Vibrio parahaemolyticus VopA Is a Potent Inhibitor of CellMigration and Apoptosis in the Intestinal Epithelium ofDrosophila melanogaster

Liping Luo,a Jason D. Matthews,b Brian S. Robinson,b Rheinallt M. Jonesa

aDepartment of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USAbDepartment of Pathology, Emory University School of Medicine, Atlanta, Georgia, USA

ABSTRACT Animal models have played a key role in providing an understanding ofthe mechanisms that govern the pathophysiology of intestinal diseases. To expandon the repertoire of organisms available to study enteric diseases, we report on theuse of the Drosophila melanogaster model to identify a novel function of an effectorprotein secreted by Vibrio parahaemolyticus, which is an enteric pathogen found incontaminated seafood. During pathogenesis, V. parahaemolyticus secretes effectorproteins that usurp the host’s innate immune signaling pathways, thus allowing thebacterium to evade detection by the innate immune system. One secreted effectorprotein, VopA, has potent inhibitory effects on mitogen-activated protein kinase(MAPK) signaling pathways via the acetylation of critical residues within the catalyticloops of mitogen-activated protein kinase kinases (MAPKKs). Using the Drosophilamodel and cultured mammalian cells, we show that VopA also has potent modulat-ing activity on focal adhesion complex (FAC) proteins, where VopA markedly re-duced the levels of focal adhesion kinase (FAK) phosphorylation at Ser910, whereasthe phosphorylation levels of FAK at Tyr397 and Tyr861 were markedly increased.Cultured cells expressing VopA were also impaired in their ability to migrate and re-populate areas subjected to a scratch wound. Consistently, expression of VopA inDrosophila midgut enterocytes disrupted the normal enterocyte arrangement. Finally,VopA inhibited apoptosis in both Drosophila tissues and mammalian cultured cells.Together, our data show that VopA can alter normal intestinal homeostatic pro-cesses to facilitate opportunities for V. parahaemolyticus to prolong infection withinthe host.

KEYWORDS Drosophila melanogaster, FAK, Vibrio parahaemolyticus, VopA,antiapoptotic, genetic models

Drosophila melanogaster is widely used as a genetically tractable animal model fordiscovering the mechanisms of diseases. Many fundamental physiological pro-

cesses are conserved between Drosophila and mammals, with a homolog of about 75%of human disease-associated genes being present in the fly (1). In identifying themechanisms whereby pathogenic bacteria elicit enteric diseases, the traditional meth-odology, mainly performed in rodent models, has employed the approach of infectingmice with either a wild-type or a mutated strain of pathogenic bacteria (2–4). Alterna-tively, the genetic tractability of Drosophila allows for a more reductionist approach todiscovering the mechanism of bacterial pathogenesis. In the fly, the open readingframes of bacterial virulence factors can be inserted into the Drosophila chromosome,thereby creating a transgenic fly stably harboring the bacterial virulence factor. Byspecifically expressing the virulence factor in Drosophila tissue with determinategrowth, such as the eye or wing, novel mechanisms of bacterial protein modulation of

Citation Luo L, Matthews JD, Robinson BS,Jones RM. 2019. Vibrio parahaemolyticus VopAis a potent inhibitor of cell migration andapoptosis in the intestinal epithelium ofDrosophila melanogaster. Infect Immun87:e00669-18. https://doi.org/10.1128/IAI.00669-18.

Editor Shelley M. Payne, The University ofTexas at Austin

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Rheinallt M. Jones,rjones5@emory.edu.

Received 7 September 2018Returned for modification 9 October 2018Accepted 22 December 2018

Accepted manuscript posted online 7January 2019Published

CELLULAR MICROBIOLOGY:PATHOGEN-HOST CELL MOLECULAR INTERACTIONS

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innate immunity have been revealed (5, 6). However, to date, few studies have takenthe approach of ectopically expressing bacterial virulence factors in Drosophila intes-tinal epithelial cells. This is an overlooked opportunity because the Drosophila andhuman intestine share impressive transkingdom functional conservation, where the flygut harbors many of the same cell types as the mammalian gut and common cellsignaling pathways function in intestinal homeostasis and response to injury (7, 8). Inaddition, flies and mammals share conserved mechanisms of host cell and commensalmicrobe interactions (9, 10).

The gut pathogen Vibrio parahaemolyticus is a causal agent of gastroenteritis,typically following ingestion of contaminated seafood. Symptoms associated withdisease include vomiting, abdominal cramping, and diarrhea, although illness is com-monly acute and self-limiting (11, 12). V. parahaemolyticus harbors two type III secretionsystems (T3SS), one on chromosome 1 (T3SS1) and the other on chromosome 2 (T3SS2)(13), with the genes on each of the secretion systems contributing to pathogenicinfection (14). One secreted protein coded on T3SS2 is VopA (also known as VopP).Biochemical analysis of the VopA function revealed that it is a potent inhibitor ofmitogen-activated protein kinase (MAPK) signaling (15, 16). However, the pathologicaloutcome of VopA activity remains unclear. VopA is a member of the YopJ-like family ofbacterial effector proteins, which includes YopJ from Yersinia enterocolitica (17), AvrA ofSalmonella enterica serovar Typhimurium (2, 6, 18), and AopP of Aeromonas salmonicida(5, 19), all of which are known enteropathogens. VopA shares amino acid sequencesimilarity with YopJ at 45%, with AvrA at 48%, and with AopP at 51% over the wholelength of the protein, with a highly conserved region of �90% amino acid similarityspanning a 20-amino-acid region that aligns with residues 175 to 195 of AvrA (Fig. 1A).This region harbors a conserved cysteine residue essential for the catalytic activity ofeach effector protein. VopA has been shown to have inhibitory effects against theextracellular signal-regulated kinase (ERK), p38, and Jun N-terminal protein kinase (JNK)(MAPK) pathways by a mechanism that involves the acetylation of the Ser, Thr, and Lysresidues of the catalytic and activation loops of mitogen-activated protein kinasekinases (MAPKKs), residues which are normally phosphorylated during pathway acti-vation (16). Acetyltransferase activity has also been shown in other YopJ-like proteins,including YopJ and AvrA (6, 20), although it is known that each family member hasevolved a specificity of activity against particular members of the mammalian MAPKKand I�B kinase (IKK) superfamily (Fig. 1B) (21). Furthermore, the specificity of theYopJ-like protein against MAPK or NF-�B has the potential to influence cell fate duringinfection. For example, YopJ induces apoptosis and dampens cytokine production dueto its broad inhibition of the MAPK and NF-�B pathways (22). AopP is proapoptotic dueto it being a potent inhibitor of NF-�B signaling (5), whereas AvrA is antiapoptoticdue to its inhibitory activity against the JNK pathway (2, 6). This property of AvrA hasrecently been exploited in the generation of nanoparticles harboring AvrA for thetreatment of inflammatory bowel disease (23).

MAPKs have been shown to function in many cellular processes, including prolifer-ation, oncogenesis, differentiation, inflammation, and stress responses. Recently, MAPKsignaling has been reported to play a role in regulating cell migration, with both theERK and JNK pathways being reported to function as mediators of extracellular signalsthat induce cell movement by interactions with proteins of the focal adhesion complex(FAC) (24, 25). Because VopA is coded within T3SS2 in V. parahaemolyticus and thus maybe secreted by V. parahaemolyticus into cells of the epithelium, including enterocytes,we reasoned that VopA may have modulatory influences on gut epithelial cell migra-tion and normal homeostasis in the intestine. Herein, we report on the outcome ofVopA activity in enterocytes using a novel approach in Drosophila. Consistent with itsknown activity in cultured mammalian cells, VopA potently inhibited ERK pathwayactivity in Drosophila enterocytes, showing transkingdom conservation of VopA activity.Ectopic VopA expression in Drosophila intestinal epithelial cells resulted in increasedorganismal mortality, disrupted epithelial cell arrangement, and resulted in fewerapoptotic cells in the Drosophila midgut. Consistent with these observations, in cul-

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FIG 1 VopA inhibits ERK pathway signaling in developing Drosophila tissues. (A) Alignment of the amino acid sequences of VopA, AvrA, AopP,and YopJ over a highly conserved region present in each protein that aligns with residues 175 to 205 of the AvrA sequence. The asterisk denotesthe conserved cysteine residue essential for the enzymatic activity of each of the aligned proteins. Black shading denotes residues similar in allfour proteins, dark gray denotes residues similar in three of the aligned proteins, and light gray denotes residues similar in only two of the alignedproteins. (B) Recognition of bacterial products by TLRs results in the activation of innate immune pathways that function to eliminate the bacterialthreat. At the same time, some pathogens secrete effector proteins, including VopA, AvrA, AopP, or YopJ, which usurp signaling pathways thattransduce innate immune alarm messages from the cell surface to the nucleus. The blockade of innate immune signaling allows enteropathogensto persist undetected in the mucosa and evade host innate immunity. (C) Expression of the vopA and mvopA transgenes in flies of the indicated

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tured mammalian cells, VopA disrupted focal adhesion complex (FAC) dynamics byaltering the phosphorylation states of critical regulatory residues within focal adhesionkinase (FAK), Paxillin, and Src. In addition, transfection of VopA significantly slowed themovement and migration of cultured enterocytes. Together, these results suggest thatV. parahaemolyticus has evolved the VopA protein to have refined biochemical activitythat inhibits the migration of enterocytes and the apoptotic response that enterocytesundergo when sensing an irreversible pathogenic threat, thus highlighting the delicatemodification of host responses that some bacteria can engender.

RESULTSVopA inhibitory activity is conserved in Drosophila. A limitation of investigating

V. parahaemolyticus pathogenicity is that an oral infection-based animal model of thehuman disease is still in development. This paucity was recently addressed by investi-gators using infant rabbit, porcine, and murine models, with some encouraging ad-vancements in disease models being seen (14, 26, 27). Alternatively, our research grouphas successfully used the Drosophila animal model to study the function of bacterialeffector proteins secreted by pathogens (5, 6). The utility of the Drosophila system isthat it allows us to undertake initial discoveries that may then be confirmed in awell-developed and relevant mammalian model. To establish the feasibility of usingDrosophila to study host/pathogen interactions in the midgut, we assessed whether theVopA inhibitory profile is conserved in the fly. We created transgenic Drosophila fliesharboring either upstream activation sequence (UAS)-myc-vopA or the catalyticallyinactive UAS-myc-mvopA (C181A) mutant form. Because a functional ERK pathway isnecessary for the normal development of the Drosophila wing, disruption of ERKpathway signaling results in markedly altered phenotypes (28–30). We first detectedexpression of the transgenes by crossing fly lines harboring UAS-myc-vopA and UAS-myc-mvopA to the heat shock (hs)-GAL4 driver line, which expresses the Saccharomycescerevisiae yeast transactivator GAL4 in all cells upon incubation of Drosophila at 30°C(Fig. 1C). A tissue that requires a functional ERK pathway for development is the wing,where inhibition of ERK pathway signaling results in disruption of wing vein formation(29, 30). Consistently, VopA expression under c765-GAL4 or with engrailed-GAL4 (en-GAL4), which targets expression to the posterior compartment of the wing, resulted inthe complete loss of wing vein formation from the posterior wing (Fig. 1D). Immuno-staining of developing wing imaginal disks expressing VopA under en-GAL4 revealed amarked decrease in the levels of phosphorylated ERK in the posterior wing, whereasphosphorylated ERK was detected in the wing margin on the anterior portion of thesame wing or in the entire wing margin tissue expressing a mutated and catalyticallyinactive version of VopA (mVopA) under en-GAL4 (Fig. 1E). Overlay of VopA- andmVopA-expressing wings revealed that VopA expression results in about a 30% de-crease in wing size (Fig. 1F and G). However, enumeration of wing bristles within theposterior wing revealed about a 30% denser number of bristles per unit area of theVopA-expressing wing tissue (Fig. 1H and I). Furthermore, the arrangement of bristlesin VopA-expressing wing tissue was noticeably altered, where bristles are not formedin a regular diagonal arrangement, as is evident in the wild type of mVopA-expressing

FIG 1 Legend (Continued)genotypes following 24 h of heat shock of third instar larvae. Each lane was loaded with 20 �g total protein extracted from larval whole bodies.Note the presence of a band following immunoblotting with myc antibody signifying expression of myc-VopA or myc-mVopA following heatshock. The blot is representative of blots from experiments done in triplicate. (D) Phenotypes of Drosophila adult wings expressing VopA ormVopA under c756-GAL4 (top) or engrailed-GAL4 (en-GAL4) (bottom). (E) Immunostain analysis of Drosophila third instar larvae wing disksexpressing VopA or mVopA under en-GAL4 with antibodies against phosphorylated ERK and myc. Note the VopA-mediated loss of p-ERK activityin the posterior wing disk (white arrows). (F) Superimposition of adult Drosophila wings of phenotypes en-GAL4::vopA (smaller wing) anden-GAL4::mvopA, presented in panel D. The smaller-wing phenotype was observed at 100% penetrance in en-GAL4::vopA flies. (G) Quantificationof the size of the adult wing posterior to the L3 wing vein from the phenotypes described in panel D. The average measured wing size of thecontrol en::mvopA fly was taken as 100%, and the area for the en::vopA fly is presented as a value relative to that for the en::mvopA fly. Statisticalanalysis was calculated by Student’s t test (n � 5). ***, P � 0.001. (H) Wing bristles within the posterior adult wings of the indicated phenotypes.(I) Quantification of the number of wing bristles within the selected area of the posterior adult wing in panel F. Statistical analysis was performedby one-way ANOVA (n � 5). ***, P � 0.001.

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wing tissue (Fig. 1H). Together, these data indicate that ectopic VopA activity in theDrosophila wing tissue results in ERK pathway inhibition and altered tissue phenotypescharacteristic of the inhibition of tissue development. Furthermore, although VopAreduced the wing size by about 30%, the increased numbers of bristles per unit area ofthe same wing indicate that VopA-expressing cells are smaller but have no detectabledifferences in proliferation during development.

VopA disrupts epithelial homeostasis in the Drosophila intestine. To model theinfluence of VopA in isolation in an in vivo model, we ectopically expressed VopA underthe myoIA-GAL4 driver, which specifically expresses GAL4 in fly enterocytes but not inproliferating intestinal stem cells (ISCs) in the fly midgut (10). Many articles comparingthe development of the fly and mammalian gut and detailing the fly’s relevance as amodel for human intestinal physiology have been published (1, 31–33). Briefly, theDrosophila midgut is made up of an epithelial monolayer interspersed with hormone-producing enteroendocrine cells. Adult Drosophila midgut cells are continuously re-plenished by a population of pluripotent ISCs. The ISCs adjoin the intestinal basementmembrane, their progenitors, and differentiated enterocytes (34, 35) (Fig. 2A). We firstobserved a significant loss of organismal viability following expression of VopA in theadult Drosophila midgut enterocytes, indicating that VopA may influence gut physiol-ogy and/or homeostasis (Fig. 2B). Importantly, inhibition of JNK pathway signaling bythe constitutive expression of a dominant negative form of Basket (Bsk) did notinfluence survival, indicating that the loss of viability may be due to the inhibition ofERK pathway signaling (Fig. 2B). Because the decrease in survival is as a result of gutenterocyte-specific expression of VopA, we anticipated that the loss of viability may beas a result of an impairment to gut function and/or barrier integrity. Therefore, weinvestigated the effects of VopA expression of the gut tissue architecture and cellsignaling pathways. Immunostain analysis of 7-day-old adult Drosophila distal midgutfor phosphorylated ERK (p-ERK) revealed strong staining in the nucleus of enterocytesin control flies, whereas no p-ERK was detected in enterocytes expressing VopA (Fig. 2C,top). In addition, expression of VopA in enterocytes resulted in marked changes in cellarrangement in the distal midgut, with the loss of the regular enterocyte patterningcompared to that in the controls being observed (Fig. 2C and D). Furthermore,Drosophila enterocytes in the distal midgut are normally arranged as a layer of singleepithelial cells overlaying a basement membrane and circular muscle. A transversesection through the Drosophila gut by confocal z-stack analysis revealed that a disrup-tion of the normal architecture was altered in VopA-expressing enterocytes (Fig. 2D,bottom). The movement of enterocytes in the gut is controlled, in part, by the activityof proteins that form the focal adhesion complex (FAC), including focal adhesion kinase(FAK) (24, 25). Immunostaining of the Drosophila midgut using an antibody againstphospho-FAK Tyr397 revealed a marked increase in the fluorescent signal in VopA-expressing enterocytes (Fig. 2E). Finally, pulse analysis of 5-ethynyl-2=-deoxyuridine(EdU) incorporation into midgut cells revealed no marked difference in the totalnumber of cells which had incorporated EdU over a 24-h period but did revealdifferences in their distribution. Here, EdU incorporates into ISCs, which then matureand differentiate into enterocytes. Enterocytes organize in the fly midgut to distinctspecial patterning, as seen following expression in control, myoIA::w1118, or mutantmyoIA::mVopA cells. However, enterocyte-specific expression of VopA results in EdU-positive cells being arranged in distinct clusters (Fig. 2F). Because VopA is not expressedin ISCs, the results point to VopA-mediated enterocyte disorganization occurring as aresult of the inability of enterocytes to spatially organize, which may be as a result ofVopA-mediated inhibition of ERK and FAC protein signaling. Together, we show thatVopA alone can decrease p-ERK levels, increase FAK phosphorylation at Tyr397, andalter the normal enterocyte arrangement in the Drosophila intestine.

VopA alters the phosphorylation state of FAC proteins. To establish if VopAalters the activity of focal adhesion complex (FAC) proteins in cultured mammaliancells, plasmids harboring either wild-type VopA or a mutated and catalytically inactive

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version of VopA (mVopA) with a cysteine-to-alanine transition at position 181 weretransfected into HEK293 cultured cells. Consistent with previous established reports (15,16), VopA activity resulted in reduced levels of phosphorylated ERK at steady state (Fig.3). Strikingly, VopA markedly elevated the levels of phosphorylation of FAK at Tyr397

FIG 2 VopA disrupts epithelial homeostasis in the Drosophila intestine. (A) Schematic depiction of a transverse section of an adult Drosophila midgut. ISC,intestinal stem cell; EC, enterocyte; EB, enteroblast; CM, circular muscle; EE, enteroendocrine cells. Note that the enteroblast in adult Drosophila flies isequivalent to transit-amplifying cells in mammals. ISCs proliferate to generate enteroblasts. The enteroblasts then differentiate to either enterocytes or EEs,which form the gut epithelium in adult Drosophila flies. (B) Life span of adult Drosophila expressing VopA, mVopA, or dominant negative Basket (dnBsk)under myoIA-GAL4. Temperature-sensitive GAL80 (tsGAL80) was also included in each genetic background. Drosophila flies were propagated at 18°C untilthey were 2-day-old adults, whereupon they were moved to 29°C. Percent survival was recorded at regular intervals. Data are for 100 flies per group. Dataare representative of those from experiments undertaken in triplicate. (C) Immunostaining of 7-day-old adult Drosophila midguts of the indicated genotypesin panel B using an antibody against p-ERK. Images were captured by confocal microscopy at a �40 magnification. (D) Staining of DNA within the distalmidgut of Drosophila expressing VopA or mVopA under myoIA-GAL4. (A=) Enface image; (A�) transverse section generated by z-stack analysis of imagescaptured by confocal microscopy. (E) Immunostaining of 7-day-old adult Drosophila posterior midguts dissected from the indicated genotypes from panelB using an antibody against FAK phosphorylated at Tyr397 (p-FAKTyr397). Images captured by confocal microscopy at a �100 magnification. (F) Detectionof proliferating cells in the adult Drosophila posterior midgut by chase analysis. Seven-day-old adult Drosophila flies were fed EdU for 24 h, and cellular EdUincorporation was detected by confocal microscopy at a �20 magnification. For the images presented in panels C to F, the VopA-mediated phenotype wasdetected with 100% penetrance, and the images presented are representative of 10 intestinal dissections (n � 10) per group.

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and Tyr861, whereas, in contrast, VopA mediated reduced levels of FAK phosphoryla-tion at Ser910 (Fig. 3). In addition, VopA caused elevated levels of phosphorylation ofPaxillin at Tyr118, SHP-2 phosphorylation at Tyr542, and Src phosphorylation at Tyr416(Fig. 3). Together, these data show that VopA-mediated inhibition of ERK has markeddownstream influences on the activity of proteins that make up the FAC and associatedsignaling pathways. Importantly, these changes were detected at steady state and notfollowing serum starvation and pathway induction, as is commonly carried out to revealeffector protein activity on the cell.

VopA inhibits migration of cultured enterocytes. Focal adhesion complexes(FACs) are macromolecular protein assemblies that, through their dynamic assemblyand disassembly, mediate enterocyte migration (36). FAC assembly and disassembly arecontrolled by the phosphorylation and protein complex constituents, including FAK.We thus reasoned that VopA may have modulatory influences on enterocyte migration.Indeed, cultured SKCO15 cells, which are cells of a human intestinal epithelial cell (IEC)line, transfected with plasmids harboring vopA had a significantly reduced capacity torepopulate an area of the confluent cell monolayer damaged by a needle-inflictedscratch wound compared to cells transfected with the mutant and catalytically inactiveform, mvop (Fig. 4A to C). SKCO15 cells have a modest transfection efficiency, which weestimated to be about 30% in this experiment (Fig. 4C). Nevertheless, this transfectionrate was sufficient to elicit a potent inhibitory influence on the closure of a scratchwound in the scratch wound assay. Consistent with the findings presented in Fig. 3,immunostain analysis of transfected HEK293 cells revealed decreased levels of phos-phorylated FAK at Ser910 in VopA-expressing HeLa cells, showing that the activity ofVopA is not specific to one cell type (Fig. 4D). Together, these data show that VopAactivity has measurable influences on cell movement.

VopA inhibits JNK pathway activity and apoptosis in Drosophila. Our previousinvestigations showed that the S. enterica serovar Typhimurium-secreted protein AvrAdampens apoptosis by inhibiting JNK pathway signaling (2, 6). VopA has also beenshown to be a potent inhibitor of JNK signaling in cultured mammalian cell systems

FIG 3 VopA alters the phosphorylation state of proteins that make up the focal adhesion complex (FAC).The results of immunoblot analysis of lysates from cultured HEK293 cells transfected with plasmid vectorpCMV (control) or pCMV harboring myc-vopA or a mutant and catalytically inactive species of vopA,myc-mvopA (C181A), for 24 h are shown. Cell lysate (0.5 �g) was loaded into each well, and proteinabundance was assayed using antibodies against the indicated proteins. The data presented are represen-tative of those from three independent experiments (n � 3). p-Paxillin, phosphorylated Paxillin.

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(15). We thus investigated the extent to which VopA influences apoptosis in the fly. Tothis end, we induced JNK pathway activation and tissue apoptosis by constitutiveexpression of Eiger, the Drosophila ortholog of mammalian tumor necrosis factor alpha(TNF-�) (37). Eiger expression in Drosophila eye tissue caused an extreme-small-eyephenotype and elevated levels of phosphorylated JNK in eye imaginal disks of thirdinstar larvae (Fig. 5A), findings which are consistent with past reports (6, 37). Coexpres-sion of VopA with Eiger markedly rescued the small-eye phenotype (Fig. 5A) andinhibited the Eiger-induced phosphorylation of JNK in the developing eye, whereasmVopA expression exhibited no phenotypic change (Fig. 5A, bottom). A central regu-lator of JNK signaling is the mitogen-activated protein kinase kinase kinase (MAPKKK)family member Drosophila TAK-1 (dTAK-1). Constitutive expression of dTAK-1 is pro-apoptotic in Drosophila tissue. Similar to the findings obtained with Eiger, eye-specificexpression of dTAK-1 also resulted in a strong small-eye phenotype (Fig. 5B). Coex-pression of VopA with dTAK-1 markedly suppressed the small-eye phenotype, whereascoexpression of mVopA with dTAK-1 did not (Fig. 5B). These data indicate that VopAmediates its suppressive activity at the level of or downstream of dTAK-1. dTAK-1 is aMAPKKK that functions as a Jun N-terminal protein kinase kinase kinase (JNK-KK).Hemipterous (Hep) (mitogen-activated protein kinase kinase 4/7 [MKK4/7] in mammals)is the downstream MAPKK (Jun N-terminal protein kinase kinase [JNK-K]) in the JNKpathway. Expression of Hep or of Basket (Drosophila JNK) individually does not alter theeye phenotype. However, concurrent expression of Hep and Basket in the eye duringdevelopment is lethal at the pupal stage. Importantly, expression of VopA with Hep andBasket in the eye resulted in a viable adult Drosophila fly with a markedly rescued eye

FIG 4 VopA inhibits migration of cultured enterocytes. (A) Images of scratch wounds inflicted by a needle onconfluent monolayers of SKCO15 cultured cells transfected with plasmid vector pCMV (control) or with pCMVharboring myc-vopA or a mutant and catalytically inactive species of vopA, myc-mvopA (C181A). Wounds wereinflicted at 24 h posttransfection (time zero [T0]) (top). Repopulation of the wound area was assessed at 24 h afterwound infliction (time 24 h [T24]) (bottom). (B) Quantification of the percent closure of wounds inflicted by aneedle scratch shown in panel A. Data are represented as mean � standard error of the mean (nonparametricunpaired t test; ****, P � 0.0001; n � 8). (C) Immunostain analysis of cells at the wound edge in the assays describedin the legends to panels A and B using antibodies against myc to detect the transfection of myc-VopA, andmyc-mVopA. (D) Immunostain analysis of cultured HeLa cells transfected with pCMV-myc-vopA or pCMV-myc-mvopA using an antibody against FAK phosphorylated at Ser910 and an antibody against the myc tag. Whitearrows point to transfected cells expressing VopA or mVopA.

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FIG 5 VopA inhibits JNK/Bsk phosphorylation resulting from constitutive Eiger or dTAK expression. (A) Adult Drosophila eye phenotypes resulting fromconstitutive Eiger expression or from constitutive coexpression of Eiger and VopA, with the genotypes indicated (top), and immunostain analysis of larvaleye disks from the indicated phenotypes using antibodies against phosphorylated JNK (bottom). (B) Phenotypes of adult Drosophila eyes resulting fromconstitutive expression of dTAK-1 and from constitutive coexpression of dTAK-1 and VopA or mVopA, with the genotypes indicated. (C) Phenotypes ofadult Drosophila eyes resulting from constitutive expression of a constitutively active form of Hemipterous [hep(act)] with VopA, from constitutivecoexpression of Hemipterous (hep) and Basket (bsk), and from constitutive coexpression of Hemipterous, Basket, and VopA. (D) TUNEL analysis for thedetection of apoptotic cells in the posterior midgut of flies of the indicated phenotypes. (E) Numeration of TUNEL-positive cells in panel D. ****, P � 0.0001(n � 5). For panels A to C, representative images were observed in all eclosed adult flies of the indicated phenotype, indicating that the inhibitory effectof VopA on Eiger-induced JNK signaling in the eye occurred with 100% penetrance.

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phenotype (Fig. 5C). Furthermore, a constitutively active allele of Hemipterous [UAS-hep(act)] (with S326D and T330D amino acid replacements in the kinase activationloop) also mediated a rough-eye phenotype. This phenotype could not be reversed byVopA expression (Fig. 5C), indicating that VopA inhibits the JNK pathway at the level ofHep (MKK4/7). Similar to the natural loss of enterocytes at the tip of mammalianintestinal villi through anoikis, the Drosophila midgut cells undergo apoptosis (35).Consistent with these reports and as part of normal homeostasis in the Drosophilamidgut, sporadic terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick endlabeling (TUNEL)-positive cells were observed to be interdispersed between midgutenterocytes in control flies or flies with enterocyte-specific expression of mVopA (Fig.5D and E). In contrast, few or no TUNEL-positive cells were detected in VopA-expressingenterocytes (Fig. 5D and 5E). Thus, consistent with the observation that VopA inhibitsEiger-induced apoptosis in Drosophila eye tissue, VopA expression in Drosophila alsoinhibits natural anoikis apoptosis in the Drosophila midgut.

VopA inhibits apoptosis in cultured mammalian cells. To corroborate the anti-apoptotic activity detected in flies in mammalian cells, we activated the JNK pathwayin human cultured cells with plasmids harboring JNK1 and MKK4, which togetherinduce JNK phosphorylation and the ensuing cleavage of the apoptotic markerpoly(ADP-ribose) polymerase (PARP). Cotransfection of plasmids harboring vopA totallyabolished both MKK4-mediated JNK phosphorylation and PARP cleavage (Fig. 6A), thusindicating that VopA inhibits JNK activation and apoptosis at the level of MKK4 and/orJNK. In addition, cultured HeLa cells transfected with vopA had markedly less detectablecleaved PARP, which is a marker of apoptosis following the induction of cytotoxicity bythe addition of actinomycin D and TNF-� for 24 h into the culture medium (Fig. 6B).These data indicate that the VopA protein, in isolation, not only can suppress the stresssignaling JNK pathway but also can actively reduce markers of apoptosis in mammaliancells.

DISCUSSION

Here, we show the utility of the Drosophila model to discover potential phenotypeselicited by the VopA effector protein in the intestinal epithelia. We show that theactivity of VopA at inhibiting ERK MAPK signaling is conserved across kingdoms, i.e.,between the fly and cultured mammalian cells. Examination of the phenotype elicited

FIG 6 VopA protects mammalian cultured cells from apoptosis. (A) Immunoblot analysis of lysates fromcultured HEK293 cells transfected with the indicated plasmids, using antibodies against phosphorylatedJNK or myc. (B) Immunoblot analysis of lysates from cultured HeLa cells transfected with the indicatedplasmid before stimulation with actinomycin D (Ac.D) and TNF-� for 24 h, using an antibody againstcleaved PARP or myc. The data presented are representative results from three independent experiments(n � 3).

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by VopA expression in Drosophila enterocytes, together with the known cross talkbetween ERK signaling and the FAC, pointed toward examining the effect of VopA onproteins involved in cell movement. To this end, we show that VopA has a modulatingeffect on FAC proteins, which was confirmed by immunoblot analysis of culturedmammalian cells transfected with VopA and functionally in a scratch wound assaymeasuring cell migration. The Drosophila model also revealed that VopA can elicitpotent antiapoptotic activity in Drosophila tissue, which was also corroborated incultured mammalian cells.

To date, VopA-related studies have focused on identifying the inhibitory spectrumand biochemical mechanisms of VopA, with less attention being given to the patho-logical outcome of VopA activity within cells. This in part is due to the lack of asatisfactory system to model V. parahaemolyticus infection, although advances wererecently made to establish a mammalian model of disease (14, 26). The approach takenin many models is to infect subjects with the wild type or mutant variants of pathogens.The strength of using the Drosophila animal system is that a single protein can beectopically expressed in target model tissue in the context of a whole organism (38).This reductionist approach has already been utilized to identify the pathogenic out-come of other pathogen-secreted proteins on host signaling networks (5, 6, 39–41). Weshow that VopA expression in the Drosophila developing wing leads to altered tissuephenotypes, including the loss of wing vein formation and reduced cell growth,consistent with the known function of ERK signaling in wing development (30).However, the Drosophila wing is a tissue of determinate size and is thus not asatisfactory model for tissues that undergo constant regeneration and homeostasis, asoccur in the metazoan gut. The recent availability of a midgut enterocyte-specificmyoIA-GAL4 driver fly enabled us to directly express a secreted protein from an entericpathogen within the gut enterocytes of Drosophila. Furthermore, Drosophila is asuitable model organism due to the physiological similarities and developmentalsimilarities between the fly midgut and the mammalian intestine (31).

The VopA protein is secreted by V. parahaemolyticus, which is predominantly anextracellular pathogen, as determined by strong supporting data from an infant rabbitmodel of infection (26), although other reports suggest that it may also actively invadenonphagocytic cells (42). A critical step in its pathogenesis is the initial binding to thehost cell, where adhesion factors on the V. parahaemolyticus surface contact with hostenterocytes to facilitate secretion of effector proteins from the bacteria to the host cell.These adhesion factors include the constitutively expressed bacterial protein multiva-lent adhesion molecule 7 (MAM7), which is conserved in many Gram-negative patho-gens and which functions in establishing the immediate contact of V. parahaemolyticuswith host cells, before the upregulation of other adhesion proteins and the subsequentsecretion of virulence factors during pathogenesis (43, 44). At the same time, host cellsexpress surface pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs),that function as sentinels that detect microbe-associated molecular patterns (MAMPs).TLR-mediated sensing of specific MAMPs triggers MyD88- and TAK-1-dependent innateimmune NF-�B and JNK pathway activation, which induces the secretion of proinflam-matory cytokines or induces apoptosis, depending on the strength and duration of theMAMP signal (45). Therefore, strategies evolved by bacteria to subvert innate immunitywill enhance their chances of prolonged survival within the intestine and opportunitiesfor pathogenic activity.

Secreted effector proteins are one strategy evolved by pathogens to dampen theinnate immune response, and these effector proteins are known to inhibit the activa-tion of a number of cellular signaling pathways (46). As mentioned, members of theYopJ-like family of proteins have differential inhibitory effects on substrates at theMAPKK level (Fig. 1B). Moreover, MAPK pathways are known to cross talk with manyother signaling pathways, including the FAK pathway (47). We confirmed the secondaryeffects of ERK pathway blockade on Src-FAK signaling both in cultured cells and in theDrosophila midgut and thus show the effects of an effector protein on factors thatinfluence cell movement. This is significant because during intestinal homeostasis

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enterocytes of the intestine rapidly migrate from the bottom of the crypt to the villustip, with a transit time of about 5 days (48). Once at the villus tip, enterocytes undergoanoikis, where cells detach from the surrounding extracellular matrix (ECM) and areeliminated into the fecal stream. By extension, any bacterium that is attached to theenterocyte or that has invaded the enterocyte is also eliminated. We speculate thatinhibition of enterocyte migration would allow V. parahaemolyticus more time toremain attached to the enterocyte, thus increasing the chances of establishing a nichewithin the host. Similar to AvrA, which is secreted by S. enterica serovar Typhimurium(2, 5), VopA also has antiapoptotic activity both in cultured cells and in Drosophilaenterocytes. Our data show that the VopA-mediated inhibition of apoptosis blocks theapoptotic fail-safe mechanism signaled through the JNK pathway that cells mayundergo when they encounter an irreversible threat. Therefore, similar to slowing cellmigration, inhibition of enterocyte apoptosis also increases the chances of V. parahae-molyticus establishing a niche in the host. Collectively, our data show the ability ofVopA to influence specific MAPKK-mediated signaling, apoptosis, and cellular migrationin a eukaryotic in vivo system.

As well as VopA, the V. parahaemolyticus type III secretion system on chromosome2 (T3SS2) is known to secrete other effector proteins that are essential for colonizationand pathogenicity. Another V. parahaemolyticus effector protein is VopZ, which wasreported to inhibit the signaling of both the JNK and NF-�B pathways via its inhibitoryactivity on TAK-1 (49). The TAK-1 protein is a MAPKKK and is the upstream kinase thatphosphorylates the JNK pathway intermediates MKK4/7, also known as Hemipterous inDrosophila. Both VopA and VopZ therefore exhibit activity that inhibits functionallyrelated elements involved in the activation of JNK signaling. Interestingly, whereas theclosest homologues to VopA, namely AvrA, YopJ, and AopP, all have reported inhibitoryactivity on NF-�B at the level of IKK� (5, 6, 20), VopA, in contrast, apparently does notinhibit NF-�B pathway signaling (Fig. 1B). Therefore, V. parahaemolyticus may havenecessarily evolved the VopZ effector protein to target NF-�B. Indeed, many of the V.parahaemolyticus-secreted effector proteins likely function synergistically during patho-genesis, independently targeting key cell signaling pathway intermediates to subvertthe innate immune response and avoid detection and elimination by the host.

The utility of the Drosophila system is to make initial discoveries that may then betested in a relevant mammalian model. Thus, a limitation of the current study is that theactivity of VopA was not corroborated in a mammalian in vivo infection model, whichneeds to be performed. Nevertheless, by elucidating these VopA-mediated phenotypesin the fly and cultured cells, investigators now have a scientific premise on which toassay for them in a mammalian model. In addition, in light of the report suggesting thatV. parahaemolyticus actively invades nonphagocytic cells (42), it is probable thatinvasion of enterocytes lining the mammalian intestine villus by V. parahaemolyticusdoes not occur in each enterocyte. Rather, invasion likely occurs only in a small numberof cells, making such an analysis challenging and invasion difficult to detect in amammalian in vivo infection model. Finally, the information generated in this studymay be valuable, in light of the recent report of a study in which investigators exploitedthe inhibitory activity of AvrA on innate immunity in nanoparticles to treat inflamma-tory bowel disease (23). The nanoparticle study performed using AvrA revealed thatexpression of this class of protein at steady state in cells can by sufficient to dampengut inflammation. Identification of enteric or other diseases that can be treated bymodulating cell movement and apoptosis with VopA may point to a function for VopAin therapeutics.

MATERIALS AND METHODSPlasmids and constructs. Plasmids harboring the vopA or mvopA coding sequence were a gift from

Kim Orth. The vopA and mvopA coding sequences were cloned into pCMV-myc, creating pCMV-myc-vopAand pCMV-myc-mvopA, respectively. The DNA amplicon myc-vopA or myc-mvopA was cloned intopP[UAST] (a gift from Kevin Moses), creating pP[UAST]-myc-vopA or pP[UAST]-myc-mvopA, respectively.The creation of pCMV-mycJNK1 and pCMV-mycMKK4 was described previously (6).

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Drosophila lines. The vectors pP[UAST]-myc-vopA and pP[UAST]-myc-mvopA were microinjectedinto w1118 embryos, creating fly lines harboring UAS-myc-vopA and UAS-myc-mvopA, respectively. Otherfly stocks used included UAS-dTAK (50), UAS-eiger (37), UAS-hep(act), UAS-hep, and UAS-Bsk, all of whichwere obtained from the Bloomington Drosophila Stock Center. Driver line glass multiple reporter(GMR)-GAL4 (which specifically expresses GAL4 in omatidial cells during development) was a gift fromKevin Moses, c765-GAL4 (which expresses GAL4 in the wing pouch during development) was a gift fromDaniel Marenda, and engrailed-GAL4 (which expresses GAL4 in the posterior compartment of the wing)was obtained from the Bloomington Drosophila Stock Center. myoIA-GAL4 was a gift from ShigeoTakashima (51).

Antibodies and reagents for Drosophila immunostaining and immunohistochemistry. For im-munostaining procedures, third instar larvae imaginal eye or wing disks were dissected in phosphate-buffered saline and fixed in 4% paraformaldehyde for 20 min. The tissues were washed 3 times for 10 mineach time in 0.1% Triton X-100 and then placed in blocking solution (1% goat serum in 0.1% Triton X-100)for 30 min. The tissues were incubated in primary antibody overnight at 4°C with gentle rocking, beforebeing washed 3 times for 10 min each time in 0.1% Triton X-100 and incubated in the secondaryantibody for 1 h at ambient temperature. The antibodies used for Drosophila tissue immunostainingincluded anti-phospho-p44/42 MAPK (catalog number 4370), anti-phospho-FAK (Tyr397) (catalog num-ber 3283), and �-tubulin (9F3) rabbit monoclonal antibody (catalog number 2128) (all from Cell SignalingTechnology, Danvers, MA) and monoclonal anti-�-actin (clone AC-74; catalog number A2228; Sigma-Aldrich). DNA was stained with SYTO24 and TO-PRO-3 (Life Technologies, Grand Island, NY). Secondaryantibody incubations were done using goat anti-rabbit immunoglobulin-Cy5 or goat anti-mouseimmunoglobulin-fluorescein isothiocyanate (Jackson ImmunoReserach, West Grove, PA).

Immunoblot and immunostain analysis. For immunoblot analysis, human embryonic kidney(HEK293) cells grown in tissue culture to 50% confluence in 24-well plates were transfected with 0.5 �gof plasmid pCMV-myc, pCMV-myc-vopA, or pCMV-myc-mvopA, using 0.75 �l of the Lipofectamine 2000reagent per well. Control wells were transfected with a plasmid expressing green fluorescent protein inorder to quantify the transfection efficiency. At 24 h posttransfection, the transfected cells wereharvested in 100 �l Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004%bromphenol blue, 0.125 M Tris HCl; pH, approximately 6.8) per well. The amount of protein wasquantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce) before 0.5 �g ofprotein was loaded per well. The presence of specific proteins transferred to immunoblots was detectedusing the following antibodies: from Cell Signaling Technology (Danvers, MA), anti-phospho-p44/42MAPK (catalog number 4370), anti-phospho-FAK (Tyr397) (catalog number 3283), anti-FAK (catalognumber 3285), anti-phospho-Src (Tyr416) (catalog number 2101), anti-Src (catalog number 2109), antiphospho-Paxillin (Tyr118) (catalog number 2541), anti-Paxillin (catalog number 2542s), anti-phospho-SHP(Tyr542) (catalog number 3751s), and anti-SHP-2 (catalog number 3752); from BD Biosciences, anti-human c-Myc (catalog number 631206); and from Sigma-Aldrich, anti-phospho-FAK (pTyr861) (catalognumber F9176) and monoclonal anti-�-actin (clone AC-74; catalog number A2228). Immunoreactivespecies were detected using anti-rabbit immunoglobulin-horseradish peroxidase (HRP) or anti-mouseimmunoglobulin-HRP, followed by visualization with an ECL chemiluminescence detection reagent (GEHealthcare Biosciences, Piscataway, NJ). Experiments were conducted in triplicate, and the averagedensity of the band was quantified using ImageJ software. For immunofluorescence analysis, HeLa cellswere grown on coverslips submerged in cell culture dishes to 70% confluence, whereupon they weretransfected with 0.5 �g of plasmid pCMV-myc, pCMV-myc-vopA, or pCMV-myc-mvopA. At 24 h post-transfection, the coverslips were recovered from the dishes and the cells were fixed in 4% paraformal-dehyde. The levels of phosphorylated FAK at serine 910 within the cells were detected using antibodyanti-FAK Ser910 (44-596G; Bio-source Inc.) and anti-human c-Myc (catalog number 631206; BD Biosci-ences).

Detection of markers of apoptosis and cytotoxicity in cultured mammalian cells. HEK293 cellsgrown in tissue culture to 50% confluence in 12-well plates were transfected with combinations of 0.5 �geach of plasmid pCMV-myc, pCMV-myc-vopA, pCMV-myc-mvopA, pCMV-mycJNK1, or pCMV-mycMKK4,using 2.5 �l of the Lipofectamine 2000 reagent per well. The total amount of DNA transfected per wellwas normalized by the addition of pCMV-myc, to make a total of 1.5 �g DNA in the transfection mix. At24 h posttransfection, the transfected cells were harvested in 200 �l Laemmli sample buffer per well. Theamount of protein was quantified using a BCA protein assay kit (Thermo Scientific Pierce) before 0.5 �gof protein was loaded per well. The presence of specific proteins transferred to immunoblots wasdetected using the following antibodies: antibodies against cleaved PARP (Asp214) (human specific;catalog number 9541), phospho-SAPK/JNK (Thr183/Tyr185) (catalog number 9251), and anti-humanc-Myc (catalog number BD631206) (all from BD Biosciences). In addition, to measure the effect of VopAon cytotoxicity, HeLa cells were grown in 12-well dishes to 70% confluence, whereupon they weretransfected with 0.5 �g of plasmid pCMV-myc, pCMV-myc-vopA, or pCMV-myc-mvopA. At 24 h post-transfection, actinomycin D (final concentration, 1 �g/ml) and TNF-� (final concentration, 200 ng/ml)were included for a further 24 h. Cells were then harvested in 200 �l Laemmli sample buffer per well,protein was quantified using a BCA protein assay kit (Thermo Scientific Pierce), and 0.5 �g of protein wasloaded per well. The presence of specific proteins transferred to immunoblots was detected usingantibodies against cleaved PARP (Asp214) antibody (human specific; catalog number 9541; Cell SignalingTechnologies) and anti-human c-Myc (catalog number BD631206; BD Biosciences).

Cell migration assay. For the cell migration assay, human SKCO15 intestinal epithelial cells at 85 to90% confluence in 24-well plates were transfected with 0.5 �g of plasmid pCMV-myc, pCMV-myc-vopA,or pCMV-myc-mvopA, using 0.75 �l of the Lipofectamine 2000 reagent per well. At 48 h posttransfection,

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the monolayer of epithelial cells was scratched with a pipette tip connected to a vacuum line, and theinitial width of the scratch wound was photographed by microscopy, with a marked line being used asthe reference point. On the following morning (elapsed time, approximately 18 h), the scratch woundwas imaged again at the reference line and the covered area (the initial area subtracted from the finalarea) was determined using ImageJ software. The covered area was averaged across 10 replicates pertransfected plasmid and was represented as the percent scratch wound closure.

EdU incorporation assay. The EdU incorporation assay was done as described by Luo et al. (52).Briefly, pellets of dried yeast were made into a paste with water containing 1 mg/ml EdU. Aliquots of thepaste were put into vials containing fly food. After 24 h, Drosophila intestines were dissected and EdUincorporation into chromosomal DNA was detected using Click-iT EdU cell proliferation assays (LifeTechnologies, Grand Island, NY) according to the manufacturer’s protocol.

ACKNOWLEDGMENTSR.M.J. is supported in part by NIH grant R01DK098391. J.D.M. is funded by the

Crohn’s and Colitis Foundation of America (CCFA). B.S.R. is funded by training grantT32DK108735-03.

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