Identification of Vibrio cholerae Type III Secretion System Effector Proteins

Abstract

AM-19226 is a pathogenic O39 serogroup Vibrio cholerae strain that lacks the typical virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) and instead encodes a type III secretion system (T3SS). The mechanism of pathogenesis is unknown, and few effector proteins have been identified. We therefore undertook a survey of the open reading frames (ORFs) within the ∼49.7-kb T3SS genomic island to identify potential effector proteins. We identified 15 ORFs for their ability to inhibit growth when expressed in yeast and then used a β-lactamase (TEM1) fusion reporter system to demonstrate that 11 proteins were bona fide effectors translocated into HeLa cells in vitro in a T3SS-dependent manner. One effector, which we named VopX (A33_1663), is conserved only in V. cholerae and Vibrio parahaemolyticus T3SS-positive strains and has not been previously studied. A vopX deletion reduces the ability of strain AM-19226 to colonize in vivo, and the bile-induced expression of a vopX-lacZ transcriptional fusion in vitro is regulated by the T3SS-encoded transcriptional regulators VttR_A and VttR_B. An RLM1 yeast deletion strain rescued the growth inhibition induced by VopX expression, suggesting that VopX interacts with components of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway. The collective results show that the V. cholerae T3SS encodes multiple effector proteins, one of which likely has novel activities that contribute to disease via interference with eukaryotic signaling pathways.

Vibrio cholerae is the etiologic agent of the severe and potentially lethal diarrheal disease called cholera (31, 49). Although over 200 different serogroups of V. cholerae have been identified, only O1 and O139 serogroup strains cause epidemic and pandemic disease (5, 6, 16). In pathogenic O1 and O139 serogroup isolates, the virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) are essential, and their expression is controlled by a transcriptional cascade regulated primarily by the transmembrane protein ToxR (36). Strains belonging to other serogroups can also cause disease and are collectively referred to as non-O1/non-O139 serogroup strains. Although they are typically associated with sporadic diarrheal disease, reports of increased incidence of non-O1/non-O139-associated disease suggest that these strains warrant increased attention and may be an emerging threat (17, 47). However, the pathogenic mechanisms of non-O1/non-O139 strains are not as well studied as those of O1 and O139 strains. Recent reports suggest that a type III secretion system (T3SS) is present in a subset of non-O1/non-O139 isolates and represents an important virulence mechanism for some V. cholerae strains (4, 8, 12, 56).

T3SSs are commonly found in pathogenic, Gram-negative bacteria, such as Yersinia, Salmonella, Shigella, enteropathogenic/enterohemorrhagic Escherichia coli (EPEC/EHEC), and Pseudomonas. However, T3SS conservation among species is typically limited to the components of the structural apparatus (19, 48). The proteins delivered through the apparatus to the eukaryotic host cell (effector proteins) are dissimilar in their amino acid sequence; each species manufactures a different set of effector proteins that constitute a “pathogen-specific” repertoire of virulence factors. Once translocated, effector proteins can perform a wide variety of functions to promote pathogenesis, including modulation of host cell signaling pathways, reorganization of the host cell cytoskeleton, bacterial attachment, invasion of the host cell, and evasion of immunological responses (9, 26). Disease thus results from the concerted action of multiple effector proteins that subvert host cellular machineries and disrupt homeostasis (9).

Some T3SS-positive bacteria synthesize as many as 30 effector proteins, although others seem to possess just a few (13, 18, 23, 37, 42, 43, 58). To date, only one effector, VopF, has been identified and studied in V. cholerae. The VopF protein is a unique effector protein containing both formin homology 1-like (FH1-like) and WASP homology 2 (WH2) domains. The translocation of VopF into host cells alters the actin cytoskeletal organization, and both FH1-like and WH2 domains are required for actin nucleation and polymerization activity (56). Interestingly, parts of the V. cholerae T3SS genomic island are most similar in gene organization and protein coding content to the T3SS2 in Vibrio parahaemolyticus strain RIMD2210633, and a VopF homolog (VopL) has been identified in V. parahaemolyticus (35). Both vopF and vopL are located in the central “core” region of the T3SS island that encodes the structural proteins of the apparatus, this region is conserved in V. parahaemolyticus, and numerous open reading frames (ORFs) encoding hypothetical proteins are interspersed among the structural genes. The 5′ and 3′ flanking sequences are less conserved between the two species, and many of the encoded products are hypothetical proteins. In other pathogens, the T3SS effector proteins are encoded within the same genomic region or locus as the T3SS structural components. It is therefore likely that the V. cholerae T3SS island encodes other effector proteins (in addition to VopF), both within the core region and in the species-specific flanking regions.

Pathogen-specific functions are therefore typically mediated via unique effector proteins. In some cases, conserved domains may be present and provide a clue to effector protein function (e.g., VopF). However, effector proteins characteristically share little or no homology to other proteins in current databases, thus presenting a challenge in their identification. To identify proteins that belong to the effector repertoire of V. cholerae, we used Saccharomyces cerevisiae yeast as a surrogate host for effector protein expression. Lesser and Miller reported in 2001 that the expression of bacterial effector proteins in S. cerevisiae can serve as a model system to study the molecular mechanisms of effector functions (34). Others have reported similar results, and yeast provides significant benefits over mammalian cell culture, given its genetic tractability and well-studied signaling pathways (7, 10, 46, 51, 53-55). We focused our studies on strain AM-19226, a clinically isolated O39 serogroup strain of V. cholerae whose genomic sequence analysis led to the identification of the V. cholerae T3SS, and screened a subset of ORFs found within the T3SS island for their ability to induce growth defects when expressed in S. cerevisiae. AM-19226 proteins whose expression resulted in a growth defect in yeast were then tested for their ability to be translocated into mammalian cells in vitro in a T3SS-dependent manner, which would identify them as bona fide effector proteins. We present data indicating that the AM-19226 T3SS island encodes more than 10 effector proteins that are part of the effector protein repertoire of V. cholerae, and we focused additional experiments on a novel effector, annotated as A33_1663 and herein named VopX.

MATERIALS AND METHODS

Strains, growth conditions, and in silico analysis.

Bacterial and yeast strains and plasmids used in this study are listed in Table 1. Table S1 in the supplemental material details the constructs used for yeast and translocation studies to screen AM-19226 ORFs. E. coli and V. cholerae strains were maintained at −80°C in Luria-Bertani (LB) broth containing 20% glycerol. Yeast strains were maintained at −80°C in complete yeast extract-peptone-dextrose (YPD) medium or synthetic complete dextrose (SCD) medium containing 15% glycerol. Ampicillin (Amp) and streptomycin (Str) were each used at 100 μg/ml. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) was added to LB agar at 20 to 30 μg/ml. The preparation of bile (bovine bile, product no. B-3883; Sigma) has been previously described (1). Clone Manager Professional Suite, version 9, was used for basic sequence analyses and manipulations. Basic protein BLAST (NCBI) was applied to find protein similarities, and the Clustal W program was used to perform multiple sequence alignments (33).

TABLE 1.

Strains and plasmids

Strain or plasmida	Genotype and descriptionb	Reference or source
Strains
E. coli
DH5αF′	F′ endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1 ΔU169(lacZYA-argF) (φ80dlacΔM15)	Laboratory stock
SM10λpir	thi thr leu tonA lacY supE recA RP4-2-Tc::M(λpir) Kanr	Laboratory stock
DH5αλpir	F− (φ80dlacΔM15)(lacZYA-argF ΔU169)endA1 recA1 hsdR17 deoR thi-1 supE44 gyrA96 (Nalr) relA1 λpir	Laboratory stock
DH5α	F− (φ80dlacΔM15)(lacZYA-argF ΔU169)endA1 recA1 hsdR17 deoR thi-1 supE44 gyrA96 (Nalr) relA1	Laboratory stock
V. cholerae
MD992	AM19226 Strr R− M+	Laboratory stock
MD996	AM19226 Strr R− M+ ΔlacZ	Laboratory stock
AAC40	MD992 ΔvttRB	1
AAC228	MD992 ΔvttRA	1
MD1069	MD992 ΔvttRA ΔvttRB	1
AD10	MD992 ΔtoxR	1
AAC477	MD992 ΔvopX	This study
AAC155	MD992 Δhap ΔhlyA ΔrtxA	This study
AAC330	MD992 Δhap ΔhlyA ΔrtxA ΔvcsN2	This study
S. cerevisiae
BY4742	MATα his3-Δ1 leu2-Δ0 lys2-Δ0 ura3-Δ0	Laboratory stock
Plasmids
pCVD442umcs	Suicide vector with unique multiple cloning site, sucrose sensitive; Ampr	Laboratory stock
pEH3	Allelic-exchange suicide vector based on pCVD442 to integrate of lacZY transcriptional fusions into V. cholerae lacZ; Ampr	1
pCVD442umcs-ΔvopX	VopX deletion plasmid; Ampr	This study
pVTM30	pDSW204-Bla, used to construct Bla fusions, IPTG inducible; Ptrc Ampr	56
pBG1805	Gateway cloning yeast-E. coli shuttle vector, URA3, PGAL1, S. cerevisiae 2μm plasmid; Ampr	21
pYCplac111	Yeast-E. coli shuttle vector, centromere (CEN4) based; LEU2 Ampr	22

^aumcs, unique multiple cloning site introduced in pCVD442 (MD1003).

^bAmp^r, ampicillin resistant; Str^r, streptomycin resistant; Kan^r, kanamycin resistant; R⁻, type II restriction endonuclease deletion; M⁺, methyltransferase positive.

Yeast growth inhibition assay.

Yeast strains were grown in SCD-Ura medium (SCD medium lacking uracil) at 30°C on a roller drum to an optical density at 600 nm (OD₆₀₀) of 1 to 1.5. After adjusting the OD₆₀₀ to 1.0, 10-fold serial dilutions to 10⁻⁵ were made, and 7 μl of each of the dilutions was spotted onto SCD-Ura or synthetic complete galactose lacking uracil (SCGal-Ura) plates. The plates were incubated at 30°C and photographed at 72 h (day 3). Caspofungin was added to plates to a final concentration of 30 to 80 ng/ml. The concentration added represents the calculated, active amount of the drug in the resuspended preparation, and frozen aliquots were stored at −20°C for a maximum of 6 months. All yeast deletion mutations were verified by PCR analysis using gene-specific primers.

Strain and plasmid constructions.

Nucleic acid manipulations were performed using standard molecular biology techniques (50). Primer sequences are available upon request. Strain AM-19226 ORFs of interest, designated by their A33_XXXX locus tags, were amplified by PCR using primers containing att sites compatible with the Gateway cloning system, and the resulting products were recombined into the entry vector pDONR201 (Invitrogen). ORFs from the entry plasmid were then moved into the Gateway-compatible yeast expression vector pBG1805 (21) (a gift from Elizabeth Grayhack). Correct inserts were verified by PCR analysis and DNA sequencing. The resulting pBG1805-A33_ORF plasmids were used to transform yeast strain BY4742. Briefly, the plasmid of interest and calf thymus DNA (as a carrier) were added to yeast cells resuspended in 1× Tris-EDTA, pH 8.0, and 1× lithium acetate (TE-LiAc). After the addition of 1 ml of sterile 1× polyethylene glycol 3350-TE-LiAc, the cell suspension was mixed well and incubated for 30 min at 30°C with gentle rotation. Cells were heat shocked at 42°C for 15 min, centrifuged, and resuspended in water. Finally, the cells were plated onto selection agar.

Nonpolar, in-frame deletions of genes were constructed using overlapping PCR (splicing by overlap extension) and standard allelic-exchange methods (11, 25). The A33_1663 deletion left sequences coding for 11 amino acids, the rtxA deletion left 1,311 amino acids, and the hlyA deletion left 55 amino acids. The vcsN and hap deletions have been previously reported (56).

The RLM1 gene and 500 bp of 5′ and 3′ flanking sequences were cloned into the centromere-based plasmid pYCplac111 (22) and used to transform wild-type or rlm1-Δ yeast strains containing pBG1805 or pBG1805-A33_1663.

Translocation assay.

AM-19226 ORFs encoding candidate effector proteins were cloned into pVTM30 (56) downstream from an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible Trc promoter and in frame with the gene encoding β-lactamase. HeLa cells were seeded at ∼5.0 × 10⁴ cells/ml in Lab-Tek/Nunc 8 chamber cover slides in 500 μl minimal essential medium (MEM) with 10% fetal bovine serum (FBS) and allowed to grow for ∼24 h before coculture. Bacteria were grown overnight in LB with streptomycin and ampicillin, subcultured 1:100 in LB with streptomycin, ampicillin, and 0.8 mM IPTG, and grown at 37°C for 2 h. HeLa cells were washed three times with Hanks' balanced salt solution (HBSS) plus Ca plus Mg and infected with ∼5 × 10⁵ bacteria (multiplicity of infection [MOI] of ∼10 to 40) in MEM medium containing 1% FBS, streptomycin, ampicillin, and 0.8 mM IPTG for 3 h. Cells were washed three times with HBSS and then incubated with CCF2-AM (GeneBLAzer detection kits; Invitrogen) for 1 to 1.5 h. Microscopy was conducted using an Olympus FV1000 confocal microscope at ×20 magnification.

Preparation of yeast cell extracts.

To verify the expression of bacterial proteins in yeast, 15 μl of a culture grown to saturation in SC-Ura plus 2% raffinose was used to inoculate 2 ml of SC-Ura plus 2% raffinose. After 16 h of incubation at 30°C, 1 ml of 3× yeast extract-peptone-galactose (YPGal) was added to induce protein expression, and cultures were incubated at 30°C for an additional 5 h on a roller drum. Cells were then centrifuged, and the supernatant was aspirated. Cell pellets were resuspended in buffer (10 mM HEPES, pH 7.9, 150 mM KCl, 1.5 mM MgCl₂, 0.1 mM dithiothreitol, and 2 μl/ml fungal protease inhibitors [Sigma]) and vortexed (3 to 5 times for 1 min each) in the presence of glass beads, with the sample cooled on ice in between times. Proteins in the supernatant of cell lysates were precipitated by adding trichloroacetic acid to a final concentration of 3 to 4%, washed with acetone, and then resuspended in 100 μl of SDS-PAGE loading buffer.

Preparation of bacterial cell extracts.

To confirm the expression of β-lactamase protein fusions by strains used in the translocation assay, overnight cultures were diluted 1:100 in fresh LB containing Str, Amp, and 0.8 mM IPTG. Cultures were grown for 2 h, and cell pellets were resuspended in protein loading buffer.

Western blot analysis.

Cell extract proteins were separated by SDS-PAGE and then transferred to a nitrocellulose membrane. pBG1805-based constructs resulted in C-terminal His₆ and hemagglutinin (HA) epitopes, and proteins produced from these constructs were detected by using a mouse monoclonal anti-HA antibody (Sigma). ORF-Bla fusion proteins produced from the pVTM30-based constructs were detected by using an anti-β-lactamase antibody (QED Bioscience). In all cases, antibody detection was carried out using a Western Lightning ECL enhanced chemiluminescence substrate kit (PerkinElmer).

lacZY transcriptional reporter construction and β-galactosidase assay.

A single-copy lacZ transcriptional fusion to the putative promoter region of A33_1663 was constructed by PCR amplification of 274 bp upstream of the A33_1663 coding sequence, followed by cloning of the product into pEH3 (1). The lacZY reporter construct was integrated into the chromosome of AM-19226 strains MD992 (wild type), AAC40 (ΔA33_1675), AAC228 (ΔA33_1664), MD1069 (ΔA33_1675 ΔA33_1664), and AD10 (ΔtoxR). Integration of the reporter constructs at the V. cholerae lacZ locus was confirmed by PCR analysis. β-Galactosidase assays were performed on strains grown overnight in LB alone or LB with 0.4% bile, following the protocol previously described (1).

Infant mouse competition assay.

Competition assays using 4- to 5-day-old CD-1 mice were performed as previously described, using ∼10⁵ bacteria in the inoculum (1, 20). A lacZ⁺ strain carrying an in-frame deletion in the A33_1663 gene was competed against a strain that was wild type at the vopX locus but lacZ deficient. The competitive index (CI) was calculated based on the input and output ratios of bacteria for each strain, where CI = (mutant output/wild-type output)/(mutant input/wild-type input).

RESULTS

Selection of T3SS-encoded proteins for screening as putative effector proteins.

Our goal was to identify the effector proteins encoded within the AM-19226 T3SS pathogenicity island. As described below, several genes were predicted to encode proteins associated with the structural apparatus or virulence in other strains and were therefore excluded from our survey (Table 2). Because effector proteins typically do not share sequence similarity across species, we considered any protein having little or no homology to known proteins as a candidate effector protein to be screened in the S. cerevisiae model system. We used VopF, the sole effector protein identified by conserved WH2 and FH1 domains, as a positive control in our studies. Figure 1 A shows the genetic organization of the T3SS island of V. cholerae AM-19226, including the previously described genes encoding the structural components of the apparatus (vcsRTCNS2, vcsVUQ2, vspD, and vcsJ2), the vttR_A and vttR_B genes encoding two regulatory proteins, and vopF (1, 12). Genes shaded in light gray in the figure were not included in our screen because they are predicted to encode proteins having sequence similarity to known virulence factors or cytoplasmic proteins. For example, the AM-19226 TRH (thermostable direct hemolysin [TDH]-related hemolysin) predicted protein includes a potential signal sequence for the type II secretion system (analyzed by SignalP; data not shown); similarly, in V. parahaemolyticus, the TDH and TRH proteins are secreted in a T3SS-independent manner (24). A33_1701 and A33_1698 show ∼77% and ∼58% amino acid similarity with V. cholerae strain N16961 proteins AcfD (VC0845) and AcfC (VC0841), respectively, which are proteins involved in colonization (14, 15, 27, 45). An ORF identified in our own annotation but missing from the NCBI annotation is predicted to encode an AcfA (VC0844) homolog upstream of A33_1662, having ∼50% amino acid sequence similarity to AcfA encoded by strain N16961. Therefore, we did not include this ORF in our screen for effector proteins but have indicated it on the map along with the other acf genes (Fig. 1). A33_1660 is predicted to encode a P4 integrase and, hence, a cytoplasmic protein, and A33_1692 shows sequence similarity to two component response regulators.

TABLE 2.

Summary of T3SS island genes

Group (no. of ORFs in group)a	Gene name(s), ORF designations, or protein encoded
Known genes and ORFs
Structural proteins (10)	vcsJ2, vspD, vcsVUQ2, vcsRTCNS2
Transcriptional regulators (3)	vttRA, vttRB, A33_1692 (putative)
Known effectors (1)	vopF
Known virulence factors (4)	trh, acfD, acfC, acfA
Known function other than virulence (1)	P4 integrase
Newly identified ORFs
ORFs showing yeast inhibition without
stressors (11)	A33_1662, A33_1663, A33_1665, A33_1668, A33_1680, A33_1684, A33_1687, A33_1695, A33_1697, A33_1699, A33_1706b
ORFs showing yeast inhibition with
stressors (4)	A33_1679, A33_1690, A33_1700, A33_1678c
Translocated effector proteins (11)	A33_1662, A33_1663, A33_1678, A33_1680, A33_1684, A33_1687, A33_1690, A33_1697, A33_1699, A33_1700, A33_1704

^aApproximately 46 open reading frames (ORFs) were identified within the 49.7-kb region encoding the T3SS. Functional assignments for ORFs were based on protein similarities and previous studies. The remaining ORFs were considered candidate effector ORFs and were assayed for yeast inhibition and translocation. There were 27 candidate ORFs for yeast screening and 15 ORFs showing yeast inhibition.

^bThe A33_1696 protein (VopF), which shows yeast inhibition without stressors, has been previously reported.

^cThe strains carrying A33_1679, A33_1690, and A33_1700 were inhibited with caspofungin as the stressor (see Fig. 2), and the strain carrying A33_1678 was inhibited with caffeine as the stressor (data not shown).

FIG. 1.

(A) Schematic representation of the T3SS island found in strain AM-19226. The region shown is ∼49.7 kb. Genes encoding T3SS apparatus proteins are shown as dark gray arrows, two transcriptional regulators (vttR_A and vttR_B) and a putative transcriptional regulator are shown as black arrows, the gene encoding the VopF effector protein (A33_1696) is checkered, non-T3SS-related genes encoding proteins presumably involved in virulence and the P4 integrase are shown in light gray, and genes selected for and tested in yeast are shown as white arrows. (B) Yeast growth inhibition assay. Yeast strains carrying AM-19226 T3SS ORFs (designated by the A33 locus number) cloned in pBG1805 were grown in SCD-Ura to saturation, serially diluted 10-fold, and then spotted onto SCD-Ura or SCGal-Ura plates. The strain harboring pBG1805 served as the negative control (showing no inhibition of yeast growth), whereas the strain carrying A33_1696, encoding the VopF protein, served as the positive control (showing inhibition of yeast growth). Experiments were repeated three times with similar results.

We therefore chose 27 ORFs, shown as white arrows in Fig. 1 and listed in Table 3, as candidates for our effector protein screen. We included an ORF identified from our own annotation (ORF58) that was not identified in the NCBI annotation. In Table 3, we also note any discrepancies between the NCBI annotation and our annotation that resulted in our testing an ORF containing a different number of amino acids than predicted by NCBI. In general, we have included additional sequences upstream of the NCBI annotation of a particular ORF if our annotation indicated that an initiation codon upstream was proximal to a predicted Shine-Dalgarno sequence. Although most of the ORFs encode hypothetical proteins, any conserved domains identified by recent BLAST analyses are indicated. The protein encoded by A33_1662 has a conserved Rho- GTPase domain; we and others have named this protein VopE by analogy with the Yersinia sp. YopE effector (V. C. Tam, M. Suzuki, and J. J. Mekalanos, personal communication).

TABLE 3.

Summary of yeast and translocation assay results^a

A33 locus tag or ORFb	Productc	Yeast inhibition without stressorsd,e	% of HeLa cells showing translocatione,f	Effector proteine,g
A33_1662	HP (VopE)	++	3	Y
A33_1663	HP (VopX)	+++	30	Y
A33_1665	CHP	+	Y/N	N
A33_1667	CHP	N	NT	—
A33_1668	HP	+	0	N
A33_1671	CHP	N	NT	—
A33_1674	HP	N	0.06	Nh
A33_1677	HP	N	NT	—
A33_1678	HP (VopH)	N	0.02	Y
A33_1679	HP	N	NT	—
A33_1680	CHP (VopA)	++	0.05	Y
A33_1683	HP	N	NT	—
A33_1684	HP (VopM)	+	31	Y
A33_1685	CHP	N	NT	—
A33_1687	CHP (VopI)	+	0.03	Y
A33_1688	CHP	N	NT	—
A33_1690	HP (VopW)	N	0.08	Y
A33_1691	HP	N	NT	—
A33_1694	CHP	N	NT	—
A33_1695	HP	+	0	N
A33_1696	VopF	++++	∼0.05 (PC)	Y
A33_1697	HP (VopG)	+	11	Y
A33_1699	HP (VopK)	++++	0.23	Y
A33_1700	CHP (VopY)	N	0.13	Y
A33_1703	CHP	N	0	N
A33_1704	HP (VopZ)	N	7	Y
ORF58	HP	N	NT	—
A33_1706	CHP	++++	0	N

^aTwenty-seven candidate ORFs were tested in yeast for their ability to inhibit growth. Those showing growth inhibition were then screened for translocation using a FRET-based assay as described in the text. ORFs are listed as their A33 locus tag, except for ORF58, which was identified by our own annotation.

^bOur annotation of the AM-19226 T3SS island identified alternative start sites for some genes. Differences compared to the NCBI annotation are noted as follows. A33_1665 has an additional 243 bp upstream of the NCBI annotation. A33_1668 has an additional 160 bp upstream of the NCBI annotation. A33_1671 has an additional 15 bp upstream of the NCBI annotation. A33_1677 has an additional 6 bp upstream of the NCBI annotation. A33_1679 has an additional 45 bp upstream of the NCBI annotation. A33_1680 has an additional 39 bp upstream of the NCBI annotation. Due to DNA sequence differences, our annotation predicts that A33_1683 starts 12 bp upstream of the NCBI annotation; our annotation is consistent with the slightly larger protein annotated for Vibrio parahaemolyticus strain RIMD2210633. A33_1685 has an additional 465 bp upstream of the NCBI annotation. A33_1687 has an additional 21 bp upstream of the NCBI annotation. A33_1691 begins 66 bp downstream of the NCBI-annotated translational start site. A33_1694 has an additional 36 bp upstream of the NCBI annotation. A33_1695 has an additional 9 bp upstream of the NCBI annotation. A33_1703 has an additional 60 bp upstream of the NCBI annotation. A33_1704 has an additional 33 bp upstream of the NCBI annotation. ORF58 is our annotation of a 255-bp ORF; A33_1705 is annotated by NCBI as a 120-bp ORF in the same region but does not overlap with ORF58.

^cORFs that we designated as effector proteins were assigned “Vop” names as shown in parentheses. HP, hypothetical protein; CHP, conserved hypothetical protein.

^dGrowth defects were scored as mild (+), moderate (++), strong (+++), and severe (++++).

^eY, yes; N, no.

^fY/N, translocation was variable; NT, not tested; PC, positive control.

^gEffector protein designation (yes/no) was based on the results of the translocation assay. —, ORF was not tested for translocation.

^hA33_1674 is translocated independently of the T3SS.

Screening putative effector proteins in yeast.

Yeast growth inhibition has been shown to be a reliable model for assessing the ability of effector proteins to detrimentally interact with eukaryotic host cell processes (2, 10, 51, 54). We used pBG1805 to construct plasmids that would express AM-19226 ORFs in yeast in a galactose-inducible manner (21). Plasmids expressing each of the 27 candidate effector proteins and VopF as a control were introduced individually into yeast strain BY4742. The resulting strains were grown to stationary phase under conditions that repress expression from the gal promoter (2% dextrose), and 10-fold serial dilutions were spotted onto media containing either 2% dextrose (repressing conditions) or 2% galactose (inducing conditions). As shown in Fig. 1, all yeast strains grew well on media containing dextrose. However, we observed a growth defect for some strains upon induction of AM-19226 protein expression on galactose media. As a control for the phenotype induced by a known V. cholerae effector protein, we included the A33_1696 locus, encoding VopF. The yeast strain that expresses VopF shows a strong growth defect, with an ∼1,000-fold reduction in the ability to form colonies when grown on galactose medium, scored as “++++” in Table 3. Similar results of growth inhibition by VopF expression in yeast have been reported by Tripathi et al. (57). Several other strains also show strong growth defects when grown on galactose. The strains expressing the proteins encoded by loci A33_1699 and A33_1706 showed growth inhibited ∼1,000-fold compared to the growth of the strains grown on dextrose, and the strain expressing A33_1663 showed an ∼100-fold growth defect. Ten-fold defects in growth were observed for strains expressing the A33_1662 and A33_1680 proteins. Finally, six strains showed very mild growth defects (e.g., strains expressing A33_1665, A33_1668, A33_1684, A33_1687, A33_1695, and A33_1697), and these were scored as a single “+” in Table 3. Other ORFs did not produce an observable growth defect when expressed in yeast. Western blot analysis confirmed that all constructs produced proteins of expected sizes when the strains were grown under inducing conditions, except for A33_1684, A33_1685, and A33_1699 (data not shown; see Discussion).

The results of previous studies indicated that effector proteins may not produce a growth defect phenotype in yeast if they target cellular processes that are not normally rate limiting for yeast growth (54). One method to increase the range of detection is to stress, or sensitize, yeast using compounds that perturb signaling pathways yet do not inhibit growth when used at certain concentrations. For example, the addition of caspofungin to the medium at subinhibitory concentrations (for wild-type strains) can sensitize strains to the activity of some effectors by perturbing the mitogen-activated protein kinase (MAPK) pathway that is important for maintaining cell wall integrity. We therefore tested whether some AM-19226 ORFs could affect yeast growth when strains were plated on galactose medium that included 30 to 80 ng/ml caspofungin. A yeast BCK1 gene deletion strain was used as a control, since this mutation impairs the cell wall integrity signaling pathway, thus sensitizing the strain to growth in the presence of caspofungin. As shown in Fig. 2, the bck1-Δ strain could not grow in the presence of caspofungin but grew normally in the absence of this stress. In contrast, the wild-type yeast strain with pBG1805 alone or pBG1805 expressing a dubious yeast open reading frame (YAL069W) grew well in the presence or absence of caspofungin. As shown in Fig. 2, when the ORFs with locus tags A33_1679, A33_1690, and A33_1700 were expressed in yeast and grown on medium containing galactose and caspofungin, a growth defect was observed compared to the growth of the control strain. (The strain expressing A33_1704 did not show a growth defect in the presence of caspofungin.) The growth defect was not observed when strains were grown on galactose alone (Fig. 1). In addition, the expression of ORF A33_1678 yielded a mild growth defect when expressed in yeast in the presence of caffeine, an additional cell wall biosynthesis stressor (data not shown). The remaining ORFs did not cause a growth defect when expressed in yeast under the stress conditions we tested (data not shown). In summary, these assays revealed that the expression of 11 putative effectors inhibits yeast growth and that the addition of caspofungin or caffeine to the medium sensitizes cells to the expression of an additional 4 putative effectors. These findings suggest that 15 proteins encoded by the AM-19226 T3SS island might specifically inhibit pathways required for eukaryotic cell growth.

FIG. 2.

Yeast growth inhibition screen in medium containing caspofungin. Strains carrying either vector alone (pBG1805), pBG1805 expressing a dubious yeast ORF (YAL069W), or pBG1805-based plasmids encoding V. cholerae strain AM-19226 putative effector proteins were grown in noninducing selective synthetic medium containing dextrose. Ten-fold serial dilutions of yeast strains were spotted onto selective medium containing 2% galactose and 30 to 80 ng/ml of caspofungin. The yeast strain carrying the bck1-Δ mutation is sensitive to caspofungin and serves as a control for caspofungin activity. Experiments were repeated three times with similar results.

Identification of bona fide effector proteins.

We next employed a fluorescence resonance energy transfer (FRET) translocation assay to test the validity of our hypothesis that these proteins represent true T3SS effectors. In this assay, an intact CCF2-AM molecule (a coumarin-fluorescein FRET pair linked by a beta-lactam ring) can freely diffuse into HeLa cells and produces FRET-based fluorescein fluorescence. However, the presence of beta-lactamase activity (provided by the fusion protein) can cleave CCF2-AM, thus resulting in only coumarin fluorescence. In addition to the 15 putative effectors identified in the yeast assays, we included 3 additional proteins in the FRET screen even though their expression in yeast did not result in a growth defect. These are A33_1703 and A33_1704, included on the basis of ORF characteristics such as size, location, and lack of amino acid similarity to other known proteins, and ORF A33_1674, due to its location as the first gene in the operon encoding the structural components VcsRTCNS2. To determine which of the 17 candidates are bona fide effector proteins, we constructed β-lactamase fusions to the carboxy-terminal domains of the AM-19226 proteins and conducted FRET-based assays as previously described (3, 56). Coding regions for candidate effector proteins were cloned into the vector pVTM30 (56) and transferred to V. cholerae strain AAC155 (Δhap hlyA rtxA) or AAC330 (Δhap hlyA rtxA vcsN). (The Δhap hlyA rtxA triple deletion strain was used to eliminate any cytotoxic effects that might complicate extended coculture, as previously described [56].) Again, we used VopF as a positive control. Protein expression was confirmed by Western blot analysis using a monoclonal antibody against β-lactamase (TEM1). Fusion proteins of the expected size were produced by all strains except that expressing A33_1678, in which a 45-kDa protein was detected instead of the predicted 68-kDa size (data not shown). However, we still scored the protein as translocated since it gave clear results in the translocation assay (see below).

The FRET-based assay has been previously described and relies on the activity of β-lactamase fusion proteins that can be translocated from the bacterial cell to the eukaryotic cell during coculture. Figure 3 shows the results of the translocation assay for AM-19226 ORFs that appear to be bona fide effector proteins, with the results of assays conducted in a T3SS-competent strain shown in panel A and four representative examples of the results observed using a T3SS-incompetent strain (ΔvcsN) shown in panel B. A V. cholerae strain carrying the empty pVTM30 plasmid (no fusion protein) was used as a negative control for translocation; all HeLa cells cocultured with this strain emitted green fluorescence, consistent with an intact FRET reaction. We used a VopF-Bla fusion protein as a positive control in our assay, since VopF has been previously shown to be a translocated effector (56). Figure 3A shows one cell in the field emitting blue fluorescence, consistent with the activity of a translocated VopF-β-lactamase fusion protein. Similarly, Bla fusions with the products of ORFs with locus tags A33_1662 (VopE), A33_1663, A33_1678, A33_1680, A33_1684, A33_1687, A33_1690, A33_1697, A33_1699, A33_1700, and A33_1704 also showed translocation (Fig. 3A and data not shown). The results indicated that the proteins were dependent on the T3SS for translocation, since we were unable to detect cells emitting blue fluorescence when fusions were expressed in V. cholerae strains with the deletion of the vcsN gene that encodes the ATPase essential for T3SS function (Fig. 3B and data not shown). The Bla protein fused with the products of A33_1678 and A33_1687 showed infrequent translocation in this assay, but the results were reproducible over the course of several experiments. We also found that, although the expression of A33_1706 in yeast consistently resulted in strong growth inhibition, we were unable to detect translocation of the protein in the in vitro FRET assay. Another candidate effector, the product of A33_1665, showed variable translocation; we have reported the result as “Y/N” in Table 3 since the results of four experiments were inconclusive. The A33_1674 product-reporter fusion appeared to be translocated in both the T3SS-competent and the ΔvcsN (T3SS-deficient) background. We therefore do not consider it an effector protein at this time. We have not yet tested the product of A33_1679 for translocation, although its expression did produce a phenotype in yeast when caspofungin was present. Table 3 therefore reports the translocation data calculated as the percentage of blue cells visualized in a given well (based on ∼10⁴ cells/well) and then reports our determination of effector protein status based on whether translocation was both visible and present only in the strain with a wild-type T3SS. It is important to note that the percentage of blue cells varies among the different fusion proteins, and we interpret this to be due to multiple in vitro parameters that affect the translocation and cleavage of the reporter fluorophore (e.g., protein stability and ability to be translocated in the in vitro system) but not a reflection on the inherent translocation efficiency in vivo.

FIG. 3.

Identification of the bona fide effector proteins. Confocal microscopic images showing the translocation of Bla fusion proteins by the T3SS into HeLa cells. HeLa cells were cocultured with AM-19226 strains expressing different Bla fusion proteins and then observed for fluorescence following excitation at 409 nm. Emission due to FRET can be viewed as green fluorescence at 520 nm, whereas disruption of FRET by Bla fusion protein activity results in emission at 447 nm (blue fluorescence). The white bars represent 50 μm. One representative field is shown for each strain, and each AM-19226 protein was tested for translocation at least twice. (A) Results of experiments conducted using the wild-type T3SS AM-19226 strain (competent for translocation). (B) Representative examples of assays conducted using the T3SS-ΔvcsN strain (translocation deficient).

We assigned names to the effectors identified in our study, as shown in Table 3. As described earlier, VopE has also been named by others, and we have adopted the names assigned by others for VopM and VopA (Tam et al., personal communication).

A33_1663 homologs are present only in T3SS-positive non-O1/non-O139 V. cholerae and V. parahaemolyticus.

Although the protein encoded by A33_1663 does not show sequence similarity to proteins encoded by bacteria from other genera, BLAST analysis using A33_1663 as the query did identify similar proteins in other non-O1/non-O139 V. cholerae strains and in V. parahaemolyticus. As expected, all Vibrio strains carrying an A33_1663 homolog encode a T3SS. A33_1663 product homologs present in V. cholerae strains V51, TMA21, and 12129 (1) show 98 to 99% amino acid sequence identity. V. cholerae strains 623-39 and 1587 and two V. parahaemolyticus strains (AQ4037 and TH3996) encode homologs with only ∼38% amino acid identity to the product of A33_1663. The sequence divergence is consistent with the data presented by Okada et al., suggesting distinct Vibrio T3SS lineages based on the sequence of genes encoding the apparatus components (40). Nonetheless, the A33_1663 gene appears to encode a novel T3SS effector protein that is present only in Vibrio species. We therefore chose the effector protein encoded by A33_1663 for further experiments and hereinafter refer to the protein as VopX.

The vopX mutant strain shows reduced colonization in the infant mouse model.

Previous results demonstrate that the T3SS is essential for colonization in vivo, since a strain with a deletion of the essential ATPase (ΔvcsN) was unable to colonize when tested in the infant mouse model (56). To assess whether the VopX protein has a role in colonization, an in-frame deletion of the vopX gene was introduced into the AM-19226 background by allelic replacement, leaving sequences coding for 11 amino acids. The resulting ΔvopX strain has the same growth characteristics as the strain carrying a wild-type vopX gene and was competed against an isogenic strain (MD996) to determine its ability to colonize the infant-mouse small intestine. The results shown in Fig. 4 demonstrate that the ΔvopX mutant strain has an ∼5-fold defect in colonization compared to that of the parent strain. Although the defect is less than that observed for the ΔvcsN strain (an ∼1,000-fold defect [56]), the results suggest that the VopX protein is important for the full colonization capability of strain AM-19226.

FIG. 4.

Growth curves and mouse competition assay. (A) OD₆₀₀ recorded for the VopX-positive (solid line; n = 5) and VopX-negative (dashed line; n = 5) strains in a 96-well plate after 1:200 dilutions of overnight cultures in LB. (B) Competition assays in CD-1 infant mice were performed using a lacZ deletion derivative of strain AM-19226 (MD996) and ΔvopX strain AAC477. The results of a single experiment are shown, where each symbol represents the competitive index (CI) from a single animal (n = 8). The bar indicates the mean CI. The experiment was repeated with similar results.

Suppression of the VopX-induced growth defect in S. cerevisiae.

Others have shown that the expression of effector proteins in yeast strains with deletions of signaling pathway components can restore wild-type growth if the gene deleted is a target of effector protein activity (2). We therefore chose four different MAPK pathways in yeast (pheromone response/mating, filamentous growth, hyperosmotic growth/glycerol, and cell wall integrity [CWI]) and obtained 14 strains with deletions in specific, nonessential gene components (7, 46). We expressed VopX in each of the yeast mutant strains and scored for observable growth defects. We did not see suppression of yeast growth inhibition by deletions in the nonessential genes encoding the components of pheromone response/filamentous growth (FUS3, STE20, STE11, STE7, and KSS1), Rho GTPases (RHO2, RHO4, RHO5), and the hyperosmotic growth/glycerol (HOG) pathway (SHO1, PBS2, and HOG1). However, we found that the VopX-mediated yeast growth inhibition phenotype was partially suppressed in the rlm1-Δ strain (Fig. 5 A and B). Rlm1 is the transcriptional regulatory protein that is responsible for modulating gene expression in response to cell wall stress. Notably, deletions in genes upstream of RLM1 in the CWI pathway (BCK1 and SLT2) did not rescue the growth defect induced by VopX expression, suggesting that the specific absence of Rlm1 restored the ability of yeast to grow.

FIG. 5.

Genetic approach in yeast for identification of VopX functional target. (A) The components of the cell wall integrity pathway in yeast. (B) Deletion of the RLM1 gene suppresses yeast growth inhibition by VopX. Strains with the wild-type CWI or a bck1-Δ, slt2-Δ, or rlm1-Δ deletion carrying pBG1805, pBG1805-YAL069W, pBG1805-VopF, or pBG1805-VopX were plated onto SCD-Ura or SCGal-Ura. (C) Complementation of the RLM1 gene in the rlm1-Δ strain. Strains with the wild-type CWI or the rlm1-Δ deletion and carrying pBG1805 and pYCplac111-based plasmids were plated onto SCD-Ura-Leu or SCGal-Ura-Leu (lacking uracil and leucine).

We next performed complementation analyses to examine whether the restoration of yeast growth in an rlm1-Δ mutant strain is truly due to the absence of the RLM1 gene (Fig. 5C). The RLM1 coding sequence and 500 bp of upstream and downstream flanking sequence were cloned into the centromere-based plasmid pYCplac111, and the resulting plasmid was introduced into the yeast strain carrying the pBG1805-A33_1663 (VopX) ORF plasmid. When VopX was expressed (by galactose induction), yeast growth was again inhibited. Although the growth inhibition was not as severe as that observed in the wild-type strain expressing VopX (Fig. 5C), the result supports the finding that the absence of Rlm1 may be responsible for the suppression phenotype.

Rlm1 is a member of the family of MADS box transcriptional regulators. Yeast has four MADS box transcription factors: Mcm1 and Arg80, which are similar to the mammalian SRF proteins, and Rlm1 and Smp1, which are similar to Mef2-like mammalian proteins (38). To further investigate whether VopX activity was specifically linked to Rlm1, we expressed VopX in the arg80-Δ and smp1-Δ mutant strains. We did not observe suppression of the VopX-induced growth defect, suggesting that VopX activity is specific for the Rlm1 MADS box transcription factor (data not shown). MCM1 is an essential gene in yeast, so we were unable to assay the effect of VopX expression in an mcm1-Δ background.

Bile-induced expression of VopX is regulated by VttR_A and VttR_B.

VttR_A and VttR_B are T3SS island-encoded transcriptional regulators that were shown to modulate the expression of the genes encoding the T3SS structural components when V. cholerae strains were grown in the presence of bile and purified bile salts (1). To determine whether bile might also stimulate the expression of VopX, we constructed a strain carrying a chromosomally integrated vopX-lacZ transcriptional reporter fusion. Strains carrying the vcsRTCNS2 (structural gene operon)-lacZ fusion and the promoterless reporter fusions were used as positive and negative controls for bile-regulated expression. After overnight growth in LB, the vcsRTCNS2-lacZ fusion showed ∼20 units of activity in the wild-type strain background, as previously reported (Fig. 6) (1). However, the vopX-lacZ fusion showed ∼180 units of activity in the same background, indicating that VopX is expressed in stationary phase in LB alone. When the wild-type strain carrying the vopX-lacZ reporter fusion was grown overnight in the presence of 0.4% bile, the expression increased ∼7-fold compared to the expression levels obtained during growth in LB medium alone. This suggests that bile increases the expression of the vopX-lacZ reporter fusion. The vcsRTCNS2-lacZ reporter fusion also showed increased expression, as previously described (1).

FIG. 6.

vopX-lacZ reporter fusion expression is modulated by ToxR homologs in the presence of bile. β-Galactosidase activity levels were measured in strains carrying a single-copy chromosomal lacZ gene fused to putative vopX promoter sequences. Strains carrying the vcsRTCNS2 reporter fusion and the promoterless reporter were used as control strains. Wild-type background strains expressing the reporter fusion were grown overnight in LB medium without (light gray bars) or with 0.4% bile (checkered bars). Reporter fusion strains with the following deletions were simultaneously assayed in four additional genetic backgrounds after growth in LB plus 0.4% bile overnight: ΔvttR_A (dark gray bars), ΔvttR_B (vertical striped bars), ΔvttR_A ΔvttR_B (narrow hatched bars), and ΔtoxR (wide hatched bars). The experiment was repeated, with similar results, and the data shown represent the results of a single experiment using three individual colonies of each strain. Units, μmol o-nitrophenyl-β-d-galactopyranoside hydrolyzed/min/optical density at 600 nm.

The expression of genes regulated by ToxR and ToxR-like proteins can be modulated by specific environmental signals in vitro, and previous experiments identified bile as an inducing condition for the VttR_A- and VttR_B-regulated expression of T3SS structural genes in strain AM-19226 (1, 36). We therefore next determined the role of ToxR-like proteins in the regulation of VopX expression by integrating the vopX-lacZ transcriptional fusion into the lacZ locus of four AM-19226-derived strains containing in-frame deletions of vttR_A alone, vttR_B alone, vttR_A and vttR_B, and toxR. The strains carrying vcsRTCNS2-lacZ and promoterless lacZ fusions in four different deletion backgrounds were also included. All strains were grown overnight in the presence of bile, and the assay results are shown in Fig. 6. As described above, the vcsRTCNS2-lacZ fusion showed decreased expression in the vttR_A and vttR_B deletion strains when grown in the presence of bile. Similar results were obtained when the vopX-lacZ fusion was expressed in the ΔvttR_A and ΔvttR_B strains, suggesting that both the VttR_A and VttR_B protein contribute to the positive regulation of VopX expression. The expression of vopX-lacZ was also decreased in the ΔtoxR strain. Together, the data show that the bile-induced expression of VopX is regulated largely by VttR_A and VttR_B and suggest that ToxR may also contribute to the bile-induced expression of VopX.

DISCUSSION

One of the major challenges in understanding T3SS-mediated disease is effector protein identification. However, knowing the arsenal of proteins that bacteria have at their disposal is an essential prerequisite to begin studying how disease occurs at the molecular level. In general, non-O1/non-O139 serogroup-associated cholera presents similarly to infection by O1 and O139 serogroup strains, with minor exceptions (e.g., a slight inflammatory component). Yet, most T3SS-positive non-O1/non-O139 strains do not encode TCP or CT. It is therefore presumed that the coordinated action of multiple effector proteins works to promote bacterial colonization in the intestinal epithelium, followed by disruption of cellular homeostasis, resulting in choleralike diarrhea. We therefore set out to survey the proteins encoded within the V. cholerae AM-19226 T3SS island for potential effector protein activity, using yeast growth inhibition and T3SS-dependent translocation into human cells as assays. It is important to note that the inability to cause a yeast growth defect does not necessarily exclude a protein as an effector, since some effector proteins might act cooperatively or in a coordinated manner with other effectors in vivo. In such a case, we would expect that the expression of multiple effectors in yeast would be required to produce a detrimental phenotype. However, we chose to begin our analysis by evaluating the potential activities of individual V. cholerae proteins.

A total of 15 proteins demonstrated the ability to inhibit growth when expressed in yeast, including four that required yeast-sensitizing agents to uncover the phenotype. The strongest inhibition was due to the expression of VopF (a known effector protein) and the product of A33_1706. VopF has also been shown by others to inhibit yeast growth and may function early in infection to reorganize the host cell actin network (56, 57). The expression of A33_1699 and A33_1663 (whose product is herein named VopX) also resulted in strong growth inhibition when expressed in yeast. The remaining proteins demonstrated mild to moderate inhibition. Collectively, our results indicate that yeast can demonstrate a range of growth defects and serve as a suitable system for screening V. cholerae effector protein activity. This is consistent with the use of yeast to screen effector proteins from other bacterial T3SSs.

We used a FRET-based in vitro coculture assay to identify bona fide effector proteins that were translocated into eukaryotic host cells in a T3SS-dependent manner. We tested a total of 17 AM-19226 proteins for translocation: 14 identified by the yeast screen and an additional three that we chose for reasons discussed above. All were expressed as β-lactamase fusion proteins in V. cholerae cells that were cocultured with HeLa cells in the translocation assay. Eleven were detected as translocated proteins (Fig. 3 and Tables 2 and 3). Interestingly, we detected various degrees of translocation with different A33_ORF-β-lactamase fusion proteins, with VopX consistently showing translocation to >50% of the cell population and VopF being one of the proteins demonstrating limited translocation. Because the MOI for this assay was similar for all assays (between 4 and 40), we conclude that the variation in reporting might be due to different translocation efficiencies in vitro for the fusion proteins or abilities of the fusion proteins to function once inside the HeLa cell. Western blot analysis suggested that all fusion proteins were made to similar levels, although the stability of each once inside eukaryotic cells was not determined. Certainly, it might be expected that some effector proteins are prioritized in their translocation, and given that all other effectors were present in addition to the highly expressed β-lactamase fusion protein, it is reasonable to speculate that the sensitivity of the FRET system facilitates detection under nonideal conditions where protein expression and levels might be dysregulated. Nonetheless, the assay provides a high level of confidence that all 11 proteins are bona fide effectors that depend on the T3SS for their injection into host cells.

We were surprised to find that A33_1706, one of the proteins producing the strongest growth defect when expressed in yeast, was not translocated in our assay. The predicted protein is larger than 450 amino acids and encodes two conserved domains having sequence similarity to bacterial IgG-like domains found in adhesins such as intimin. We therefore predict that A33_1706 encodes a protein that, rather than functioning as an effector, is important for the bacteria to colonize the intestinal epithelium in a TCP-independent manner. It is unclear at this time why the expression of the protein in yeast results in a strong growth defect, but based on sequence analysis using the SignalP algorithm, A33_1706 is predicted to encode a signal peptide (data not shown). We speculate that A33_1706 encodes a surface-expressed protein that may interact with eukaryotic cells and might have the capacity to interact with eukaryotic signaling pathways to promote colonization.

Two proteins that displayed relatively weak growth defects when expressed in yeast did not show translocation: A33_1668 and A33_1695. Our annotation suggested that the A33_1668 protein might begin ∼160 bp upstream of the start site annotated at NCBI, and so we included the additional sequences in our β-lactamase fusion protein. The additional sequences could account for the lack of translocation, due to protein misfolding. However, we favor the interpretation that because the expression of A33_1668 has a weak phenotype in yeast, it is not a translocated effector. Additional studies using a fusion protein constructed based on the NCBI sequences are expected to resolve any remaining questions. The sequence of the A33_1695-encoded protein in NCBI matches our own annotation very closely (we included 9 additional base pairs at the 5′ end of the ORF), and so we expect that the results of the translocation assay indicate that A33_1695 is not an effector protein. The A33_1665-encoded protein also showed mild inhibition of yeast growth. Translocation assay results with the β-lactamase fusion to this protein were inconclusive: the protein appeared to be translocated in only two of the four assays that we conducted. Again, technical parameters of the assay that depend on protein stability and translocation efficiency may explain the inability to demonstrate consistent translocation.

We also conducted bioinformatic analysis of small T3SS-encoded ORFs that did not have a phenotype when expressed in yeast but were candidates for encoding chaperones. While T3SS chaperones do not exhibit primary amino acid sequence similarity, they often are small, have an acidic pI, and may possess a C-terminal amphipathic helix. Class I chaperones associate with effector proteins, while class II chaperones associate with the translocator proteins (44). Class I chaperones share similar structures and contain five beta strands and three alpha helices, while class II chaperones often possess tetratricopeptidelike repeats in an all-alpha-helical array (28, 41, 44). CesA is a chaperone in EPEC that has a unique structure, consisting of helices shaped like a hairpin, and has a pI of 9.5 (61). CesA is sometimes referred to as a class IV chaperone (39). Class V chaperones interact with the needle subunit in Yersinia and Pseudomonas and also with a translocator in Salmonella (39). The class V chaperone SsaE in Salmonella contains three alpha helices, one of which is predicted to be amphipathic (39). We have identified putative chaperones based on secondary-structure prediction (PSIPRED) and determination of the theoretical pI (ExPASy) of small, hypothetical proteins in the AM-19226 T3SS island (data not shown). Secondary-structure prediction did not reveal any proteins with similarities to the class I chaperones. The majority of the proteins predicted to have an acidic pI are mostly alpha helical in nature. The A33_1668 and A33_1694 proteins are almost entirely helical, with a small coiled region between two helices, most closely resembling the class V chaperones. The A33_1671 protein possesses two alpha helices which are connected by ∼15 residues that are predicted to be coiled and a small beta strand. The A33_1683 protein is slightly large for a chaperone, at ∼200 amino acids, but is predicted to have a pI of 5.10 and an alpha helix of almost 150 residues, followed by two beta strands and a small alpha helix.

We focused additional experiments on VopX as an effector protein because it encoded novel sequences and produced a strong phenotype when expressed in yeast. In addition, deletion of the vopX gene resulted in a strain that showed a diminished ability to colonize the infant mouse intestine when competed against the isogenic strain encoding a wild-type VopX (Fig. 4). The colonization defect was relatively mild compared to that observed for epidemic strains with deletions of TCP or even compared to that of an AM-19226 strain carrying a deletion in vopF or vcsN2 (56), so we postulate that VopX plays an accessory role in colonization or a role in maintaining colonization during infection or interacts with other effectors to promote full colonization. Because the model is limited to reporting on the colonization ability of a strain, we cannot say at this time whether VopX may play a more significant role in disease progression or in eliciting a diarrheal response.

While none of the other 13 yeast deletion strain we tested could restore the ability of yeast to grow in the presence of VopX, the deletion of the RLM1 gene suppressed the growth defect induced by VopX expression. Complementation of the RLM1 deletion reversed this suppression, resulting in a yeast strain that was again sensitive to the expression of VopX. These results further support the finding that the VopX-induced growth defect is mediated, either directly or indirectly, through Rlm1. We observed that growth inhibition was demonstrably weaker in the rlm1-Δ-complemented strain than in the strain with an intact, chromosomal RLM1 gene. The relatively weaker inhibition could be due to the cloning of limited sequences flanking the RLM1 gene (∼500 bp upstream and downstream), which may have inadvertently omitted elements present in the chromosomal sequence that are necessary for proper RLM1 expression. Also, RLM1 is complemented on a plasmid. Therefore, the expression level of the gene may vary in comparison to its expression in the wild-type strain.

Rlm1 is a MADS box transcriptional factor regulating genes involved in cell wall biogenesis and maintenance. Rlm1 activity is regulated by the protein kinase C-mediated cell wall integrity (CWI) MAPK pathway (59, 60). The finding that the deletion of RLM1 suppresses the toxicity of VopX suggests several possible interpretations. First, based on our analyses, VopX activity may be involved in only one of the four MAPK pathways tested, likely the CWI pathway. Second, the negative effect of VopX may require its interaction with a gene product(s) whose expression depends on activation by Rlm1. Third, VopX may interact directly with Rlm1, causing deregulation of CWI components downstream of Rlm1 and resulting in inhibition of cell growth. Finally, it is possible that the expression of VopX activates the CWI pathway upstream of Bck1 and Slt2, the MAP kinases tested for suppression in our assay (Fig. 5A). Previous results showed that overexpression or overactivation of the Rho1 or Pkc1 inputs into the CWI pathway results in constitutive activity that is lethal for bck1-Δ or slt2-Δ strains but not for rlm1-Δ strains (29, 30, 32). Accordingly, the bck1-Δ and slt2-Δ mutations would fail to suppress VopX upstream activation of the pathway, while the rlm1-Δ mutation would succeed. Further experiments are required to distinguish between these alternatives.

Rlm1 is one of four MADS box transcription factors present in yeast and is thought to function most similarly to the mammalian Mef2 family of proteins. As transcriptional regulators, Mef2-like proteins can be activated during the inflammatory process by phosphorylation as a result of the activity of the p38 MAP kinase (46). Mef2 proteins have been shown to mediate smooth muscle cell differentiation but have recently been recognized as having a role in cell survival in response to cellular stress. It is therefore interesting to speculate that VopX interaction with Rlm1 and the CWI pathway in yeast may affect cell survival by disabling mechanisms important for cellular structure and that similar mechanisms of interaction may interfere with mammalian cell integrity and survival during V. cholerae infection in the human intestine.

VopX expression appears to be coregulated with the genes encoding the T3SS structural apparatus. Its expression was detectable when the strain carrying the vopX-lacZ promoter fusion was grown in LB alone and was strongly induced when the strain was grown in the presence of bile. VttR_A and VttR_B deletions markedly reduced the expression levels in LB plus bile, similar to the effect observed for reporter fusions for T3SS structural genes. It is unclear at this time whether all genes within the T3SS pathogenicity island are regulated similarly. However, previous results from reporter assays looking at vopF expression suggest that at least some effector proteins are controlled by a regulatory network or set of signals that may vary from those governing the expression of the genes encoding the structural apparatus (1, 52).

The yeast screen proved to be both sensitive and effective, since we were able to detect growth defects of various degrees induced by V. cholerae protein expression. We also tested several V. cholerae proteins for their ability to inhibit yeast growth in the presence of stressors and discovered an additional four proteins that produced a phenotype in yeast. Because we did not employ stressors in looking for a phenotype for every A33_ORF or express putative effectors in combination in yeast, it is possible that we have not identified the complete assembly or repertoire of effector proteins encoded by AM-19226. However, the identification of 11 proteins in addition to VopF that are part of the V. cholerae T3SS arsenal provides an important first step in beginning to understand the pathogenic capabilities of T3SS-positive non-O1/non-O139 V. cholerae strains that cause diarrheal disease. Considering that epidemic strains rely on the toxin-coregulated pilus and cholera toxin as the main components of effecting colonization and diarrhea, it will be interesting to unravel the molecular mechanisms of the multiple effector proteins that lead to a choleralike disease in the absence of TCP and CT.