Gliding motility protein LIMP promotes optimal mosquito midgut traversal and infection by Plasmodium berghei

Molecula & Biochemical Pa asi ology 241 (2021) 111347 A ailable online 18 Decembe 2020 0166-6851/© 2020 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). Sho communica ion Gliding mo ili y p o ein LIMP p omo es op imal mosqui o midgu a e sal and in ec ion by Plasmodium be ghei Saskia Ega e a , 1 , Jo ge M. San os b , 1 , 2 , Jessica Keh e a , Julia Sa le a , F ied ich F ischknech a , Gunna R. Mai a , b , c , * a Pa asi ology, Depa men o In ec ious Diseases, Heidelbe g Uni e si y Medical School, Heidelbe g, Ge many b Ins i u o de Medicina Molecula Jo˜ ao Lobo An unes, Faculdade de Medicina da Uni e sidade de Lisboa, Edi ício Egas Moniz, A . P o . Egas Moniz, Lisbon, Po ugal c Iowa S a e Uni e si y, Biomedical Sciences, Ames, IA, Uni ed S a es ARTICLE INFO Keywo ds: Gliding mo ili y LIMP Ookine e T ansmission T ansla ional ep ession DOZI ABSTRACT Subs a e-dependen gliding mo ili y is key o mala ia ansmission. I media es hos cell a e sal, in asion and in ec ion by Plasmodium and ela ed apicomplexan pa asi es. The 110 amino acid-long cell su ace p o ein LIMP is essen ial o P. be ghei spo ozoi es whe e i is equi ed o he in asion o he mosqui o’s sali a y glands and he li e cells o he oden hos . He e we de ine an addi ional ole o LIMP du ing mosqui o in asion by he ookine e. limp mRNA is p o ided as a ansla ionally ep essed mRNP (messenge ibonucleop o ein) by he emale game ocy e and he p o ein ansla ed in he ookine e. Pa asi es deple ed o limp (Δlimp) de elop ooki- ne es wi h appa en no mal mo phology and no de ec du ing in i o gliding mo ili y, and ye display a p o- nounced educ ion in oocys numbe s; compa ed o wild ype 82 % mo e Δlimp ookine es emain wi hin he mosqui o blood meal explaining he dec ease in oocys s. As in he spo ozoi e, LIMP exe s a p o ound ole on ookine e in ec ion o he mosqui o. Gliding mo ili y o mala ia pa asi es is equi ed o mosqui o and mammalian hos in ec ion. Despi e hei di e en mo phologies, all in asi e li e cycle s ages o he mala ia pa asi e – me ozoi e, ookine e and spo ozoi e – ely on a conse ed gliding mo ili y machine y o a- e se di e se hos cell ba ie s in o de o es ablish hos cell in ec ions and con inue he li e cycle [1]. Many o he mo ili y ac o s a e sha ed wi h ela ed apicomplexan pa asi es such as Toxoplasma gondii. The pea -shaped mala ia me ozoi e in ades ed blood cells. The ookine e is a banana-shaped li e cycle s age ha is o med om a e ilised emale game e (zygo e) in he mosqui o midgu and equi ed o es ablish an in ec ion in he mosqui o; in o de o do so, he ookine e c osses he blood meal sac as well as he mosqui o pe i ophic ma ix and midgu epi helium o ans o m in o a sessile oocys . Spo ozoi es a e inally o med in a pe iod o 2 weeks and a e dis inc ly slende -shaped; hey mo e signi ican ly as e han ookine es and mig a e om he hemocoel h ough he sali a y gland epi helium in o he gland lumen as well as h ough he skin and ac oss he blood essel endo helium o ul ima ely in ade hepa ocy es. The 110 amino acid-long cell su ace p o ein LIMP is exp essed in ookine es and spo ozoi es, bu no me ozoi es. In he spo ozoi e, LIMP de ines he pa asi e’s abili y o popula e he mosqui o’s sali a y glands and is essen ial o he es ablishmen o an in ec ion o he oden li e . In he ookine e, a dele ion o he LIMP gene p oduces a obus , 50 %- educ ion in oocys numbe s [2]. The cause o his de ec is howe e unknown. He e we sough o add ess he ole o LIMP a his c ucial bo leneck in he mala ia pa asi e’s li e cycle ocusing on key aspec s o ookine e biology: de elopmen , mo ili y and in ec i i y. The o ma ion o he P. be ghei ookine e om he e ilized emale game e (zygo e) depends on ma e nally p o ided gene p oduc s (mRNA) ha a e p oduced in he emale game ocy e, bu a e only ansla ed a e e ilisa ion. Hund eds o mRNAs – some 50 % o he de ec ed an- sc ip ome – a e p o ided in ansla ionally silen mRNPs (messenge ibonucleop o eins) [3]. When ansla ed in o p o ein hey a e ins u- men al in many de elopmen al p ocesses o he ookine e and can e en a ec he o ma ion o oocys -de i ed spo ozoi es [4–8]. E alua ing he p o ein exp ession p o ile h ough C- e minal GFP- agging o LIMP in * Co esponding au ho a : Iowa S a e Uni e si y, Biomedical Sciences, Ames, IA, Uni ed S a es E-mail add ess: [email p o ec ed] (G.R. Mai ). 1 Au ho s con ibu ed equally. 2 P esen add ess: Depa men o Immunology and In ec ious Diseases, Ha a d T.H. Chan School o Public Heal h, Bos on, MA, Uni ed S a es. Con en s lis s a ailable a ScienceDi ec Molecula & Biochemical Pa asi ology jou nal homepage: www.else ie .com/loca e/molbiopa a h ps://doi.o g/10.1016/j.molbiopa a.2020.111347 Recei ed 29 June 2020; Recei ed in e ised o m 4 Decembe 2020; Accep ed 15 Decembe 2020 Molecula & Biochemical Pa asi ology 241 (2021) 111347 2 limp::g p mu an s by li e luo escence mic oscopy ound no ansla ion in asexual s age pa asi es no game ocy es; limp::g p is ansla ed in he ookine e s age and he p o ein is hen mainly isible nea o in he c ys alloid, a ansien and pu a i e s o age o ganelle o he ookine e [9] (Fig. 1A and p e iously shown in [2]). We obse ed no ansla ion o limp in sexual p ecu so cells (game ocy es), despi e he ansc ip being eadily de ec ed by RT-PCR in his li e cycle s age (see limp inpu lanes labelled wi h ed a ows in Fig. 1B). Using pa asi e clones exp essing GFP- agged ansla ional ep esso s DOZI (a homolog o yeas DHH1 DEAD-box RNA helicase) and CITH (a homolog o yeas ep esso o ansla ion ini ia ion p o ein SCD6) [3,10] we iden i ied limp mRNA o be associa ed wi h bo h p o eins by RNA-immunop ecipi a ion (RIP) ollowed by RT-PCR de ec ion o he bound ansc ip . Monoclonal an i-GFP an ibodies and a p o ein G sepha ose a ini y eagen we e used o immunop ecipi a e DOZI::GFP and CITH::GFP and any bound mRNA om en iched game ocy e popula ions (lanes IP- α GFP); con ols included he use o an un ela ed an i-cmyc IgG (lanes IP- α cmyc) o omission o IgG al oge he (lanes IP beads). Like p25 and p28 – bo h a e hallma k ansla ionally ep essed ansc ip s encoding ookine e su ace p o eins and hus impo an and p omising an imala ial ans- mission-blocking accine a ge s [11–13] – limp mRNA is bound by DOZI and CITH (Fig. 1B). As expec ed, mRNA encoding he DNA/RNA-binding p o ein ALBA-3, which is ansla ed in he game o- cy e, does no co-IP wi h DOZI o CITH. The RIP da a explain he lack o LIMP exp ession in he game ocy e; limp mRNA is a bona ide ma e nally p o ided mRNA ha is kep ansla ionally quiescen in a complex con aining DOZI and CITH in game ocy es. Once ansla ed, LIMP a - ec s in i o ansmission om he in ec ed expe imen al mouse hos o Anopheles emale mosqui oes; limp gene dele ion mu an s expe ience a 50 % educ ion in oocys numbe s [2]. This dec ease could esul om de ec s in ookine e de elopmen , mo ili y, adhesion o in asion, bu he unde lying cause is no clea . In o de o es whe he hei mo ili y was a ec ed, we pe o med in i o ookine e cul u es om game ocy e-in ec ed blood. Knock-ou pa asi es p oduced appa en ly no mal ookine es as e alua ed by di e en ial in e e ence con as (DIC) mic oscopy (Fig. 1C); hey showed no de ec in gliding mo ili y; a d op o pu i ied ookine es in Roswell Pa k Memo ial Ins i u e (RPMI) 1640 medium was placed on a glass slide and imaged using an in e ed Zeiss Axio e 200 M wide ield mic oscope wi h an XBO75 xenon lamp, he AxioVision 4.7.2 so wa e, a 63X objec i e and a CoolSNAP™ HQ2 high esolu ion CCD came a (Pho ome ics); images we e cap u ed e e y 20 s o 15 min; he mo ing ookine es we e inally analysed using he Fig. 1. LIMP is ma e nally p o ided o he ookine e whe e i ensu es op imal mosqui o in ec ion. (A) Li e luo escence o limp:g p blood s age game ocy e shows he absence o LIMP::GFP exp ession, while imaging o blood meal- e ie ed ookine es highligh s LIMP::GFP localisa ion o disc e e oci (*) a and nea he c ys alloid. Scale ba =5 μ m. (B) RT-PCR analyses o DOZI::GFP and CITH::GFP RNA immunop ecipi a ion (RIP) elua es demons a e ha limp co-p ecipi a es wi h bo h ansla ional ep esso s. Inpu : o al game ocy e mRNA; IP- α GFP: IP wi h an i-GFP an ibody; IP- α cmyc: IP wi h an i-c-myc an ibody; IP beads: no an ibody used o IP. (C) Rep esen a i e images o wild ype and Δlimp ookine es isola ed om mosqui o midgu s. Scale ba =5 μ m. (D) Quan i ica ion o pa asi e mo ili ies o wild ype and Δlimp ookine es. Mann-Whi ney es ; means wi h SEM a e indica ed. 3 independen ookine e cul u es.(E) Oocys numbe s om wild ype and Δlimp- in ec ed mosqui oes. Mann-Whi ney es ; means wi h SEM a e indica ed. 2 independen memb ane eeding assays. Midgu s wi h no pa asi es we e emo ed om he g aph. Mean p e alence o in ec ion was 74 % o bo h g oups (Chi-squa e es , p =0.8877). (F) Numbe o wild ype and Δlimp ookine es e ained in he mosqui o blood meal bolus 24 h a e in ec ion. Ra io pai ed es is indica ed. 4 independen memb ane eeding assays. Lines connec da a poin s om he same epli- ca e expe imen . S. Ega e e al. Molecula & Biochemical Pa asi ology 241 (2021) 111347 3 manual acking plug-in o ImageJ [14] e ealing a speed o jus below 5 μ m×min −1 o mu an and wild ype pa asi es (Fig. 1D). Knock-ou ookine es appea ed o show a sligh inc ease in gliding speed. Howe - e , his inc ease is only ma ginal (p =0.0460) and we belie e i does no bea any biological ele ance. We nex es ed he abili y o in i o-cul- u ed ookine es o in ec he mosqui o ec o in a con olled s anda d memb ane eeding assay (SMFA). To his end, wild ype and mu an ookine es we e i s pu i ied, hen mixed wi h 500 μ l naï e mouse blood and inally o e ed o emale Anopheles s ephensi mosqui oes o 30 min in wo independen eeding expe imen s (ei he o e ing 1.6 o 6 million ookine es a a concen a ion o 3200 o 12,000 ookine es × μ l −1 ). On day 10 pos -in ec ion, oocys numbe s we e quan i ied ollowing me cu och ome s aining. The gene dele ion mu an ep oduced he 50 % educ ion in oocys numbe s obse ed in ou p e ious publica ion using di ec mosqui o eeds on in ec ed mice [2] (Fig. 1E). P e alence o in ec ion was as ollows: eplica e 1–58% wild ype s 68 % knock-ou (Chi-squa e es , p =0.4272) – and eplica e 2–89% wild ype s 79 % knock-ou (Chi-squa e es , p =0.2898). Mean p e alence o in ec ion calcula ed by combining bo h eplica es was 74 % o bo h wild ype and knock-ou pa asi es (Chi-squa e es , p =0.8877). By bypassing he in i o di e en ia ion o ookine es, ou esul s show ha he lowe numbe o oocys s p oduced by knock-ou pa asi es canno be a ibu ed o an in i o de ec in zygo e- o-ookine e ans o ma ion. To cha ac e ize he knock-ou ’s ailu e o p oduce high oocys numbe s in mo e de ail, we inally quan i ied ookine es in he blood meal a e 24 h o ou inde- penden SMFA. The e o e, midgu s om in ec ed mosqui oes we e collec ed in RPMI and he blood meal isola ed; he numbe o e ained ookine es was quan i ied ia haemocy ome e . We ound he educ ion in oocys numbe s o co ela e wi h an accumula ion o ookine es in he mosqui o blood meal bolus. On a e age, we obse ed an 82% inc ease in e ained knock-ou ookine es when compa ed o he wild ype con ols (Table 1 and Fig. 1F), indica ing a ole o LIMP in a achmen /- a e sal/in asion o he mosqui o midgu by he ookine e. Gi en he obse ed 50 % educ ion in knock-ou oocys numbe s, one could expec o ind wice as many ookine es e ained in he blood meal bolus (i.e., 200 % o wild ype numbe s, o a 100 % inc ease). Ins ead, we obse e, on a e age, an 82 % inc ease om wild ype in he numbe o e ained ookine es. This is sligh ly lowe han he p edic ed 100 % inc ease. Howe e , i is concei able ha some s ochas ic e en s migh ha e esul ed in an unde es ima ion o e ained ookine es (e.g., loss o ooki- ne es du ing sample p ocessing o pa ial exc e ion o he blood bolus p io o gu collec ion). The e o e, i is plausible o an icipa e ha , wi h u he eplica ion o his expe imen , he a e age pe cen inc ease would p og essi ely ge close o he heo e ical alue o 100 %.In summa y, ou da a show ha LIMP p o ein is p oduced om ansla- ionally ep essed, ma e nally p o ided mRNA in he ookine e whe e i helps o op imize mala ia pa asi e in ec ion o he mosqui o; limp-de- ple ed zygo es de elop in o mo ile ookine es wi h appa en no mal mo phology, and ye display a de ec in escaping he mosqui o blood meal and eaching he midgu basal lamina whe e hey can ans o m in o he spo ozoi e-p oducing oocys s age and con inue he li e cycle. The accumula ion o Δlimp mu an s in he midgu lumen is ep oducible and s ong, bu no absolu e, allowing he es ablishmen o oocys s in a labo a o y en i onmen whe e P. be ghei ypically can p oduce hund eds o oocys s in an in ec ed mosqui o. In he ield on he o he hand, absence o he conse ed P. alcipa um LIMP p o ein migh well cause a comple e ansmission blockade o human mala ia pa asi es. The much smalle numbe o P. alcipa um game ocy es p esen in mala ia-posi i e indi iduals – 450 a he han 10,000 ound in he labo a o y o he oden model P. be ghei – ansla es in o ield mosqui oes ypically hos ing, on a e age, a ound 6 ookine es, and jus 2 oocys s pe in ec ed mosqui o; in addi ion, only 1 / 3 o mosqui oes ac ually do become in ec ed [15]. The game ocy emia numbe s unde labo a o y condi ions can each 10 % o he o al pa asi e popula ion in he oden model; i is hus ha we obse e la ge numbe s o oocys s p oduced in wild ype condi ions. The absence o LIMP causes a 50 % educ ion in oocys numbe s. In he con ex o he high baseline in ec ion his is no enough o block he li e cycle; his occu s ul ima ely du ing sali a y gland and li e cell in ec ion by he spo ozoi e [2]. The accumula ion o mo phologically no mal ookine es in he mosqui o blood meal is also documen ed o mu an s deple ed o he pe o in-like p o eins PPLP3/MAOP, PPLP4 and PPLP5 [16–19], o he cell- a e sal-p o ein- o -ookine es-and-spo ozoi es (CelTOS) [20,21]; he e, he block in P. be ghei ansmission is almos absolu e. Ookine e a e sal o he mosqui o midgu epi helium is no comple ely unde - s ood and i has no been p o en unequi ocally whe he he pa asi e p edominan ly pene a es he cells o he epi helial issue o he midgu – pe haps wi h he help o po e- o ming PPLPs and CelTOS – and/o mo es be ween hese cells in o de o each he basal lamina o he dis al side o he midgu whe e hey es ablish oocys s ha go on o p oduce spo ozoi es [22]. The midgu in asion de ec o Δlimp pa asi es could esul om a de ec in adhesion o he pe i ophic ma ix, he luminal side o he midgu epi helium o a ailu e o a e se he epi helial cell laye . These scena ios would be consis en wi h ou p e ious indings o he spo ozoi e, whe e bo h adhesion and a e sal p ocesses ely on LIMP [2]. Pe haps his dual ole o LIMP could be used o in e e e wi h bo h mala ia pa asi e ansmission s ages: ookine e and spo ozoi e. Decla a ions o Compe ing In e es None. Acknowledgemen s We hank Mi iam Reinig (Uni e si y o Heidelbe g Medical School) as well as Ana Pa ei a and Leono Pinho (iMM Jo˜ ao Lobo An unes) o mosqui o ea ing. This s udy was suppo ed by Fundaç˜ ao pa a a Ciˆ encia e a Tecnologia g an s o GRM (PTDC/BIA-BCM/105610/2008 and PTDC/SAU-MIC/122082/2010) and JMS (SFRH/BD/63849/2009); he Na ional Ins i u es o Alle gy and In ec ious Diseases o GRM (1R21AI139579-01A1); he Ho izon 2020 F amewo k P og amme Ma ie Sklodowska-Cu ie g an ag eemen No 660211 o SE; as well as a Human F on ie Science P og am G an (RGY/0071/2011) o FF. The unde s had no ole in s udy design, da a collec ion and in e p e a ion, o he decision o submi he wo k o publica ion. The au ho s ha e no compe ing in e es s o decla e. Re e ences [1] J. Baum, e al., Hos -cell in asion by mala ia pa asi es: insigh s om Plasmodium and Toxoplasma, T ends Pa asi ol. 24 (12) (2008) 557–563. [2] J.M. San os, e al., Mala ia pa asi e LIMP p o ein egula es spo ozoi e gliding mo ili y and in ec i i y in mosqui o and mammalian hos s, Eli e (2017) 6. [3] G.R. Mai , e al., Uni e sal ea u es o pos - ansc ip ional gene egula ion a e c i ical o Plasmodium zygo e de elopmen , PLoS Pa hog. 6 (2) (2010) e1000767. [4] J.M. San os, e al., Ma e nally supplied S-acyl- ans e ase is equi ed o c ys alloid o ganelle o ma ion and ansmission o he mala ia pa asi e, P oc Na l Acad Sci U S A 113 (26) (2016) 7183–7188. [5] J.M. San os, e al., The Plasmodium palmi oyl-S-acyl- ans e ase DHHC2 is essen ial o ookine e mo phogenesis and mala ia ansmission, Sci. Rep. 5 (2015) 16034. Table 1 Da a summa y o ookine e e en ion expe imen s. Numbe o e ained ooki- ne es is plo ed in Fig. 1F. 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