) or immunoglobulin G (IgG). Dealing with physical barriers Acidic pH Bacteria belonging to the genus Salmonella usually invade their hosts through oral ingestion of contaminated food or water. Lysozyme found in saliva is not a significant barrier for these pathogens since, like many other Gram-negative bacteria, they are capable of resisting it thanks to their outer membrane shield around the PG layer (Van Kesteren et al., 1942; Masschalck and Michiels, 2003). Therefore, the first real challenge that these bacteria have to face upon ingestion is the acidic pH of the stomach of the host (Ramos-Morales, 2012b) due to gastric secretions and hydrochloric acid. The pH value of the stomach is variable: a pH of 1.3 was observed in healthy humans in the fasted state and of 4.9 after meal ingestion (Russell et al., 1993). Values of 3.9 and 4.0 were measured in rats and mice, respectively, in the fasted state (McConnell et al., 2008). This low gastric pH can quickly kill enteric bacteria, but different bacterial species have developed mechanisms to survive during passage through the stomach. When compared with E. coli, that resists extreme acid stress and can survive at pH 2 for hours, S. enterica is much less acid resistant (Koutsoumanis and Sofos, 2004; Lin et al., 1995). However, S. Typhimurium possesses at least three mechanisms of acid tolerance response (ATR) (Audia et al., 2001): (i) the exponential phase ATR is induced at pH 4.5–5.8 and allows subsequent survival at pH 3; (ii) the RpoS dependent stationary phase ATR is pH independent and is part of a general stress response; (iii) the RpoS independent stationary phase ATR is induced by exposure to pH 4.5 and provides longer tolerance to pH 3 than the RpoS dependent system. These tolerance systems require acid shock proteins whose synthesis is regulated by RpoS, the iron regulatory protein Fur or the two-component systems PhoQ/PhoP or EnvZ/OmpR (Audia et al., 2001; Foster, 1993). Small RNAs (sRNAs) have recently been identified as major regulators of stress response networks. For instance, the sRNA DsrA influences the ATR and virulence in S. Typhimurium (Ryan et al., 2016). Bacteria that are able to survive the passage through the stomach must face new challenges in the intestine before establishing an intracellular niche. This includes mucus, bile, antimicrobial peptides and the resident microbiota (Fig. 6.1). Mucus Epithelial cells in the human gut are covered with mucous secretions that protect the epithelium from the environment. The mucus barrier is composed of at least two glycan layers, which in turn incorporate multiple layers of glycoproteins (mucin) and complex oligosaccharides (glycocalyx) that protect cells from the local environment and infection. Microbes are able to use the host glycan as nutrients that regulate bacterial infection and virulence (Marcobal et al., 2013). Intestinal intracellular pathogens have to penetrate the mucin layers and subsequently gain access to the cell membrane. In order to achieve this, Salmonella uses a set of glycosyl hydrolases (GHs) that are able to degrade glycans. Recently, Arabyan et al. (2016) investigated the specific enzymes used by Salmonella to degrade the glycan. They Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 138 | identified two specific glycan-degrading enzymes in Salmonella, NanH and MalS, as new virulence factors. These enzymes are expressed during in vitro infection of human colonic epithelial cells (Caco-2) and degrade the glycocalyx layer produced by these cells. The authors also analysed the host response to glycan degradation and they observed induced expression of host genes that hydrolyse mannose, fucose, and N-acetylneuraminic acid (Neu5Ac) from the glycan; as well as sialyltransferases, suggesting host glycan remodelling during infection (Arabyan et al., 2016). Bile Bile is a secretory and excretory fluid produced in the liver whose composition varies depending on the individual’s diet (Coleman, 1987). Secretory functions include: delivery to the intestinal tract of bile salts to aid fat digestion and absorption/secretion of polymeric IgA to prevent infection in the biliary and upper intestinal tracts. In addition, bile is the excretory vehicle of liver-derived metabolites of potentially toxic materials. Bile contains inorganic ions, bile salts, lipids (phosphatidylcholine, cholesterol) and proteins (plasma proteins, liver-specific proteins, polymeric IgA). It is a bactericidal agent that disrupts Figure 6.1 Schematic representation of Salmonella strategies to circumvent host physical barriers. After ingestion of contaminated food or water, Salmonella crosses the stomach where it is able to resist to acidic pH activating an acid tolerance response. Then Salmonella reaches the intestine where it has to face different chemical challenges, like the bile produced in the liver or antimicrobial peptides secreted by Paneth cells in the gut. In addition to that, Salmonella has to compete with the resident intestinal microbiota for essential nutrients. Interestingly, Salmonella triggers an inflammation response in the gut that offers different nutrients or adhesion receptor sites that can be exploited only by pathogens. Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 139 bacterial cell membranes due to the detergent activity of bile acids (Begley et al., 2005), causes DNA damage (Prieto et al., 2004, 2006) and alters the conformation of proteins. Salmonella, however, resists bile through different mechanisms. First, the bacterial cell envelope acts as a barrier to membrane-active agents like bile salts. Two components of the outer membrane are important for bile resistance: the LPS (Crawford et al., 2012; Gunn, 2000) and the enterobacterial common antigen (ECA) (Ramos-Morales et al., 2003). Recently, it has been shown that exposure to the bile salt sodium deoxycholate is associated with changes in the structure of the second layer of the cell envelope, the PG, and that PG remodelling contributes to bile resistance (Hernández et al., 2015). Once bile enters the cell, additional mechanisms are activated. Efflux pumps mediate the export of bile salts from the bacterial cytoplasm to the outside medium and are responsible for resistance to many different toxic compounds (Nishino et al., 2006). AcrAB-TolC is the best characterized efflux system in enteric bacteria (Ma et al., 1993, 1995; Nikaido et al., 2008; Zgurskaya and Nikaido, 1999). AcrB is an inner-membrane protein, TolC is the outer membrane component and AcrA is located in the periplasm. The genes encoding these proteins are regulated by the transcriptional regulator RamA, which is synthesized in response to bile (Baucheron et al., 2014). In addition, bile-induced DNA damages can be repaired by Dam-directed mismatch repair and by base excision repair, and the impairment of DNA replication rescued by SOS-dependent translation DNA replication and RecBCD-dependent recombinational repair (Prieto et al., 2006). Finally, Salmonella is also able to adapt to grow in extremely high bile concentrations if previously exposed to sublethal doses. This is a transitory state that involves multiple changes in gene expression (Prouty et al., 2004). The RpoS-dependent general stress response plays a crucial role in the adaptation of Salmonella to bile (Hernández et al., 2012). Antimicrobial peptides Different families of antimicrobial peptides protect the intestine. Defensins and cathelicidins are of special interest in the context of gastrointestinal infections (Wehkamp et al., 2007). They are produced in the intestinal epithelium either constitutively or in an inducible manner. For instance, Paneth cells of the small intestine express the most abundant, constitutively expressed defensins. Human cathelicidin is expressed by epithelial cells in the stomach, small bowel and colon. In this context, butyrate, a short-chain fatty acid produced in the colon by bacterial fermentation of dietary fibres, has been shown to induce expression of the human cathelicidin gene. Salmonella is able to sense sublethal concentrations of antimicrobial peptides and induce several mechanisms of resistance that involve modification of the LPS or sequestering, efflux and proteolytic degradation of antimicrobial peptides (Matamouros and Miller, 2015). Regulatory proteins involved in this process include the alternative sigma factor RpoE and the two-component or phosphorelay systems PhoQ/ PhoP, PmrA/PmrB and Rcs. PhoQ is an inner-membrane histidine kinase that senses antimicrobial peptides (Bader et al., 2003, 2005) and phosphorylates the response regulator PhoP. Activation of PhoP prevents dephosphorylation of PmrA (Kato and Groisman, 2004). This protein directly controls expression of genes involved in LPS modifications necessary for resistance to antimicrobial peptides. The Rcs system is a phosphotransfer cascade that responds to antimicrobial peptides through RcsF (Farris et al., 2010). This system controls the synthesis of colanic acid capsule and the transcription of other genes necessary for resistance (Detweiler et al., 2003). Modifications of the bacterial surface that contribute to antimicrobial peptide resistance include: Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 140 | 1 Regulation of O-antigen length by the PmrA/PmrB system (Farizano et al., 2012; Pescaretti et al., 2011). 2 Modification of the anionic phosphate groups in the core and lipid A regions of the LPS by addition of aminoarabinose and phosphoethanolamine. PmrA is involved in the regulation of these modifications as well as in the inhibition of the activity of LpxT, which is responsible for increasing the negative charge of LPS by adding an additional phosphate. Together these changes lead to a reduction of the negative charge of the outer membrane and of the electrostatic interactions with antimicrobial peptides (Gunn et al., 1998; Herrera et al., 2010; Jones et al., 2008a; Lee et al., 2004; Zhou et al., 2001b). 3 Decrease in the fluidity of the outer membrane by the incorporation, catalysed by PagP, of an additional palmitate to lipid A and to phosphatidylglycerol (Bishop et al., 2000; Dalebroux et al., 2014; Guo et al., 1998). Additional resistance mechanisms involve binding, efflux or degradation of antimicrobial peptides. The two PhoP-regulated proteins Mig-14 and VirK are suggested to bind antimicrobial peptides to prevent their penetration into Salmonella (Brodsky et al., 2005). The sapABCDF operon encodes an ABC transporter system involved in resistance to antimicrobial peptides (Parra-Lopez et al., 1993). It has been proposed that the SapABCDF transporter system functions as a complex with SapG and SapJ to mediate both peptide and K+ transport (Parra-Lopez et al., 1994). Finally, PgtE is a post-transcriptionally regulated component of the PhoQ/PhoP regulon with protease activity against alpha-helical cationic antimicrobial peptides (Guina et al., 2000). Intestinal microbiota Before invading appropriate host cells and becoming an intracellular pathogen, Salmonella has to compete with the resident intestinal microbiota. The lumen of the human gastrointestinal tract is rich in nutrients and provides an appropriate niche for a diverse community of bacteria (Bäckhed et al., 2005). They increase in number from the stomach to the colon, where they reach concentrations around 1012 per gram of luminal content. Most colonic bacteria are members of the Firmicutes and Bacteroidetes phyla, whereas Proteobacteria, Actinobacteria, Fusobacteria and Verrucomicrobia are less abundant (Eckburg et al., 2005). This microbiota helps to prevent infection by providing a physical barrier for the attachment of bacterial pathogens to the mucosal surfaces and competing with pathogens for essential nutrients (van der Waaij et al., 1971). Salmonella, however, can exploit the host innate immune response and its own metabolic capacities to overcome, at least temporarily, this barrier (Khan, 2014). First, the initial growth of Salmonella in the gut is powered by Hyb hydrogenases that enable the bacteria to use hydrogen produced by the resident microbiota as a central intermediate of metabolism (Maier et al., 2013). Using a mouse colitis model, it has been shown that inflammatory host responses triggered by S. Typhimurium virulence factors (T3SS effectors) shift the competition in favour of the pathogen (Stecher et al., 2007). The mechanisms leading to this result are not completely understood and different explanations have been proposed: (i) inflammation involves release of antibacterial factors that may kill or delay the growth of specific members of the microbiota that would normally inhibit Salmonella growth; and (ii) the inflamed intestine could offer different Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 141 nutrients or adhesion receptor sites that can be exploited only by pathogens. In particular, reactive oxygen species generated by neutrophils during inflammation can react with endogenous thiosulfate to form tetrathionate. The ttrRSBCA locus, located in SPI2, confers to S. Typhimurium the ability to use tetrathionate as a terminal electron acceptor in anaerobic respiration (Winter et al., 2010a). This confers a growth advantage because, unlike most bacteria in the gut, S. Typhimurium can use ethanolamine as a carbon source in the presence of tetrathionate as a respiratory electron acceptor (Thiennimitr et al., 2011). It has also been suggested that the ability of S. Typhimurium to degrade fucose and anaerobically degrade 1,2-propanediol, probably using tetrathionate as a terminal electron acceptor, provides an advantage to this pathogen during competition with the microbiota (Staib and Fuchs, 2015). In addition, some S. Typhimurium isolates carry the gene sopE in a prophage. The product of this gene increases the severity of intestinal inflammation and drives the host to generate an additional terminal electron acceptor, nitrate, that suppresses genes responsible for utilization of energetically inferior electron acceptors such as tetrathionate (Lopez et al., 2012). Stecher et al. (2012) described in an interesting study the discovery of a mechanism driving efficient conjugation between Enterobacteriaceae (S. Typhimurium and E. coli) in the host’s intestine. Using a mouse colitis model, the authors observed parallel blooms of pathogenic S. Typhimurium and resident commensal E. coli. This phenomenon, probably due to dysbiosis of the anaerobic microbiota caused by Salmonella infection, led E. coli to account for > 80% of the total intestinal bacteria. In this scenario, the authors observed conjugative horizontal gene transfer efficiencies of 100%. This suggests that bacterial blooms occur after Salmonella infection fuels the reassortment of genetic material between different Enterobacteriaceae. This further implies that infected patients might enhance the spread of plasmid-encoded fitness, virulence- and antibiotic resistance determinants (Stecher et al., 2012). Evasion of the innate immune system Salmonellae that are able to deal with the aforementioned physical barriers can invade the gut epithelium, preferentially in the distal ileum, using several routes. They usually invade microfold cells (M cells) that overlie Peyer’s patches, but they can also induce their own uptake by epithelial cells or be engulfed by CD18+ phagocytes, probably monocytes or dendritic cells (Watson and Holden, 2010). In a systemic infection, Salmonella can disseminate to internal organs [mesenteric lymph nodes (MLNs), liver and spleen]. Access to other cell types, like macrophages, could be a consequence of cell death induction (Fink and Cookson, 2007) and subsequent phagocytosis. Both intracellularly and during the transient extracellular passage from one host cell to another, Salmonella has to face and resist components of the innate immune system. Salmonella finds cationic antimicrobial peptides not only in the intestinal lumen but also inside macrophages. Resistance mechanisms against this innate defence were described in the previous section. Interestingly, the PhoQ/PhoP two component system, that sense these antimicrobial peptides, are also additively activated by intracellular signals (acidic pH and divalent cation limitation). However the presence of antimicrobial peptides could be the most relevant signal for systemic virulence (Hicks et al., 2015). Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 148 | The activity of this enzyme causes depletion of exogenous l-asparagine, leading to downregulation of TCR-β expression, suppression of T-cell blastogenesis, blockade of cytokine production, and, ultimately, inhibition of T-cell proliferation (Kullas et al., 2012). In agreement with this, the cytolytic capacities of CD8+ T cells have been shown to be initiated by TCR interactions (Lewinsohn et al., 2011). The phagosomal lifestyle of S. Typhimurium seems to be responsible for delayed MHC-I dependent antigen presentation and delayed expansion and contraction of the CD8+ T-cell response. Interestingly, even memory CD8+ T cells failed to undergo rapid expansion in response to infection with S. Typhimurium expressing the model antigen OVA (Luu et al., 2006). Additionally, Salmonella is able to induce apoptosis of Ag-specific CD4 T cells (Srinivasan et al., 2009). This is driven by SPI-2 virulence genes. In vivo studies by Ertelt et al. suggested that Salmonella undergoes, in a SPI-2 dependent manner, a selective culling of activated CD4+ T-cell subsets, which re-shapes the repertoire of antigen-specific T cells that persist later after infection (Ertelt et al., 2011). Non-cognate activation of T cells In addition to conventional activation by cognate peptide–MHC complexes presented by DCs, T cells can be indirectly stimulated (bystander stimulation) by Salmonella. This alternative pathway involves the well-characterized TLRs and NLRs, as well as other less known PRRs. It results in production of inflammatory cytokines that prime T cells at the site of infection (Broz and Monack, 2013b). Indeed, Salmonella has been proposed as a particularly appropriate model pathogen for study of non-cognate CD4 T-cell responses based on (i) strength of the Th1 response during infection, (ii) the requirement for CD4 T cells in bacterial clearance and (iii) the well-characterized inflammatory response to conserved molecular patterns induced by Salmonella infection (O’Donnell and McSorley, 2014). B lymphocytes’ role B lymphocytes have critical roles as positive and negative regulators of immunity. For instance, B cells have important contributions in acquired immunity against Salmonella infections, being essential for immunity during secondary challenge (McSorley and Jenkins, 2000). Several studies describe antibody dependent (Mastroeni et al., 1993; McSorley and Jenkins, 2000; Mittrücker et al., 2000) and antibody independent Salmonella specific B cells responses (Barr et al., 2010; Mastroeni et al., 2000; Ugrinovic et al., 2003). These studies suggest that B cells can work as APCs and have a key role in the production of inflammatory cytokines during Salmonella infection. The immunosuppressive role of B lymphocytes, classically associated with B cell-derived IL-10, is related to the regulation of autoimmune disease. This IL-10 also increase susceptibility to pathogens (Fillatreau, 2011; Fillatreau et al., 2008). In a recent study, Shen et al. described interleukin 35 (IL-35)-producing B cells as novel key players in the negative regulation of immunity (Shen et al., 2014). The authors showed that mice whose B cells did not express IL-35, displayed improved resistance to S. Typhimurium infection. The increased immunity found in these mice was associated to higher activation of macrophages and T cells, as well as an increased stimulatory function of B cells as APCs. The data presented in this work demonstrate that B cells can inhibit antimicrobial immunity through production of IL-35 (Shen et al., 2014). Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 149 One of the strategies developed by Salmonella to evade the action of B cells is to delay the formation of the germinal centre, which is necessary to produce high-affinity antibodies against a specific pathogen (Cunningham et al., 2007). It has been shown that during Salmonella infection germinal centre formation and affinity maturation are delayed until the second month of infection. This differs greatly with conventional 7 day functional germinal centre formation. Furthermore, high-affinity antibodies start to be produced during the second week after immunization. Persistence One important aspect of typhoid Salmonella is its capacity to establish chronic infections. After adequate treatment the majority of patients who present acute typhoid fever recover; however, a significant percentage of patients (1–6%) develop a chronic infection (Levine et al., 1982; Monack et al., 2004a). These patients become asymptomatic chronic carriers and sporadically shed bacteria in their stool, allowing the pathogen to close its life cycle and to be transmitted to new hosts (Monack et al., 2004b). Despite the importance of Salmonella chronic infections, relatively little is known about the host immune response or virulence mechanisms that characterize long-term systemic infections. Since the majority of chronically infected patients are asymptomatic, the identification of potential targets is challenging (Shpargel et al., 1985; Sinnott and Teall, 1987). However, it is interesting to note that gut persistent infections are Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2) dependent (Lam et al., 2014). One study suggested that the human carrier state may be associated to an ineffective immune response (Thompson et al., 2009). In this section we describe some recent advances with respect to S. Typhi and S. Typhimurium persistent host colonization. Biofilm formation Biofilm development is an important component of bacterial survival and a common source of persistent infections (Costerton et al., 1999). Salmonella are able to form biofilm in both biotic and abiotic surfaces. Moreover, there is a clear correlation between chronic infection and bacterial biofilm formation on the surface of gallstones (Gonzalez-Escobedo et al., 2011; Prouty et al., 2002). Epidemiological studies have revealed a strong association between chronic carriers of S. Typhi and gallstones (Schiøler et al., 1983). Although, 90% of chronically affected individuals have gallstones (Karaki and Matsubara, 1984) or other biliary track pathologies, this is not required to develop a carrier state (Monack et al., 2004b). Salmonella biofilms have been found on gallstones in both S. Typhi patients and a mouse model of chronic S. Typhimurium infection (Crawford et al., 2010), suggesting both serovars share this biofilm strategy to survive in their host. Interestingly, bile has been shown to enhance S. Typhi biofilm formation (Gonzalez-Escobedo et al., 2011). Consistently, S. Typhi exhibits specific binding to cholesterol-coated surfaces, like gallstones (Crawford et al., 2008). FliC and OmpC are two proteins that were described to have a key role on initial adhesion to cholesterol-coated surfaces (Crawford et al., 2010). A hallmark of biofilm formation is the self-production of an extracellular matrix that ensures the integrity of the biofilm. The biofilm matrix is composed of EPS (extracellular polymeric substances) and water. Some components of biofilm EPS like cellulose, colonic acid and O antigen capsule are crucial for S. Typhi persistence and biofilm development (Crawford et al., 2008; Prouty Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 150 | and Gunn, 2003; Prouty et al., 2002). Some studies show that bile present in the gallbladder induces the production of O-antigen, which facilitates S. Typhi biofilm formation on human gallstones (Crawford et al., 2008; Hall-Stoodley and Stoodley, 2009). Persistence in immune cells The study of the molecular mechanisms underlying persistent infections is limited by the lack of appropriate animal models that resist challenges using virulent strains of Salmonella. A suitable mice model was established in 2004 to study S. Typhimurium persistent infections (Monack et al., 2004b). In this model virulent S. Typhimurium strains cause long-term chronic infections. The persistent bacteria can then be monitored within macrophages in the MSNs up to 1 year post inoculation (Monack et al., 2004b). In addition to that, in vitro assays showed that hemophagocytic macrophages might represent a survival niche for Salmonella (Nix et al., 2007). Another study showed that persistent bacteria are sequestered within macrophages in systemic tissues, where they do not replicate and seem to be in a dormant-like state. This supports the idea of macrophages as a reservoir for persistent bacteria (Helaine et al., 2010). Another study published recently, showed that S. Typhimurium preferentially associates with anti-inflammatory/M2 macrophages at later stages of infection in mice (Eisele et al., 2013). This subset of macrophages does not express many of the defence mechanisms needed to eliminate invading microbes. Moreover, there are pathogens that have developed strategies to induce polarization of cells towards the M2 phenotype as a virulence mechanism (Jensen et al., 2011). In addition to macrophages, it has been shown that Salmonella remains for a long period of time within plasma cells, bone marrow B cell precursors, and all B cell subsets from the spleen (Castro-Eguiluz et al., 2009; López-Medina et al., 2015a). The ability of Salmonella to persist within B cells suggests that it developed a CD8+ T-cell response evasion mechanism. In fact, Salmonella infection results in the enhanced expression of PD-L1 in B cells (López-Medina et al., 2015a). PD-L1 is a member of the programmed death-1 (PD-1)/ programmed death-ligands (PD-Ls) pathway that can turn off or reduce TCR signalling by recruiting phosphatases like SHP-1 and SHP-2 (Chemnitz et al., 2004; Freeman et al., 2000; Sheppard et al., 2004). The activation of the PD-1 : PD-Ls axis leads to impairment of CD8 T cells during Salmonella infection (López-Medina et al., 2015b). T3SS effectors and persistence In order to shed light on the molecular mechanisms involved in persistence, Monack’s group performed a genome-wide screen to identify Salmonella genes required for persistent infection of the mice (Lawley et al., 2006). The screen revealed that SPI1 was necessary to maintain a persistent infection for at least 1 month post challenge. The authors confirmed that SPI1 effectors SipB, SipC, and SipD, which have a role on invasion or translocation of other effectors, contribute to establish a persistent infection. Similarly, another study showed that SPI1 is required for persistently infected mice to transmit S. Typhimurium to naive cage-mates (Lawley et al., 2008). Data from the genome screen mentioned above also showed that the SPI2 effector SseI was required for maintaining a long-term systemic infection. A follow-up study showed that SseI modulates macrophages and DCs by directly binding to IQGAP1, an important regulator of host cell migration (McLaughlin et al., 2009). So, SseI blocks somehow the migration of macrophages and DCs interfering with the host immune system to clear systemic bacteria. Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 151 Other elements that have been shown to contribute to persistent Salmonella infection are those that protect against host-derived antimicrobial peptides. Examples of these factors are PgtE, a modifier of the bacterial outer surface membrane (Lawley et al., 2006), and Mig-14, which provides resistance against anti-microbial peptides like VirK, RcsC, and YdeI (Erickson and Detweiler, 2006), both of them regulated by the PhoP/PhoQ system (Bader et al., 2005). Persistence and metabolism Recently, a pioneering study described how bacteria modulate the host cell metabolism to create an appropriate environment for long-term infection (Eisele et al., 2013). Firstly, the authors observed that PPARδ, which is a transcriptional factor involved in fatty acid metabolism, is up-regulated in Salmonella infected M2 macrophages. This situation produces a shift in the metabolic state of the cell that leads to an increase in the level of glucose available to bacteria. Moreover, the authors show that pharmacological activation of PPARδ increases glucose availability and enhances bacterial replication in macrophages, while Salmonella fails to persist in macrophage lines lacking PPARδ. All together, these data, support the idea that Salmonella has evolved to survive long periods of time in M2 macrophages by harnessing the unique metabolism of these cells (Eisele et al., 2013). Another relevant topic concerns genetic adaptation of bacteria during a persistent infection. A recent study describes the acquisition of adaptive mutations that facilitate persistence and survival in the host over the course of an infection (Søndberg and Jelsbak, 2016). In this work, the authors used a model of mice infected chronically with S. Typhimurium. They observed that bacteria acquire distinct single nucleotide polymorphisms (SNPs) in known regulators of metabolic and virulence genes. One such difference, the kdgR-SNP was confirmed to confer selective advantage during chronic infections and constitutes a true patho-adaptive mutation. Thus, the results provide evidence for rapid genetic adaptation to the host of S. Typhimurium during persistent infection (Søndberg and Jelsbak, 2016). A non-mutational way for Salmonella to cause persistent infections even after antibiotic exposure is through the production of persister cells. These are rare cells, produced by all bacterial populations, that transiently become multidrug tolerant. It is thought this could be due to a state of low metabolic activity that leads to dormant or slow-growing cells (Bigger, 1944; Helaine and Kugelberg, 2014; Maisonneuve and Gerdes, 2014). Salmonella persisters are part of a non-replicating population formed immediately after uptake by macrophages and are induced by vacuolar acidification and nutritional deprivation (Helaine et al., 2014). Furthermore, the majority of the 14 toxin–antitoxin (TA) modules contributed to intracellular persister formation. A more comprehensive analysis of S. Typhimurium TA modules identified multiple toxins with anti-proliferative activity A selected group of the corresponding TA modules were important for survival of intracellular bacteria inside fibroblasts (Lobato-Márquez et al., 2015). One of these toxins, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation (Cheverton et al., 2016). Transmission During systemic salmonellosis, the life cycle completed when the pathogenic bacteria leave the former host to infect a naive one. This highlights the main issue with persistent infections: the constant shedding of virulent bacteria on faeces allows for propagation and maintenance Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 152 | within a population. In a chronic infection scenario, Salmonella can persist in MLNs, bone marrow, and gallbladder, and periodically discharge Salmonella from the gallbladder to the small intestine in bile. The molecular mechanisms that control transmission of bacteria from host reservoirs are poorly understood. However, an interesting study proposed that host transmission of S. Typhimurium is controlled by virulence factors and indigenous intestinal microbiota. The authors described a model in which persistently infected mice provide a natural mode of S. Typhimurium transmission. In this context, a subgroup of mice (30%), called ‘supershedders’ was found to shed high levels of S. Typhimurium in their stools, leading to rapid transmission of the infection to naive hosts (Lawley et al., 2008). The authors showed that the development of the supershedder phenotype depends on SPI1 and SPI2. Moreover, treatment with antibiotics induced the supershedder state in mice, suggesting that the indigenous intestinal microbiota has a role in controlling this phenotype. Concluding remarks We have described recent advances concerning the strategies used by Salmonella to evade both innate and adaptive immune responses. Most of these evasion mechanisms are directly related to T3SS1 and 2 and to the different effectors injected by them (Table 6.1). In the last two decades more than 40 effectors have been identified, and the biochemical activity and host targets for some of them have been successfully characterized. However, the precise role of many of them and their contribution to the relationship between Salmonella and its hosts at the cellular and systemic level remain unknown. A better understanding of the specific functions of these effector proteins will allow us to unravel the complex network of action of these molecules and to decipher the underlying mechanisms that enable Salmonella to evade the host immune system. For instance, we have to define the role of T3SS effectors in establishing different intracellular lifestyles and decipher which signals enable Salmonella to survive intracellularly versus proliferate and escape, depending on the cellular type. Another challenge is to improve our understanding of Salmonella control of the fusion of SCV to the lysosome. The identification of new and appropriate SCV markers and the development of new imaging technologies will improve the detection of intracellular Salmonella and ultimately help us to answer these questions. One of the most significant challenges concerns host specificity achieved by different Salmonella serovars. There is an obvious shortage of appropriate models. For example, there is no suitable animal model for the typhoid human pathogen S. Typhi. Therefore, in vivo studies for this serovar are deficient. Researchers tend to use the S. Typhimurium-infected mouse model to study systemic typhoid-like disease. However, extrapolation of results obtained in S. Typhimurium to S. Typhi is not always possible. The development of new animal and cellular models are slowly helping us surmount this lack of appropriate tools. One example is the recent work of Fresnay et al., who established a controlled human infection model with wt S. Typhi using cutting-edge multichromatic flow cytometry to analyse the pre-challenge immunological status and its correlation with the subsequent clinical outcome (Fresnay et al., 2016). Specifically, the authors investigated the relationship between S. Typhi-specific CD8+ T-cell responses before exposure to wt S. Typhi and clinical outcome, i.e. whether the participants who were challenged developed disease or not. They observed higher baseline levels of multi functional S. Typhi-specific CD8+ T in patients who did not Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 153 Table 6.1 Role of T3SS effectors in Salmonella evasion of the immune system Effector T3SS Interaction with the immune system References AvrA 1Inhibits NF-κB and JNK pro-inflammatory pathways Collier-Hyams et al. (2002), Jones et al. (2008b) CigR 2 Involved in biofilm formation Yin et al. (2016) GogA Inhibits inflammation by targeting NF-κB signalling Sun et al. (2016) GogB 2Limits inflammation preventing NF-κBPilar et al. (2012) GtgA 2Inhibits inflammation by targeting NF-κB signalling Sun et al. (2016) GtgE 1/2 Counters a Rab32-dependent host defence pathway Spanò et al. (2016) PipA Inhibits inflammation by targeting NF-κB signalling Sun et al. (2016) PipB 2Role in iNOS production in chicken oviduct epithelial cells Li et al. (2009) PrgJ ? Activates caspase-1 through NLRC4 Miao et al. (2010) PipB2 1/2 Inhibits dendritic cell migration McLaughlin et al. (2014) SifA 2Inhibits dendritic cell migration McLaughlin et al. (2014) SipA/ SspA 1Elicits accumulation of PERP to the apical surface of colonic epithelial cells that leads to inflammatory responses Hallstrom et al. (2015) SipB 1 Contributes to activation and release of IL-18 Dreher et al. (2002) SipC/ SspC 1Elicits accumulation of PERP to the apical surface of colonic epithelial cells that leads to inflammatory responses Hallstrom and McCormick (2016) SipD 1Triggers cell death in macrophages Arizmendi et al. (2016) SlrP 1/2 Inhibits dendritic cell migration McLaughlin et al. (2014) SopA 1Stimulates inflammation targeting TRIM56 and TRIM65 Kamanova et al. (2016) SopB 1Inhibits production of mitochondrial superoxide ROS Activates MAPK and NF-κB signalling through stimulation of Rho-family GTPases Bruno et al. (2009), Ruan et al. (2016) SopD 1/2? Contributes to PMN migration and fluid secretion in bovine intestine Zhang et al. (2002) SopD2 2 Counters a Rab32-dependent host defence pathway Spanò et al. (2016) SopE 1Induces caspase-1 dependent pro-inflammatory responses. Activates MAPK and NF-κB signalling through stimulation of Rho-family GTPases Bruno et al. (2009), Hoffmann et al. (2010), Müller et al. (2009) SopE2 1 Activates MAPK and NF-κB signalling through stimulation of Rho-family GTPases Bruno et al. (2009) SpiC/ SsaB 2Inhibits dendritic cell migration McLaughlin et al. (2014) SptP 1Inhibits p38 MAPK signalling. Suppresses degranulation of local mast cells Choi et al. (2013), Tenor et al. (2004) Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 154 | develop the disease. Their observations also indicate that S. Typhi-specific CD8+ T migrated not only to mucosal sites, but also to secondary lymphoid tissues. These localized events, delay disease onset. They also suggested that co-production of MIP-1β with other cytokines is a key component in protection against S. Typhi (Fresnay et al., 2016). This type of studies is key to identify an early selection of novel vaccine candidates for further evaluation in clinical trials. These models are also necessary to understand the role of certain T3SS effectors that may only be relevant in certain cell types or animal species. Advances in techniques for single cell analysis allow us to study bacterial subpopulations that have different expression profiles of effectors and other virulence factors. Finally, a more precise understanding of the interactions between Salmonella and the host immune system could help us design new approaches to modify the progression of Salmonella infections, strengthening key components of the immune response. Acknowledgements We thank Sebastián Aguilar for useful discussions and critical reading of the manuscript. Work in the laboratory of FR-M is supported by Ministerio de Economía y Competitividad of Spain and the European Regional Development Fund (grants SAF2013-46229-R and SAF2016-75365-R). Effector T3SS Interaction with the immune system References SpvB 2 Contributes to gut inflammation through a T3SS2-dependent pathway Käppeli et al. (2011) SpvC 1/2 Anti-inflammatory in mice. Interferes with immunity in plants Haneda et al. (2012); Neumann et al. (2014) SpvD 1/2 Inhibits nuclear transport of NF-κB p65 Rolhion et al. (2016) SseF 2 Inhibits dendritic cell migration McLaughlin et al. (2014) SseI/ SrfH 2Modulates cell migration of macrophages and dendritic cells McLaughlin et al. (2009), Worley et al. (2006) SseK1 1/2 Interferes with NF-κB activation Li et al. (2013) SseK3 2 Interferes with NF-κB activation Yang et al. (2015) SseL 2Inhibits autophagy Mesquita et al. (2012) SspH1 1/2 Inhibits NF-κB-dependent gene expression Haraga and Miller (2003), Haraga et al. (2006) SspH2 2 Enhance SG1-dependent NLR-mediated immunity Bhavsar et al. (2013), McLaughlin et al. (2014) Inhibits dendritic cell migration SteA 1/2 Represses genes involved in immune responses Cardenal-Muñoz et al. (2014) SteB Biofilm formation Dong et al. (2008), McGhie et al. (2009) Table 6.1 Continued Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 155 References Aachoui, Y., Leaf, I.A., Hagar, J.A., Fontana, M.F., Campos, C.G., Zak, D.E., Tan, M.H., Cotter, P.A., Vance, R.E., Aderem, A., et al. (2013). Caspase-11 protects against bacteria that escape the vacuole. Science 339, 975–978. https://doi.org/10.1126/science.1230751. Alaniz, R.C., Cummings, L.A., Bergman, M.A., Rassoulian-Barrett, S.L., and Cookson, B.T. (2006). Salmonella Typhimurium coordinately regulates FliC location and reduces dendritic cell activation and antigen presentation to CD4+ T cells. J. Immunol. 177, 3983–3993. Albaghdadi, H., Robinson, N., Finlay, B., Krishnan, L., and Sad, S. (2009). Selectively reduced intracellular proliferation of Salmonella enterica serovar Typhimurium within APCs limits antigen presentation and development of a rapid CD8 T cell response. J. Immunol. 183, 3778–3787. Arabyan, N., Park, D., Foutouhi, S., Weis, A.M., Huang, B.C., Williams, C.C., Desai, P., Shah, J., Jeannotte, R., Kong, N., et al. (2016). Salmonella degrades the host glycocalyx leading to altered infection and glycan remodeling. Sci. Rep. 6, 29525. https://doi.org/10.1038/srep29525. Arizmendi, O., Picking, W.D., and Picking, W.L. (2016). Macrophage apoptosis triggered by IpaD from Shigella flexneri. Infect. Immun. 84, 1857–1865. https://doi.org/10.1128/IAI.01483-15. Audia, J.P., Webb, C.C., and Foster, J.W. (2001). Breaking through the acid barrier: an orchestrated response to proton stress by enteric bacteria. Int. J. Med. Microbiol. 291, 97–106. Bäckhed, F., Ley, R.E., Sonnenburg, J.L., Peterson, D.A., and Gordon, J.I. (2005). Host-bacterial mutualism in the human intestine. Science 307, 1915–1920. Bader, M.W., Navarre, W.W., Shiau, W., Nikaido, H., Frye, J.G., McClelland, M., Fang, F.C., and Miller, S.I. (2003). Regulation of Salmonella Typhimurium virulence gene expression by cationic antimicrobial peptides. Mol. Microbiol. 50, 219–230. Bader, M.W., Sanowar, S., Daley, M.E., Schneider, A.R., Cho, U., Xu, W., Klevit, R.E., Le Moual, H., and Miller, S.I. (2005). Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122, 461–472. Baisón-Olmo, F., Galindo-Moreno, M., and Ramos-Morales, F. (2015). Host cell type-dependent translocation and PhoP-mediated positive regulation of the effector SseK1 of Salmonella enterica. Front. Microbiol. 6, 396. Barr, T.A., Brown, S., Mastroeni, P., and Gray, D. (2010). TLR and B cell receptor signals to B cells differentially program primary and memory Th1 responses to Salmonella enterica. J. Immunol. 185, 2783–2789. https://doi.org/10.4049/jimmunol.1001431. Baucheron, S., Nishino, K., Monchaux, I., Canepa, S., Maurel, M.-C., Coste, F., Roussel, A., Cloeckaert, A., and Giraud, E. (2014). Bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through transcriptional derepression of ramA in Salmonella enterica serovar Typhimurium. J. Antimicrob. Chemother. 69, 2400–2406. Begley, M., Gahan, C.G., and Hill, C. (2005). The interaction between bacteria and bile. FEMS Microbiol. Rev. 29, 625–651. Beuzón, C.R., Méresse, S., Unsworth, K.E., Ruíz-Albert, J., Garvis, S., Waterman, S.R., Ryder, T.A., Boucrot, E., and Holden, D.W. (2000). Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 19, 3235–3249. https://doi.org/10.1093/emboj/19.13.3235. Bhavsar, A.P., Brown, N.F., Stoepel, J., Wiermer, M., Martin, D.D., Hsu, K.J., Imami, K., Ross, C.J., Hayden, M.R., Foster, L.J., et al. (2013). The Salmonella type III effector SspH2 specifically exploits the NLR co-chaperone activity of SGT1 to subvert immunity. PLOS Pathog. 9, e1003518. https://doi. org/10.1371/journal.ppat.1003518. Bigger, J. (1944). Treatment of staphylococcal infections with penicillin by intermittent sterilisation. Lancet 244, 497–500. Birmingham, C.L., and Brumell, J.H. (2006). Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles. Autophagy 2, 156–158. Birmingham, C.L., Smith, A.C., Bakowski, M.A., Yoshimori, T., and Brumell, J.H. (2006). Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole. J. Biol. Chem. 281, 11374–11383. Bishop, R.E., Gibbons, H.S., Guina, T., Trent, M.S., Miller, S.I., and Raetz, C.R. (2000). Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria. EMBO J. 19, 5071–5080. https://doi.org/10.1093/emboj/19.19.5071. Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 156 | Blancou, P., Tardif, V., Simon, T., Rémy, S., Carreño, L., Kalergis, A., and Anegon, I. (2011). Immunoregulatory properties of heme oxygenase-1. Methods Mol. Biol. 677, 247–268. https://doi. org/10.1007/978-1-60761-869-0_18. Boucrot, E., Beuzón, C.R., Holden, D.W., Gorvel, J.P., and Méresse, S. (2003). Salmonella Typhimurium SifA effector protein requires its membrane-anchoring C-terminal hexapeptide for its biological function. J. Biol. Chem. 278, 14196–14202. Brodsky, I.E., Ghori, N., Falkow, S., and Monack, D. (2005). Mig-14 is an inner membrane-associated protein that promotes Salmonella Typhimurium resistance to CRAMP, survival within activated macrophages and persistent infection. Mol. Microbiol. 55, 954–972. Broz, P., and Monack, D.M. (2013a). Noncanonical inflammasomes: caspase-11 activation and effector mechanisms. PLOS Pathog. 9, e1003144. https://doi.org/10.1371/journal.ppat.1003144. Broz, P., and Monack, D.M. (2013b). Newly described pattern recognition receptors team up against intracellular pathogens. Nat. Rev. Immunol. 13, 551–565. https://doi.org/10.1038/nri3479. Broz, P., Newton, K., Lamkanfi, M., Mariathasan, S., Dixit, V.M., and Monack, D.M. (2010). Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella. J. Exp. Med. 207, 1745–1755. https://doi.org/10.1084/jem.20100257. Broz, P., Ruby, T., Belhocine, K., Bouley, D.M., Kayagaki, N., Dixit, V.M., and Monack, D.M. (2012). Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1. Nature 490, 288–291. https://doi.org/10.1038/nature11419. Bruno, V.M., Hannemann, S., Lara-Tejero, M., Flavell, R.A., Kleinstein, S.H., Galán, J.E., H., S., Bruno, V.M., and Hannemann, S. (2009). Salmonella Typhimurium type III secretion effectors stimulate innate immune responses in cultured epithelial cells. PLOS Pathog. 5, e1000538. Bueno, S.M., González, P.A., Carreño, L.J., Tobar, J.A., Mora, G.C., Pereda, C.J., Salazar-Onfray, F., and Kalergis, A.M. (2008). The capacity of Salmonella to survive inside dendritic cells and prevent antigen presentation to T cells is host specific. Immunology 124, 522–533. Bueno, S.M., Wozniak, A., Leiva, E.D., Riquelme, S.A., Carreño, L.J., Hardt, W.-D., Riedel, C.A., and Kalergis, A.M. (2010). Salmonella pathogenicity island 1 differentially modulates bacterial entry to dendritic and non-phagocytic cells. Immunology 130, 273–287. Cardenal-Muñoz, E., Gutiérrez, G., and Ramos-Morales, F. (2014). Global impact of Salmonella type III secretion effector SteA on host cells. Biochem. Biophys. Res. Commun. 449, 419–424. https://doi. org/10.1016/j.bbrc.2014.05.056. Casson, C.N., Yu, J., Reyes, V.M., Taschuk, F.O., Yadav, A., Copenhaver, A.M., Nguyen, H.T., Collman, R.G., and Shin, S. (2015). Human caspase-4 mediates noncanonical inflammasome activation against gram-negative bacterial pathogens. Proc. Natl. Acad. Sci. U. S. A. 112, 6688–6693. Castro-Eguiluz, D., Pelayo, R., Rosales-Garcia, V., Rosales-Reyes, R., Alpuche-Aranda, C., and Ortiz-Navarrete, V. (2009). B cell precursors are targets for Salmonella infection. Microb. Pathog. 47, 52–56. https://doi.org/10.1016/j.micpath.2009.04.005. Chemnitz, J.M., Parry, R.V., Nichols, K.E., June, C.H., and Riley, J.L. (2004). SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J. Immunol. 173, 945–954. Cheverton, A.M., Gollan, B., Przydacz, M., Wong, C.T., Mylona, A., Hare, S.A., and Helaine, S. (2016). A Salmonella toxin promotes persister formation through acetylation of tRNA. Mol. Cell 63, 86–96. https://doi.org/10.1016/j.molcel.2016.05.002. Choi, H.W., Brooking-Dixon, R., Neupane, S., Lee, C.-J., Miao, E.A., Staats, H.F., and Abraham, S.N. (2013). Salmonella Typhimurium impedes innate immunity with a mast-cell-suppressing protein tyrosine phosphatase, SptP. Immunity 39, 1108–1120. Chu, Y., Gao, S., Wang, T., Yan, J., Xu, G., Li, Y., Niu, H., Huang, R., and Wu, S. (2016). A novel contribution of spvB to pathogenesis of Salmonella Typhimurium by inhibiting autophagy in host cells. Oncotarget 7, 8295–8309. https://doi.org/10.18632/oncotarget.6989. Chung, S.W., Liu, X., Macias, A.A., Baron, R.M., and Perrella, M.A. (2008). Heme oxygenase-1-derived carbon monoxide enhances the host defense response to microbial sepsis in mice. J. Clin. Invest. 118, 239–247. https://doi.org/10.1172/JCI32730. Coleman, R. (1987). Biochemistry of bile secretion. Biochem. J. 244, 249–261. Collier-Hyams, L.S., Zeng, H., Sun, J., Tomlinson, A.D., Bao, Z.Q., Chen, H., Madara, J.L., Orth, K., and Neish, A.S. (2002). Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-kappa B pathway. J. Immunol. 169, 2846–2850. Costerton, J.W., Stewart, P.S., and Greenberg, E.P. (1999). Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322. Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 157 Crawford, R.W., Gibson, D.L., Kay, W.W., and Gunn, J.S. (2008). Identification of a bile-Induced exopolysaccharide required for Salmonella biofilm formation on gallstone surfaces. Infect. Immun. 76, 5341–5349. Crawford, R.W., Rosales-Reyes, R., Ramírez-Aguilar, Mde. L., Chapa-Azuela, O., Alpuche-Aranda, C., and Gunn, J.S. (2010). Gallstones play a significant role in Salmonella spp. gallbladder colonization and carriage. Proc. Natl. Acad. Sci. U.S.A. 107, 4353–4358. https://doi.org/10.1073/pnas.1000862107. Crawford, R.W., Keestra, A.M., Winter, S.E., Xavier, M.N., Tsolis, R.M., Tolstikov, V., and Bäumler, A.J. (2012). Very long O-antigen chains enhance fitness during Salmonella-induced colitis by increasing bile resistance. PLOS Pathog. 8, e1002918. https://doi.org/10.1371/journal.ppat.1002918. Crawford, R.W., Wangdi, T., Spees, A.M., Xavier, M.N., Tsolis, R.M., and Bäumler, A.J. (2013). Loss of very-long O-antigen chains optimizes capsule-mediated immune evasion by Salmonella enterica serovar Typhi. MBio 4, 1–8. Cummings, L.A., Wilkerson, W.D., Bergsbaken, T., and Cookson, B.T. (2006). In vivo, fliC expression by Salmonella enterica serovar Typhimurium is heterogeneous, regulated by ClpX, and anatomically restricted. Mol. Microbiol. 61, 795–809. Cunningham, A.F., Gaspal, F., Serre, K., Mohr, E., Henderson, I.R., Scott-Tucker, A., Kenny, S.M., Khan, M., Toellner, K.-M., Lane, P.J.L., et al. (2007). Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection. J. Immunol. 178, 6200–6207. D’Costa, V.M., Braun, V., Landekic, M., Shi, R., Proteau, A., McDonald, L., Cygler, M., Grinstein, S., and Brumell, J.H. (2015). Salmonella disrupts host endocytic trafficking by SopD2-mediated inhibition of Rab7. Cell Rep. 12, 1508–1518. Dalebroux, Z.D., Matamouros, S., Whittington, D., Bishop, R.E., and Miller, S.I. (2014). PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane. Proc. Natl. Acad. Sci. U. S. A. 111, 1963–1968. Delves, P.J., Martin, S.J., Burton, D.R., and Roitt, I.M. (2011). Roitt’s essential immunology. Detweiler, C.S., Monack, D.M., Brodsky, I.E., Mathew, H., and Falkow, S. (2003). virK, somA and rcsC are important for systemic Salmonella enterica serovar Typhimurium infection and cationic peptide resistance. Mol. Microbiol. 48, 385–400. Dong, H., Zhang, X., Pan, Z., Peng, D., and Liu, X. (2008). Identification of genes for biofilm formation in a Salmonella enteritidis strain by transposon mutagenesis. Wei Sheng Wu Xue Bao 48, 869–873. Dreher, D., Kok, M., Obregon, C., Kiama, S.G., Gehr, P., and Nicod, L.P. (2002). Salmonella virulence factor SipB induces activation and release of IL-18 in human dendritic cells. J. Leukoc. Biol. 72, 743–751. Du, F., and Galán, J.E. (2009). Selective inhibition of type III secretion activated signaling by the Salmonella effector AvrA. PLOS Pathog. 5, e1000595. https://doi.org/10.1371/journal.ppat.1000595. Eckburg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E., Dethlefsen, L., Sargent, M., Gill, S.R., Nelson, K.E., and Relman, D.A. (2005). Diversity of the human intestinal microbial flora. Science 308, 1635–1638. Eisele, N.A., Ruby, T., Jacobson, A., Manzanillo, P.S., Cox, J.S., Lam, L., Mukundan, L., Chawla, A., and Monack, D.M. (2013). Salmonella require the fatty acid regulator PPARδ for the establishment of a metabolic environment essential for long-term persistence. Cell Host Microbe 14, 171–182. https:// doi.org/10.1016/j.chom.2013.07.010. Erickson, K.D., and Detweiler, C.S. (2006). The Rcs phosphorelay system is specific to enteric pathogens/ commensals and activates ydeI, a gene important for persistent Salmonella infection of mice. Mol. Microbiol. 62, 883–894. Ertelt, J.M., Johanns, T.M., Mysz, M.A., Nanton, M.R., Rowe, J.H., Aguilera, M.N., and Way, S.S. (2011). Selective culling of high avidity antigen-specific CD4+ T cells after virulent Salmonella infection. Immunology 134, 487–497. Farizano, J.V., Pescaretti, M. de las M., López, F.E., Hsu, F.-F., and Delgado, M.A. (2012). The PmrAB system-inducing conditions control both lipid A remodeling and O-antigen length distribution, influencing the Salmonella Typhimurium-host interactions. J. Biol. Chem. 287, 38778–38789. Farris, C., Sanowar, S., Bader, M.W., Pfuetzner, R., and Miller, S.I. (2010). Antimicrobial peptides activate the Rcs regulon through the outer membrane lipoprotein RcsF. J. Bacteriol. 192, 4894–4903. https:// doi.org/10.1128/JB.00505-10. Fillatreau, S. (2011). Novel regulatory functions for Toll-like receptor-activated B cells during intracellular bacterial infection. Immunol. Rev. 240, 52–71. https://doi.org/10.1111/j.1600-065X.2010.00991.x. Fillatreau, S., Gray, D., and Anderton, S.M. (2008). Not always the bad guys: B cells as regulators of autoimmune pathology. Nat. Rev. Immunol. 8, 391–397. https://doi.org/10.1038/nri2315. Fink, S.L., and Cookson, B.T. (2007). Pyroptosis and host cell death responses during Salmonella infection. Cell. Microbiol. 9, 2562–2570. Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 164 | Prieto, A.I., Ramos-Morales, F., and Casadesús, J. (2004). Bile-induced DNA damage in Salmonella enterica. Genetics 168, 1787–1794. Prieto, A.I., Ramos-Morales, F., and Casadesús, J. (2006). Repair of DNA damage induced by bile salts in Salmonella enterica. Genetics 174, 575–584. Prouty, A.M., and Gunn, J.S. (2003). Comparative analysis of Salmonella enterica serovar Typhimurium biofilm formation on gallstones and on glass. Infect Immun 71, 7154–7158. Prouty, A.M., Schwesinger, W.H., and Gunn, J.S. (2002). Biofilm formation and interaction with the surfaces of gallstones by Salmonella spp. Infect. Immun. 70, 2640–2649. Prouty, A.M., Brodsky, I.E., Manos, J., Belas, R., Falkow, S., and Gunn, J.S. (2004). Transcriptional regulation of Salmonella enterica serovar Typhimurium genes by bile. FEMS Immunol. Med. Microbiol. 41, 177–185. https://doi.org/10.1016/j.femsim.2004.03.002. Qu, Y., Misaghi, S., Newton, K., Maltzman, A., Izrael-Tomasevic, A., Arnott, D., and Dixit, V.M. (2016). NLRP3 recruitment by NLRC4 during Salmonella infection. J. Exp. Med. 213, 877–885. https://doi. org/10.1084/jem.20132234. Radtke, A.L., Delbridge, L.M., Balachandran, S., Barber, G.N., and O’Riordan, M.X. (2007). TBK1 protects vacuolar integrity during intracellular bacterial infection. PLOS Pathog. 3, e29. Ramos-Morales, F. (2012a). Impact of Salmonella enterica type III secretion system effectors on the eukaryotic host cell. ISRN Cell Biol. 2012, 1–36. Ramos-Morales, F. (2012b). Acidic pH: enemy or ally for enteric bacteria? Virulence 3, 103–106. https:// doi.org/10.4161/viru.19382. Ramos-Morales, F., Prieto, A.I., Beuzón, C.R., Holden, D.W., and Casadesús, J. (2003). Role for Salmonella enterica enterobacterial common antigen in bile resistance and virulence. J. Bacteriol. 185, 5328–5332. Ramu, P., Tanskanen, R., Holmberg, M., Lähteenmäki, K., Korhonen, T.K., and Meri, S. (2007). The surface protease PgtE of Salmonella enterica affects complement activity by proteolytically cleaving C3b, C4b and C5. FEBS Lett. 581, 1716–1720. Rathinam, V.A., and Fitzgerald, K.A. (2016). Inflammasome complexes: emerging mechanisms and effector functions. Cell 165, 792–800. https://doi.org/10.1016/j.cell.2016.03.046. Rémy, S., Blancou, P., Tesson, L., Tardif, V., Brion, R., Royer, P.J., Motterlini, R., Foresti, R., Painchaut, M., Pogu, S., et al. (2009). Carbon monoxide inhibits TLR-induced dendritic cell immunogenicity. J. Immunol. 182, 1877–1884. https://doi.org/10.4049/jimmunol.0802436. Riquelme, S.A., Bueno, S.M., and Kalergis, A.M. (2012). IgG keeps virulent Salmonella from evading dendritic cell uptake. Immunology 136, 291–305. https://doi.org/10.1111/j.1365-2567.2012.03578.x. Riquelme, S.A., Pogu, J., Anegon, I., Bueno, S.M., and Kalergis, A.M. (2015). Carbon monoxide impairs mitochondria-dependent endosomal maturation and antigen presentation in dendritic cells. Eur. J. Immunol. 45, 3269–3288. Riva, R., Korhonen, T.K., and Meri, S. (2015). The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H. Front. Microbiol. 6, 1–9. Rolhion, N., Furniss, R.C., Grabe, G., Ryan, A., Liu, M., Matthews, S.A., and Holden, D.W. (2016). Inhibition of nuclear transport of NF-ĸB p65 by the Salmonella type III secretion system effector SpvD. PLOS Pathog. 12, e1005653. https://doi.org/10.1371/journal.ppat.1005653. Ruan, H., Zhang, Z., Tian, L., Wang, S., Hu, S., and Qiao, J.J. (2016). The Salmonella effector SopB prevents ROS-induced apoptosis of epithelial cells by retarding TRAF6 recruitment to mitochondria. Biochem. Biophys. Res. Commun. 478, 618–623. https://doi.org/10.1016/j.bbrc.2016.07.116. Russell, T.L., Berardi, R.R., Barnett, J.L., Dermentzoglou, L.C., Jarvenpaa, K.M., Schmaltz, S.P., and Dressman, J.B. (1993). Upper gastrointestinal pH in seventy-nine healthy, elderly, North American men and women. Pharm. Res. 10, 187–196. Ryan, D., Ojha, U.K., Jaiswal, S., Padhi, C., and Suar, M. (2016). The small RNA DsrA influences the acid tolerance response and virulence of Salmonella enterica serovar Typhimurium. Front. Microbiol. 7, 599. https://doi.org/10.3389/fmicb.2016.00599. Scherer, C.A., Cooper, E., and Miller, S.I. (2000). The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection. Mol. Microbiol. 37, 1133–1145. Schiøler, H., Christiansen, E.D., Høybye, G., Rasmussen, S.N., and Greibe, J. (1983). Biliary calculi in chronic Salmonella carriers and healthy controls: a controlled study. Scand. J. Infect. Dis. 15, 17–19. Sharma, A., and Qadri, A. (2004). Vi polysaccharide of Salmonella Typhi targets the prohibitin family of molecules in intestinal epithelial cells and suppresses early inflammatory responses. Proc. Natl. Acad. Sci. U.S.A. 101, 17492–17497. Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 165 Sharma, D., and Kanneganti, T.D. (2016). The cell biology of inflammasomes: Mechanisms of inflammasome activation and regulation. J. Cell Biol. 213, 617–629. https://doi.org/10.1083/jcb.201602089. Shea, J.E., Hensel, M., Gleeson, C., and Holden, D.W. (1996). Identification of a virulence locus encoding a second type III secretion system in Salmonella Typhimurium. Proc. Natl. Acad. Sci. U.S.A. 93, 2593–2597. Shen, P., Roch, T., Lampropoulou, V., O’Connor, R.A., Stervbo, U., Hilgenberg, E., Ries, S., Dang, V.D., Jaimes, Y., Daridon, C., et al. (2014). IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases. Nature 507, 366–370. https://doi.org/10.1038/nature12979. Sheppard, K.A., Fitz, L.J., Lee, J.M., Benander, C., George, J.A., Wooters, J., Qiu, Y., Jussif, J.M., Carter, L.L., Wood, C.R., et al. (2004). PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/ CD3zeta signalosome and downstream signaling to PKCtheta. FEBS Lett. 574, 37–41. https://doi. org/10.1016/j.febslet.2004.07.083. Shpargel, J.S., Berardi, R.S., and Lenz, D. (1985). Salmonella Typhi carrier state 52 years after illness with typhoid fever: a case study. Am. J. Infect. Control 13, 122–123. Sinnott, C.R., and Teall, A.J. (1987). Persistent gallbladder carriage of Salmonella Typhi. Lancet 1, 976. Søndberg, E., and Jelsbak, L. (2016). Salmonella Typhimurium undergoes distinct genetic adaption during chronic infections of mice. BMC Microbiol. 16, 30. Spanò, S., Gao, X., Hannemann, S., Lara-Tejero, M., and Galán, J.E. (2016). A bacterial pathogen targets a host Rab-family GTPase defense pathway with a GAP. Cell Host Microbe 19, 216–226. https://doi. org/10.1016/j.chom.2016.01.004. Srinivasan, A., Foley, J., Ravindran, R., and McSorley, S.J. (2004). Low-dose Salmonella infection evades activation of flagellin-specific CD4 T cells. J. Immunol. 173, 4091–4099. Srinivasan, A., Nanton, M., Griffin, A., and McSorley, S.J. (2009). Culling of activated CD4 T cells during typhoid is driven by Salmonella virulence genes. J. Immunol. 182, 7838–7845. https://doi. org/10.4049/jimmunol.0900382. Staib, L., and Fuchs, T.M. (2015). Regulation of fucose and 1,2-propanediol utilization by Salmonella enterica serovar Typhimurium. Front. Microbiol. 6, 1116. https://doi.org/10.3389/fmicb.2015.01116. Stecher, B., Robbiani, R., Walker, A.W., Westendorf, A.M., Barthel, M., Kremer, M., Chaffron, S., Macpherson, A.J., Buer, J., Parkhill, J., et al. (2007). Salmonella enterica serovar Typhimurium exploits inflammation to compete with the intestinal microbiota. PLOS Biol. 5, 2177–2189. Stecher, B., Denzler, R., Maier, L., Bernet, F., Sanders, M.J., Pickard, D.J., Barthel, M., Westendorf, A.M., Krogfelt, K.A., Walker, A.W., et al. (2012). Gut inflammation can boost horizontal gene transfer between pathogenic and commensal Enterobacteriaceae. Proc. Natl. Acad. Sci. U.S.A. 109, 1269–1274. https://doi.org/10.1073/pnas.1113246109. Steele-Mortimer, O., Brumell, J.H., Knodler, L.A., Méresse, S., Lopez, A., and Finlay, B.B. (2002). The invasion-associated type III secretion system of Salmonella enterica serovar Typhimurium is necessary for intracellular proliferation and vacuole biogenesis in epithelial cells. Cell. Microbiol. 4, 43–54. Storek, K.M., and Monack, D.M. (2015). Bacterial recognition pathways that lead to inflammasome activation. Immunol. Rev. 265, 112–129. https://doi.org/10.1111/imr.12289. Sun, H., Kamanova, J., Lara-Tejero, M., and Galán, J.E. (2016). A family of Salmonella type III secretion effector proteins selectively targets the NF-κB signaling pathway to preserve host homeostasis. PLOS Pathog. 12, e1005484. https://doi.org/10.1371/journal.ppat.1005484. Sundquist, M., and Wick, M.J. (2009). Salmonella induces death of CD8α+ dendritic cells but not CD11c(int)CD11b(+) inflammatory cells in vivo via MyD88 and TNFR1. J. Leukoc. Biol. 85, 225– 234. https://doi.org/10.1189/jlb.0708413. Tardif, V., Riquelme, S.A., Remy, S., Carreño, L.J., Cortés, C.M., Simon, T., Hill, M., Louvet, C., Riedel, C.A., Blancou, P., et al. (2013). Carbon monoxide decreases endosome-lysosome fusion and inhibits soluble antigen presentation by dendritic cells to T cells. Eur. J. Immunol. 43, 2832–2844. Tenor, J.L., McCormick, B.A., Ausubel, F.M., and Aballay, A. (2004). Caenorhabditis elegans-based screen identifies Salmonella virulence factors required for conserved host-pathogen interactions. Curr. Biol. 14, 1018–1024. Tezcan-Merdol, D., Nyman, T., Lindberg, U., Haag, F., Koch-Nolte, F., and Rhen, M. (2001). Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB. Mol. Microbiol. 39, 606–619. Thiennimitr, P., Winter, S.E., Winter, M.G., Xavier, M.N., Tolstikov, V., Huseby, D.L., Sterzenbach, T., Tsolis, R.M., Roth, J.R., and Bäumler, A.J. (2011). Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota. Proc. Natl. Acad. Sci. U.S.A. 108, 17480–17485. https://doi.org/10.1073/pnas.1107857108. Curr. Issues Mol. Biol. Vol. 25 Bernal-Bayard and Ramos-Morales 166 | Thompson, L.J., Dunstan, S.J., Dolecek, C., Perkins, T., House, D., Dougan, G., Nguyen, T.D.H., Tran, T.H.T.P. La, Doan, C. Du, Le, T.P., et al. (2009). Transcriptional response in the peripheral blood of patients infected with Salmonella enterica serovar Typhi. Proc. Natl. Acad. Sci. U.S.A. 106, 22433–22438. Thurston, T.L., Boyle, K.B., Allen, M., Ravenhill, B.J., Karpiyevich, M., Bloor, S., Kaul, A., Noad, J., Foeglein, A., Matthews, S.A., et al. (2016). Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2-dependent antibacterial autophagy. EMBO J. 182, e201694491. Thurston, T.L., Ryzhakov, G., Bloor, S., von Muhlinen, N., and Randow, F. (2009). The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nat. Immunol. 10, 1215–1221. https://doi.org/10.1038/ni.1800. Thurston, T.L., Wandel, M.P., von Muhlinen, N., Foeglein, A., and Randow, F. (2012). Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature 482, 414–418. https:// doi.org/10.1038/nature10744. Tindall, B.J., Grimont, P.A.D., Garrity, G.M., and Euzéby, J.P. (2005). Nomenclature and taxonomy of the genus Salmonella. Int. J. Syst. Evol. Microbiol. 55, 521–524. Tobar, J.A., Carreño, L.J., Bueno, S.M., González, P.A., Mora, J.E., Quezada, S.A., and Kalergis, A.M. (2006). Virulent Salmonella enterica serovar Typhimurium evades adaptive immunity by preventing dendritic cells from activating T cells. Infect. Immun. 74, 6438–6448. Tobar, J.A., González, P.A., and Kalergis, A.M. (2004). Salmonella escape from antigen presentation can be overcome by targeting bacteria to Fc gamma receptors on dendritic cells. J. Immunol. 173, 4058–4065. Tran, Q.T., Gomez, G., Khare, S., Lawhon, S.D., Raffatellu, M., Bäumler, A.J., Ajithdoss, D., Dhavala, S., and Adams, L.G. (2010). The Salmonella enterica serotype Typhi Vi capsular antigen is expressed after the bacterium enters the ileal mucosa. Infect. Immun. 78, 527–535. Ugrinovic, S., Ménager, N., Goh, N., and Mastroeni, P. (2003). Characterization and development of T-Cell immune responses in B-cell-deficient (Igh-6(-/-) mice with Salmonella enterica serovar Typhimurium infection. Infect. Immun. 71, 6808–6819. van der Velden, A.W., Copass, M.K., and Starnbach, M.N. (2005). Salmonella inhibit T cell proliferation by a direct, contact-dependent immunosuppressive effect. Proc. Natl. Acad. Sci. U.S.A. 102, 17769–17774. van der Velden, A.W., Dougherty, J.T., and Starnbach, M.N. (2008). Down-modulation of TCR expression by Salmonella enterica serovar Typhimurium. J. Immunol. 180, 5569–5574. Van Kesteren, M., Bibby, B.G., and Berry, G.P. (1942). Studies on the antibacterial factors of human saliva. J. Bacteriol. 43, 573–583. Vogt, S.L., Peña-Díaz, J., and Finlay, B.B. (2015). Chemical communication in the gut: Effects of microbiota-generated metabolites on gastrointestinal bacterial pathogens. Anaerobe 34, 106–115. van der Waaij, D., Berghuis-de Vries, J.M., and Lekkerkerk Lekkerkerk-v (1971). Colonization resistance of the digestive tract in conventional and antibiotic-treated mice. J. Hyg. (Lond). 69, 405–411. Watson, K.G., and Holden, D.W. (2010). Dynamics of growth and dissemination of Salmonella in vivo. Cell. Microbiol. 12, 1389–1397. https://doi.org/10.1111/j.1462-5822.2010.01511.x. Wehkamp, J., Schauber, J., and Stange, E.F. (2007). Defensins and cathelicidins in gastrointestinal infections. Curr. Opin. Gastroenterol. 23, 32–38. https://doi.org/10.1097/MOG.0b013e32801182c2. Wei, L., Jin, Q., Chu, Y., Wu, S., and Huang, R. (2015). Suppression of dendritic cell and T-cell activation by the pRST98 Salmonella plasmid. Mol. Med. Rep. 11, 2306–2314. https://doi.org/10.3892/ mmr.2014.2919. Wilson, R.P., Winter, S.E., Spees, A.M., Winter, M.G., Nishimori, J.H., Sanchez, J.F., Nuccio, S.-P., Crawford, R.W., Tükel, Ç., and Bäumler, A.J. (2011). The Vi capsular polysaccharide prevents complement receptor 3-mediated clearance of Salmonella enterica serotype Typhi. Infect. Immun. 79, 830–837. Winter, S.E., Thiennimitr, P., Winter, M.G., Butler, B.P., Huseby, D.L., Crawford, R.W., Russell, J.M., Bevins, C.L., Adams, L.G., Tsolis, R.M., et al. (2010a). Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 467, 426–429. https://doi.org/10.1038/nature09415. Winter, S.E., Winter, M.G., Godinez, I., Yang, H.-J., Rüssmann, H., Andrews-Polymenis, H.L., and Bäumler, A.J. (2010b). A rapid change in virulence gene expression during the transition from the intestinal lumen into tissue promotes systemic dissemination of Salmonella. PLOS Pathog. 6, e1001060. Worley, M.J., Nieman, G.S., Geddes, K., and Heffron, F. (2006). Salmonella Typhimurium disseminates within its host by manipulating the motility of infected cells. Proc. Natl. Acad. Sci. U.S.A. 103, 17915– 17920. Wu, H., Jones, R.M., and Neish, A.S. (2012). The Salmonella effector AvrA mediates bacterial intracellular survival during infection in vivo. Cell. Microbiol. 14, 28–39. https://doi.org/10.1111/j.1462- 5822.2011.01694.x. Curr. Issues Mol. Biol. Vol. 25 Molecular Mechanisms Used by Salmonella | 167 Wynosky-Dolfi, M.A., Snyder, A.G., Philip, N.H., Doonan, P.J., Poffenberger, M.C., Avizonis, D., Zwack, E.E., Riblett, A.M., Hu, B., Strowig, T., et al. (2014). Oxidative metabolism enables Salmonella evasion of the NLRP3 inflammasome. J. Exp. Med. 211, 653–668. https://doi.org/10.1084/jem.20130627. Yachie, A., Niida, Y., Wada, T., Igarashi, N., Kaneda, H., Toma, T., Ohta, K., Kasahara, Y., and Koizumi, S. (1999). Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J. Clin. Invest. 103, 129–135. https://doi.org/10.1172/JCI4165. Yang, Z., Soderholm, A., Lung, T.W., Giogha, C., Hill, M.M., Brown, N.F., Hartland, E., and Teasdale, R.D. (2015). SseK3 is a Salmonella effector that binds TRIM32 and modulates the host’s NF-κB signalling activity. PLOS ONE 10, e0138529. https://doi.org/10.1371/journal.pone.0138529. Ye, Z., Petrof, E.O., Boone, D., Claud, E.C., and Sun, J. (2007). Salmonella effector AvrA regulation of colonic epithelial cell inflammation by deubiquitination. Am. J. Pathol. 171, 882–892. Yin, J., Xia, J., Tao, M., Xu, L., Li, Q., Geng, S., and Jiao, X. (2016). Construction and characterization of a cigR deletion mutant of Salmonella enterica serovar Pullorum. Avian Pathol. 45, 569–575. https://doi. org/10.1080/03079457.2016.1187708. Zaki, M.H., Fujii, S., Okamoto, T., Islam, S., Khan, S., Ahmed, K.A., Sawa, T., and Akaike, T. (2009). Cytoprotective function of heme oxygenase 1 induced by a nitrated cyclic nucleotide formed during murine salmonellosis. J. Immunol. 182, 3746–3756. https://doi.org/10.4049/jimmunol.0803363. Zgurskaya, H.I., and Nikaido, H. (1999). Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 96, 7190–7195. Zhang, S., Santos, R.L., Tsolis, R.M., Stender, S., Hardt, W.-D., Bäumler, A.J., and Adams, L.G. (2002). The Salmonella enterica serotype Typhimurium effector proteins SipA, SopA, SopB, SopD, and SopE2 act in concert to induce diarrhea in calves. Infect. Immun. 70, 3843–3855. Zhou, D., Chen, L.M., Hernandez, L., Shears, S.B., and Galán, J.E. (2001a). A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39, 248–259. Zhou, Z., Ribeiro, A.A., Lin, S., Cotter, R.J., Miller, S.I., and Raetz, C.R. (2001b). Lipid A modifications in polymyxin-resistant Salmonella Typhimurium: PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation. J. Biol. Chem. 276, 43111–43121. Curr. Issues Mol. Biol. Vol. 25