Identification of a conserved membrane localizationdomain within numerous large bacterialprotein toxinsBrett Geissler, Rehman Tungekar, and Karla J. F. Satchell1

Department of Microbiology-Immunology, Northwestern University, Chicago, IL 60611

Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved February 10, 2010 (received for review August 7, 2009)

Vibrio cholerae is the causativeagentof thediarrhealdiseasecholera.Many virulence factors contribute to intestinal colonization and dis-ease including the Multifunctional Autoprocessing RTX toxin(MARTXVc). The Rho-inactivation domain (RID) ofMARTXVc is respon-sible for inactivating the Rho-family of small GTPases, which leads todepolymerizationof theactin cytoskeleton. Basedonadeletionanal-ysis of RID to determine the minimal functional domain, we haveidentified a subdomain at the N terminus of RID that is homologousto themembrane targetingC1domainofPasteurellamultocida toxin.A GFP fusion to this subdomain from RID colocalized with a plasmamembranemarker when transiently expressedwithin HeLa cells andcanbefound in themembrane fractionfollowingsubcellular fraction-ation. This C1-like subdomain is present inmultiple families of bacte-rial toxins, including all of the clostridial glucosyltransferase toxinsand various MARTX toxins. GFP-fusions to these homologous do-mains are also membrane associated, indicating that this is a con-served membrane localization domain (MLD). We have identifiedthree residues (Y23, S68, R70) as necessary for proper localization ofone but not all MLDs. In addition, we found that substitution of theRID MLD with the MLDs from two different effector domains fromthe Vibrio vulnificus MARTX toxin restored RID activity, indicatingthat there is functional overlap between these MLDs. This studydescribes the initial recognitionofa familyof conservedplasmamem-brane-targeting domains found in multiple large bacterial toxins.

bacterial toxin | multifunctional autoprocessing RTX toxin | PMT | structuralmodeling | Vibrio cholerae

Bacterial pathogens use a wide array of secreted proteins tointoxicate mammalian cells and mediate disease. Many effec-

tors target a specific organelle, necessitating strategies to traffic tocertain subcellular locations after entry into the cytosol. Forexample, the cytolethal distending toxin active subunit, CdtB, uses ashortN-terminal nuclear localization sequence attached to traffic tothe nucleus (1). Similarly,manyType III secretion effectors containdistinct targeting signals:BteAuses a 130aaN-terminal sequence totarget lipid rafts (2); ExoS and YopE use internal 21 aa domains toassociate with the host membrane (3); and ExoU uses a C-terminalsequence to localize to the membrane (4). For each of theseeffectors, subcellular localization is determined by a specific aasequence separate from the catalytic site.Themultifunctional-autoprocessingRTX toxins (MARTX) are

large bacterial toxins composed of multiple effector domainsreleased by autoprocessing upon translocation into the host cellcytosol (5). The best characterized member of this family is theMARTX of Vibrio cholerae (MARTXVc), which is predicted to be4545 aa in length (6, 7). MARTXVc carries three effector domainsdelivered by autoprocessing of which two function to disrupt theactin cytoskeleton. G-actin is covalently cross-linked into oligom-ers by the MARTXVc actin cross-linking domain (ACD) (8),whereas theRho-inactivation domain (RID) reversibly inactivateshost cell RhoGTPases (9). Transfection of cells with a plasmidexpressing RID as a fusion with GFP induced cell rounding. Inaddition, when fused to the N-terminal portion of Anthrax toxinlethal factor (LFN) and delivered to cells by Anthrax toxin pro-

tective antigen (PA) (10), this domain was sufficient to induce cellrounding and Rho-inactivation (9).Proteinswith significant structural similarities toMARTXVc are

present in other pathogenic bacteria such as Vibrio vulnificus,Aeromonas hydrophila, Yersinia pestis, Clostridium difficile, Xen-orhabdus nematophila, and Photorhabdus luminescens. However,primary sequence comparisons indicate each toxin possesses adistinct collection of effector domains (reviewed in ref. 5). The V.vulnificus MARTX toxin (MARTXVv) contains four putativeeffector domains of unknown function (DUF) and a putative RIDbut no ACD (5). Three of the DUFs are homologous to otherbacterial toxin domains; DUF3 with the α/β-hydrolase family ofenzymes, DUF4 with the P. luminescensmakes caterpillars floppytoxins (11), andDUF5with theC2domain ofPasteurellamultocidatoxin (PMT) (5).In this work, we identify a conserved peptide sequence in 16

different bacterial toxins produced by both Gram-positive andGram-negative bacteria. Functional analysis of four of thesedomains reveals that these toxins use this sequence in a sharedstrategy for plasma membrane targeting, despite the variedmechanisms of host cell intoxication employed by each toxin.

ResultsDeletion Analysis of MARTXVc RID. The MARTXVc RID (RIDVc)corresponds to aa 2552–3099 of the MARTXVc holotoxin (9). Toestablish theminimum functional size ofRIDVc, a genetic deletionanalysis on the domain was performed. Based on a CLUSTALWalignment of the putative RID sequences of MARTX toxins fromV. cholerae, V. vulnificus, Vibrio anguillarum, X. nematophila, andXenorhabdus bovienii (Fig. S1), we generated threeN-terminal andthree C-terminal truncations in the eukaryotic expression plasmidpRID-GFP (Fig. 1A). The truncations were chosen to removeregions of significant (>60%) sequence divergence among thehomologs, because these regions are likely not required for RIDfunction. Transfection of pRID2650–3099-GFP, pRID2721–3099-GFP, or pRID2552–3085-GFP into HeLa cells caused cell roundingindistinguishable from pRID2552–3099-GFP but further deletionsfrom either terminus of RIDVc abolished cell rounding (Fig. 1AandFig. S2). Together, these data indicated that residues within aa2721–3085 are necessary and sufficient for RIDVc activity whenexpressed in mammalian cells as a GFP-fusion protein.In an attempt to confirm that cell rounding was due to RhoA

inactivation by the shorter RID fragment, an LFN-fusion to res-idues 2721–3099 of RIDVc was constructed and the amount ofactive GTP-bound RhoA within HeLa cells was monitored fol-lowing PA-mediated delivery of purified LFN-RID2721–3099.

Author contributions: B.G. and K.J.F.S. designed research; B.G. and R.T. performed re-search; B.G. and K.J.F.S. analyzed data; and B.G. and K.J.F.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: k-satchell@northwestern.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0908700107/DCSupplemental.

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Unlike the nearly 100% cell rounding caused by PA+LFN-RID2552–3099, incubation with PA+LFN-RID2721–3099 did notround HeLa cells even when added at a 1200× excess over theamount sufficient for LFN-RID2552–3099 toxicity (Fig. 1A and Fig.S3A). These results suggested that either the LFN fusion inhibitsthe functionality of RID2721–3099 or that the N terminus of RIDVccontains an essential element that is required for functionalactivity when RIDVc is delivered to the cell as a protein but isdispensable when the protein is overexpressed within the cell.

Residues 2561–2645 of MARTXVc Target the Plasma Membrane. Tofurther characterize the N-terminal sequence of RIDVc, the peptidesequence corresponding to aa 2552–2720 was analyzed using PSI-BLAST (12). This analysis showed that MARTXVc aa 2561–2645shares homology with the N terminus of all known RIDs from otherMARTX toxins as expected (Fig. S1), and also the N terminus ofDUF5 from MARTXVv (aa 3591–3669, hereafter referred to asVvDUF5). The DUF5 effector domain is also present withinMARTXtoxins fromVibrio splendidus,A.hydrophila,P. luminescens,and X. nematophila (5, 13). The N terminus of DUF5 has beenpreviously noted as sharing homology with the C1 domain of PMT(5). In turn, PMTC1 is known to share structural homology with theN terminus of C. difficile Toxin B (TcdB) and other closely relatedclostridial glucosyltransferase toxins (CGT) (14). A pair-wise align-ment of the 19 identified sequences showed a high degree of sim-ilarity throughout the region (44–89%), indicating that thesemay be

shared functional domains important for activity (Fig. 1C). All 19 ofthese domains were found just to the N-terminal side of three dif-ferent classes of effector domains (RID, PMT, and CGT) withinvarious large toxins.PMT C1 was previously shown to localize GFP to the plasma

membrane of 293T cells when ectopically expressed (14). To testif the C1-related domain from RIDVc also facilitates membranelocalization, aa 2561–2645 were transiently expressed in HeLacells as a fusion to either the N or C terminus of GFP. A plasmidexpressing the myristylation site and the polybasic membranetargeting sequence present within the first 15 aa of c-Src fused tomCherry (S15-mCherry) was used as a marker for the plasmamembrane (15–18). This portion of c-Src mediates a directassociation with host cell membrane components and has beenshown in macrophages to colocalize with a biomarker for phos-phatidylserine (PS) (15, 19, 20).When the plasmids expressing theN-terminal GFP-fusion (2561-2645-GFP) and S15-mCherry werecotransfected into HeLa cells, an overlap of both fluorescencesignals at the cell periphery was observed, particularly comparedto GFP alone where there was very little overlap with mCherry(Fig. 2A). Both GFP and 2561–2645-GFP showed fluorescencewithin the nucleus due to passive transport across the nuclearmembrane; however, GFP alone was rarely observed at themembrane colocalized with S15-mCherry (Fig. 2A and Table 1).Transfection of a plasmid expressing MARTXVc aa 2561–2645attached to the C terminus of GFP (GFP-2561-2645) did not

Fig. 1. Deletion analysis of RIDVc identifies a broadly conserved domain. (A) Portions of the N- and C-termini of RIDVc were deleted from pRID-GFP or LFN-RID.Each truncated RIDVc was tested for its ability to cause cell rounding after transfection (GFP-fusions) or delivery to the cell with PA (LFN-fusions). (B) Cartoon ofthe entire PMT toxin (boxed, PDB ID: 2EBF) with the PMT C1 domain (blue) overlaid with a model of MLDVcRID (red). (C) Alignment of 19 putative MLDs fromvarious bacterial toxins: see Table S1 for accession numbers and protein details. Shading shows aa conservation and residue description: green = hydrophobic;orange = polar; blue = basic; red = acidic. Arrows indicate aa mutated to either Ala or a similar residue on MLDVcRID or MLDVvDUF5.

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display any distinct localization pattern within HeLa cells oroverlap with S15-mCherry and was indistinguishable from GFPalone by microscopy (Table 1).In support of the microscopy, membrane fractionation showed

33 ± 8% of the 2561–2645-GFP fusion protein (MLD-GFP) waspresent in the membrane fraction, whereas GFP-2561–2645 (GFP-MLD) was rarely at the membrane and was not significantly differ-

ent in localization from GFP alone (Fig. 2 B and C and Table S2).Immunoblotting with anti-GFP antibody confirmed stable fusionproteins were produced for all fusions tested and thus failure tolocalize to the membrane was not due to protein degradation.Altogether, these data support that aa 2561–2645 ofMARTXVc is apeptide sequence required for plasmamembrane targeting and thusrepresents a bacterial toxin membrane localization domain (MLD).

MLD Is a Functionally Conserved Domain Found in Many BacterialToxins. To test if conserved C1-like MLDs from other toxinslikewise function in membrane targeting, HeLa cells were trans-fected with plasmids expressing the putative MLDs from VvRID(MLDVvRID), VvDUF5 (MLDVvDUF5), and TcdB (MLDTcdB)fused to GFP. Similar to MLDVcRID-GFP, cells ectopicallyexpressing theMLDVvDUF5 andMLDTcdB fusions displayed clearmembrane localization of GFP by epifluorescence microscopy(Fig. 3A) andwere identified in themembrane by immunoblottingwith anti-GFP antibody (Fig. 3 B and C). Fluorescence intensitydistribution plotting for MLDVcRID, MLDVvDUF5, and MLDTcdBshowed maximum GFP-signal at cell–cell junctions compared toGFP alone, which showed nearly uniform fluorescence through-out the cell with maximum fluorescence occurring randomly (Fig.3D and Fig. S4). This biased localization suggests that theseMLDs may preferentially target cell–cell junctions (Table 1 andFig. S4). Intriguingly, even with 88% aa identity and 93% sim-ilarity with MLDVcRID, MLDVvRID showed only weak membranelocalization and very little junction targeting (Fig. 3, Fig. S4, andTable 1). By contrast, MLDVvDUF5-GFP showed an apparenthigher affinity for the plasmamembrane (Fig. 3C andD) and wasalso localized to vesicles throughout the cells (arrows in Fig. 3A).Despite its clear association with the plasma membrane by epi-fluorescence microscopy (Fig. 3 A and B and Table 1), the per-centage of MLDTcdB found in the membrane upon fractionationwas less than MLDVcRID-GFP or MLDVvDUF5-GFP but wasstatistically greater than GFP alone (Fig. 3 C and D). These dataindicate that although each MLD has a function in targetingproteins to the plasma membrane, the relative amount of proteinlocalized, the stability of each protein at the membrane uponfractionation, the specificity for cell–cell junctions, and the pat-tern of recycling varied among the four MLDs tested.

Fig. 2. RIDVc aa2561-2645 is sufficient to drive GFP to theHeLa cell plasma membrane. (A) HeLa cells transfected witha plasma membrane marker (S15-mCherry) and either GFPor MARTXVc aa2561-2645-GFP (MLD-GFP) were imaged bydeconvolution microscopy. Arrow shows overlap at theplasma membrane of mCherry and GFP signals. (B) Repre-sentative anti-GFP immunoblot of three separate experi-ments following membrane fractionation of HeLa cellstransfected with the indicated constructs; S, soluble; M,membrane. (C) The average percentage of signal (+/− thestandard deviation) in the membrane fraction followingfractionation was determined by densitometry of immu-noblots for each MLD-GFP construct (n = 3). Tabulated rawdensitometry measurements are found in Table S2. A Stu-dent’s t test was employed to determine the statisticalsignificance of the difference between the amount of GFPversus each GFP-fusion in the membrane fractions; *, sig-nificantly different from GFP alone (P < 0.05).

Table 1. Localization of each GFP fusion to HeLa cell–celljunctions after ectopic expression

MLD constructBright

junctions*Total

junctions% Brightjunctions

GFP 3 40 8%GFP-VcRID 2 31 6%VcRID-GFP 34 38 89%VvRID-GFP 7 31 23%VvDUF5-GFP 43 43 100%TcdB-GFP 19 19 100%VcRID-GFP mutants†

Y23F 0 21 0%Y23A 2 29 7%S68T 2 21 10%S68A 1 25 4%R70K 4 49 8%R70A 3 40 8%ΔH1-2 1 15 7%

VvDUF5-GFPmutants†

Y23F 27 27 100%Y23A 0 34 0%S62T 11 71 15%S62A 7 56 13%R64K 0 36 0%

*HeLa cell–cell junctions were scored for fluorescence intensity across a linebisecting the cell–cell junction. Bright junctions exhibited a single central peakof fluorescence intensity (Fig. 3B and SI Materials and Methods). Data are theaggregate counts of the total junctions scored for at least three independentexperiments.†Representative images for site-directed mutants can be viewed in Fig. S5.

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Structural Requirements for Membrane Localization. Analysis of thePMT (PDB ID: 2EBF), TcdB (PDB ID: 2BVL), and TcsL (PDBID: 2VL8) structural models and a predicted model of MLDVcRID(Fig. 1B) shows the conservedMLDregions consist of fourα-helices(designated H1-H4) connected by three short loop regions (des-ignated L1-L3) (Fig. 1 B and C). To further test the structuralrequirements for proper membrane targeting, the first 30 aa ofMLDVcRID, corresponding to H1 and most of H2, were deletedfrom MLDVcRID-GFP and the cellular location was monitoredfollowing transfection. This deletion prevented MLDVcRID-GFPlocalization to cell–cell junctions (Table 1), suggesting that all fourhelices are required for proper membrane targeting.Fig. 1C shows that despite high divergence of sequence among

the various MLDs, nine residues are conserved in at least 90% ofthe homologs with only three residues 100% identical. Accordingto the PMT, TcdB, and TcsL structural models, Leu8, Ala12,Leu30, Leu54, Tyr61, and Ala73 (numbered according to theirlocation on MLDVcRID) lie buried within the hydrophobic coreformed by bundling of the four helices. These residues likelyhave conserved function to maintain structural integrity. Thethree 100% identical residues, Tyr23, Ser68, and Arg70, are nearor within loops L1 and L3 and are oriented near each other onthe same end of the helical bundle. Electrostatic potential pre-dictions of the known and modeled MLD structures show thatthe interface formed by L1 and L3 appears to form a positivelycharged surface possibly involved in either protein or membraneinteractions (Fig. 4).The potential importance of the three 100% identical residues

for membrane localization was tested by altering each residue onMLDVcRID and MLDVvDUF5 to either an Ala or to a closely con-served residue (Tyr-Phe, Ser-Thr, Arg-Lys). Epifluorescence andconfocal microscopy showed that substitution of any of these threeresidues on MLDVcRID abrogated GFP localization to cell–celljunctions or to themembrane (Table 1 andFig. S5B–G). Similarly,substitution of the homologous residues onMLDVvDUF5-GFPalsoabolished membrane localization (Table 1 and Fig. S5 H–M).However, MLDVvDUF5-GFP localization was decreased by muta-tion ofTyr23 toPhe (Fig. S5J). These results indicate that althoughplasma membrane localization is a conserved function of these

domains, only the Ser and Arg residues within L3 are absolutelyessential, suggesting that other requirements and specificities forlocalization are determined by nonconserved residues.

Functional Complementation Between the MLDs. The variance ofrequirements for conserved residues and the extent of sequencedivergence of exposed residues of the various MLDs suggest thateach MLD may be uniquely paired with its cognate toxin. To testif one MLD can functionally complement another, chimerictoxins were generated in which MLDVcRID on LFN-RIDVc wasreplaced with either MLDVvRID or MLDVvDUF5, resulting inLFN-MLDVvRID-RIDVc or LFN-MLDVvDUF5-RIDVc, respec-tively. When applied along with PA, either chimera roundedHeLa cells indistinguishably from LFN-RIDVc after incubationfor 3 h. (Fig. S3A).A time course of cell rounding was then performed, comparing

the percentage of HeLa cells that were round following incubationwith varying concentrations of LFN-MLDVcRID-RIDVc, LFN-MLDVvRID-RIDVc, LFN-MLDVvDUF5-RIDVc, or LFN-ΔMLD-

Fig. 3. MLDVcRID homologs localize to the HeLa cell plasma membrane and at cell–cell junctions. (A) HeLa cells were transfected with the indicated MLD-GFPplasmid then fixed and imaged by epifluorescence microscopy 24 h later. (B) Representative anti-GFP immunoblot following membrane fractionation of HeLacells transfected with the indicated constructs; S, soluble; M, membrane. (C) Densitometry was used to determine the average percentage of signal (+/− thestandard deviation) (n ≥ 3) in the membrane fraction for the indicated construct as was done in Fig. 2C. Tabulated raw densitometry measurements are foundin Table S3. (D) The average fluorescence intensity measurement of cell–cell junctions (white lines in A) was determined for each construct using ImageJ (SIMaterials and Methods) (n ≥ 2 junctions from 3 independent transfection experiments).

Fig. 4. The L1-L3 interface of each MLD displays a positive charge. (A)Cartoon representation of MLDPMT for orientation of the diagrams in B. (B)Electrostatic potential predictions for two faces of the crystallized MLDs(PMT, TcdB, and TcsL) and the modeled MLDs (VvDUF5, VcRID, and VvRID).All of the predictions indicate a net positive charge (blue) at the interfaceof L1 and L3 although the remainder of the helical bundles contain largepatches of negative charge (red).

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RIDVc. Both LFN-MLDVvRID-RIDVc and LFN-MLDVvDUF5-RIDVc were able to fully intoxicate cells similar to LFN-RIDVc atconcentrations above0.1 nMand the time required for cell roundingwas not altered (Fig. S3B). Therefore, despite the differences inmembrane localization of the individual MLD constructs whenfused to GFP, replacing MLDVcRID with another MLD had a lessthan 10-fold effect on the dynamics of LFN-RIDVc-mediated cellrounding when delivered to HeLa cells using PA.

DiscussionThe ability of a bacterial toxin to identify the location of its cellulartarget is essential for toxic activity. In this work, we describe thepresence of a conserved peptide sequence within 16 large bacterialprotein toxins capable of targeting known and putative effectordomains within these toxins to the plasmamembrane. Interestingly,the MLD is found associated with three distinct effectors: RID,typified by the RID of MARTXVc and also found in six otherMARTX toxins; PMTC2, typified by PMT and present asDUF5 infive MARTX toxins; and CGT, typified by C. difficile TcdB andfound in four other related toxins. Each of these effectors is knownto specifically target proteinspresent at themembrane.ThePMTC-terminal complex deamidates heterotrimeric G proteins via the C3domain attached to C1/C2 (21), CGTs glucosylate Rho familyGTPases (22), and RID inactivates Rho family GTPases althoughpotentially by an indirectmechanism (9). This domain appears to beunique to these toxins and is different from the known membranelocalization sequences of T3SS effectors (BteA, ExoS, YopE,ExoU) (2–4).Functional analysis of representatives of each class of MLD

identified in this study suggested that, although they show similarability to localize GFP to the plasma membrane and MLDs cansubstitute for another in a chimeric toxin, the question that arisesis whether an MLD is functional because it has specificity forlipids in membranes or because it targets host proteins that arepresent at the membrane. Previous studies support a mechanismof direct lipid association. Clostridium sordellii TcsL has beenshown to bind liposomes enriched for PS (23), an anionic lipidpreferentially associated with the cytosolic leaflet of the plasmamembrane (19). Both binding to liposomes and Rac-glucosyla-tion activity of TcsL were stimulated by PS-enriched liposomes,dependent upon the first 18 aa of the toxin, corresponding to H1and L1 of the MLD. However, TcdB showed less selectivity forPS and was active also in liposomes enriched for phosphatidyl-glycerol. In our study, TcdB showed a weaker, but significantassociation with membranes such that localization observed invivo following ectopic expression may have been dissociatedfrom the membrane during sample preparation. These dataindicate that although the MLDs may directly target lipids, theydo so with varying affinity.Further evidence supporting direct lipid association comes

from analysis of the crystal structures of PMT, TcdB, and TcsLand structural modeling of MLDVcRID and MLDVvDUF5. Thethree 100% identical residues identified as essential for properlocalization of MLDVcRID and MLDVvDUF5 are all found neareach other, with their side chains directed inward rather thanexposed to solvent. Our data suggest that the Tyr, Ser, and Argresidues may be directly involved in L1-L3 interactions requiredto maintain the overall structure of the four-helical bundle. Thefolding of MLDVvDUF5 may be less dependent on the Tyr residueitself but rather the size of the residue in its position, therebyallowing it to be substituted for Phe with little effect on mem-brane association. The defect in localization of the Ser68 andArg70 mutants in both MLDVcRID and MLDVvDUF5, even whenchanged to Thr or Lys, further emphasizes that the interactionsbetween L1 and L3 may be essential to proper localization.Assessment of the electrostatic potential of this region indicatesthat all of the MLDs with a known structure are positivelycharged at this end. Three-dimensional models constructed for

MLDVcRID, MLDVvRID, and MLDVvDUF5 also suggest theseMLDs are positively charged at the L1/L3 face (Fig. 4B). Thus,similar to the polybasic N terminus of Src, the positive surface ofthese MLDs may directly interact with the negatively chargedacid lipids such as PS, at the plasma membrane (16, 19). As afurther example, annexin, which is also known to specificallylocalize to PS lipids, is enriched in tight junctions suggestingthere is an enrichment of these lipids at this site (24), providingjustification for the observation that MLD localization isenhanced at cell–cell junctions.An alternative model to explain the membrane localization is

that the MLDs bind host membrane proteins. Because the dif-ferent classes of MLDs show apparent distinct affinity formembrane, it is possible they target different proteins. Becausethe genetic requirement for localization may be limited to Ser68and Arg70, it is possible that binding involves diverse residues atthe L1/L3 loops or structural elements elsewhere on the helicalbundle. The interface between the four helices includes a largehydrophobic patch. Similar patches in helical bundles have beenshown to participate in protein–protein interactions, for examplethe ability of focal adhesion kinase to bind paxillin (25). Alter-natively, differential affinity or binding to different proteins maydepend upon the divergent L2-H3 region of each MLD. Theamino acid composition of this region is highly variable amongthe homologs, with many of the MLDs lacking up to six residuesfrom this region alone (Fig. 1C). The composition of the MLD inthe L2-H3 region is more conserved within the subgroups ofMLDs, an analysis that would suggest that interactions areeffector related. However, this concept is contradicted by thefinding that MLDVvDUF5 and MLDVvRID were able to comple-ment the activity of MLDVcRID, suggesting that the MLDs arenot matched to their cognate toxin. Thus, although it is possiblethat protein–protein interactions are important for function atthe membrane, the proteins targeted are either similarly local-ized themselves or the interaction is with different faces of thesame target protein.Interactions between the toxin activity domains and the target

membrane protein certainly also contribute to binding specificity.Our data shows that RID2721–3085 (without an MLD) was func-tional and able to round cells, but only when produced at highlevels within the cell following transfection. Similarly, purifiedTcsLΔ18, although not toxic to cells after microinjection, wasable to glucosylate soluble Rac in vitro (23). The toxic activity ofPMT was also significantly decreased, but not completely abol-ished, when the C1 domain was deleted (14, 26). We believe thatthe high amount of RID2721–3099-GFP produced in the cell aftertransfection was sufficient to locate its cellular binding partner,despite not being directly targeted to the plasma membrane.However, the necessity of the individual MLDs for functionalityof each full-length toxin remains to be determined.The ability of the chimeric LFN toxins to fully mimic LFN-

RIDVc activity suggests that the enzymatic activity of RIDVccould be the limiting factor in the dynamics of cell rounding.Accordingly, despite a possibility of MLDVvDUF5 to enhance themembrane association of RID, cell rounding did not occur morerapidly. Furthermore, although MLDVvRID did not significantlyincrease GFP membrane targeting, a sufficient amount of LFN-MLDVvRID-RIDVc required for RID-mediated cell roundingmay have been properly localized to the membrane, therebyallowing intoxication to proceed indifferently from LFN-RIDVc.Discovery of this conservedMLDadvances our understanding of

the biology of theMARTX toxins as awhole aswell as the strategiesmany pathogenic Vibrios and emerging pathogens use to intoxicatehost cells. Because this MLD is present in multiple families ofbacterial toxins suggests a conserved mechanism for targeting pro-tein toxins to the mammalian cell plasma membrane.

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Materials and MethodsConstruction of the Rho-Inactivation Domain (RID) Deletion Plasmids. Frag-ments of V. cholerae rtxA encoding for the aa segments of RID indicated inFig. 1Awere amplified by PCR and inserted into pEGFP-N3 (Clontech) to yieldthe RID-GFP plasmids. Alignments were performed using either CLUSTAL W(27) and highlighted using TEXTSHADE through the Biology Workbench 3.2server (http://workbench.sdsc.edu) or KAlign (http://www.ebi.ac.uk/Tools/kalign/) (28) and manually fitted to match secondary structure predictions.

Construction of Membrane Localization Domain GFP-Fusions. The DNA corre-sponding to aa 2561–2645 of V. cholerae rtxA [according to the Lin sequence(7)], 2377–2461and 3591–3669ofV. vulnificus rtxA, and 1–83ofC. difficile tcdB,were amplified by PCR and inserted into either pEGFP-N3 or pEGFP-C3 (Clon-tech). Pointmutations on pMLDVcRID-GFP and pMLDVvDUF5-GFPwere generatedusing the Quikchange XL II site-directed mutagenesis kit (Stratagene).

Purification and Application of LFN-Fusion Proteins. PA, LFN-RIDVc, LFN-ΔMLD-RIDVc, LFN-MLDVvRID-RIDVc, and LFN-MLDVvDUF5-RIDVc were all purified as inSheahan and Satchell (9) using an ÄKTA Purifier. Purity of each protein wasassessed by SDS/PAGE. Intoxications were performed as in (29) with eitherPBS or 28 nmol PA and the indicated amount of LFN-fusion protein added toHeLa cells.

Microscopy.HeLa cells were grown to approximately 70% confluence on acid-washed coverslips in 12-well dishes then transiently transfected using FuGeneHD (Roche) following the manufacturer’s protocol. Coverslips were washedwith PBS and fixed in 3.7% formaldehyde in 100 mM Pipes pH 7.4 thenmounted onto glass slides (18). Slides were imaged for epifluorescence (Fig.3, and Figs. S2 and S3) with an inverted Leica DMIRE2 microscope with a CCDcamera, for deconvolution (Fig. 2) with a Deltavision microscope and soft-

ware (Applied Precision) (18), or for confocal microscopy (Fig. S5) with a ZeissLSM510-Meta. Images were then processed and compiled using AdobePhotoshop CS3 Extended. Details for cell–cell junction quantification can befound in SI Text and Fig. S4.

Membrane Fractionation and Western Blotting. Transfected HeLa cells grownin 6-well dishes were divided into cytosolic and membrane fractions asdescribed previously (9). Equivalent volumes of each fraction were separatedby 15% SDS/PAGE and transferred to nitrocellulose membranes. GFP andGFP-fusions were detected with GFP-specific antiserum conjugated tohorseradish peroxidase (Miltenyi Biotec). Membranes were developed withSupersignal West Pico chemiluminescent substrate (Pierce). Band intensitieswere determined using National Institutes of Health ImageJ 1.40g. Raw datafor densitometry measurements can be found in Tables S2 and S3.

Molecular Modeling. The MLD sequences from VcRID, VvDUF5, and VvRIDwere input into the GENO3D server (http://geno3d-pbil.ibcp.fr) for 3Dstructure modeling (30). The structures of TcdB, PMT, and TcsL were used astarget templates for all models. Structure alignments and electrostaticpotential predictions were performed using MacPymol (Delano Scientific).

ACKNOWLEDGMENTS. We thank K. Prochazkova and E. Campbell fortechnical assistance and T. Hope for the S15-mCherry plasmid and helpfulinsights in the initial stages of the project. This work was supported byNational Institutes of Health award AI051490 and an Investigator in thePathogenesis of Infectious Disease award from the Burroughs WellcomeFund (to K.J.F.S.). B.G. was supported by an Institutional National ResearchService Award Postdoctoral Research Fellowship T32-AI007476-11 and aNational Research Service Award Postdoctoral Research Fellowship F32-AI075764-01A2.

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