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Journal of Bacteriology, January 2008, p. 231-239, Vol. 190, No. 1
0021-9193/08/$08.00+0     doi:10.1128/JB.00924-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Lipidation of an FlrC-Dependent Protein Is Required for Enhanced Intestinal Colonization by Vibrio cholerae{triangledown}

David C. Morris, Fen Peng, Jeffrey R. Barker, and Karl E. Klose*

South Texas Center for Emerging Infectious Diseases and Department of Biology, University of Texas San Antonio, San Antonio, Texas 78249

Received 12 June 2007/ Accepted 15 October 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vibrio cholerae, the causative agent of cholera, has a sheathed, polar flagellum, and motility has been linked to virulence. An operon with two genes, flgO and flgP (VC2207 and VC2206), is positively regulated by FlrC, the activator of class III flagellar genes. Deletion of flgP results in a nonmotile phenotype, demonstrating the requirement of this gene for V. cholerae motility. V. cholerae {Delta}flgP cells synthesize fragile and defective flagella but transcribe flagellar genes similar to the wild-type strain. PhoA fusion analysis indicated that the putative lipoprotein FlgP is localized external to the cytoplasm, and fractionation demonstrated that it was localized to the outer membrane. Mutagenesis of the site of lipidation of FlgP (C18G) prevented [3H]palmitate incorporation and outer membrane localization. Interestingly, FlgP with the mutation C18G [FlgP(C18G)] could complement the {Delta}flgP mutant for motility, and the cells synthesized wild-type flagella. The {Delta}flgP mutant strain was defective for intestinal colonization (~20-fold), but FlgP(C18G) was unable to complement this defect, demonstrating that lipidation of FlgP is essential for its role in intestinal colonization but not flagellar synthesis. FlgP thus represents a novel V. cholerae intestinal colonization factor that is regulated by the flagellar transcription hierarchy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vibrio cholerae is a gram-negative bacterium responsible for the human disease cholera. V. cholerae is acquired by the consumption of contaminated food or water. Within the intestine, the bacteria express the toxin-coregulated pilus (TCP), which is essential for colonization of the intestinal epithelial cells (45). V. cholerae also expresses cholera toxin (CT), which leads to the copious amounts of watery diarrhea characteristic of a cholera infection (10).

V. cholerae synthesizes a sheathed polar flagellum, and motility has been linked to virulence. Studies have demonstrated that nonmotile mutants are defective for fluid accumulation and adherence in the rabbit ileal loop model (37, 40) and adherence to isolated rabbit brush borders (11); nonmotile mutants are also defective for virulence in the rabbit RITARD (removable intestinal tie adult rabbit diarrhea) model (37). Nonmotile mutants of O1 El Tor biotype strains show colonization defects in the infant mouse model (25), while nonmotile classical biotype mutants generally colonize similarly to the motile wild-type strain (14). Nonmotile mutants of live V. cholerae vaccine strains show reduced reactogenicity (disease symptoms) in human volunteers while still being able to colonize the intestine (8, 19), demonstrating a role for motility in virulence in the natural human host.

Though a number of studies have implicated motility as being important for V. cholerae virulence, the exact connection between flagellar synthesis and cholera pathogenesis is still not clear. In a study involving spontaneous mutants, Gardel and Mekalanos found that nonmotile mutants showed increased expression of CT and TCP while hypermotile mutants produced less CT and TCP than the wild-type V. cholerae, and they proposed a model where virulence and motility are inversely related (14). Recently, Silva et al. provided evidence for this model when they found that transcription of the genes encoding CT and TCP is upregulated in a V. cholerae nonmotile strain (40).

The flagellum is a complex structure made up of multiple structural subunits, and the assembly of this structure is exquisitely coordinated in a stepwise fashion, initiating inside the cell and building outward toward the flagellar tip (29). The expression of flagellar genes is also tightly regulated, and V. cholerae has a four-tiered flagellar transcription hierarchy (36). There is a single class I gene, and it encodes the master regulator of the flagellar transcriptional hierarchy, FlrA. FlrA is a {sigma}54-dependent transcriptional activator that activates the expression of class II genes, which encode primarily the MS ring and export apparatus components, as well as chemotaxis and regulatory factors, including FlrC (36). FlrC is a {sigma}54-dependent transcriptional activator that is phosphorylated and activates class III flagellar genes (7), which encode the basal body and hook, as well as some of the switch and export apparatus components and the FlaA flagellin. FliA is an alternate sigma factor ({sigma}28) that activates class IV genes, which encode the additional flagellins FlaBCDE as well as motor components (5). V. cholerae secretes an anti-sigma factor FlgM, and this allows {sigma}28 to associate with RNA polymerase and activate class IV genes.

It was previously shown that phosphorylation of V. cholerae FlrC is required for enhanced colonization in the infant mouse animal model (7), suggesting that some FlrC-dependent factor(s) might facilitate intestinal colonization. In the current study, we have identified a lipoprotein, FlgP, that is positively regulated by FlrC and that contributes to intestinal colonization; interestingly lipidation of this protein is critical for its role in colonization but not for its role in motility.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media. Luria broth (LB) was used for both liquid medium and agar plates. The LB was routinely supplemented with antibiotics (ampicillin at 50 µg/ml; streptomycin at 100 µg/ml or 250 mg/ml) or 0.1% arabinose, as required. Motility of bacterial strains was measured in 0.3% LB agar supplemented with appropriate antibiotics and 0.1% arabinose.

Plasmid construction. A complete list of plasmids and oligonucleotide primers used in this study can be found in Table 1 and Table 2, respectively. Restriction sites used in cloning are underlined in the oligonucleotides listed in Table 2. The plasmid pKEK782 was constructed by using PCR to amplify the flgO promoter (flgOp) from V. cholerae O395 with oligonucleotides 2208p Down and 2208p Up. The PCR fragment was digested with BamH1 and EcoR1 and ligated into pRS551 (41) digested with the same enzymes; the promoter fragment extends 259 bp upstream of the initiating codon of flgO.


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  		TABLE 1. Plasmids used in this study

 

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  		TABLE 2. Oligonucleotides used in this study

 
The splicing by overlap extension PCR technique (17) was used to create the {Delta}flgO and {Delta}flgP mutations, utilizing the primers 2207 Down BHI, {Delta}2207 Up RI, {Delta}2207 Down MfeI, and 2207 Up RI for {Delta}flgO and 2206 Down Bgl2, 2206 {Delta}Up, 2206 {Delta}Down, and 2206 Up EcoRI for {Delta}flgP. The {Delta}flgO and {Delta}flgP fragments were digested with BamHI or BglII and EcoRI and ligated into pKAS32 (42), which was digested with BglII and EcoRI to create pKEK902 and pKEK802, respectively. The in-frame {Delta}flgO mutation fuses the codon for amino acid 7 to the codon for 217, while the in-frame {Delta}flgP mutation fuses the codon for 7 to the codon for 139.

Expression plasmids containing flgP, flgP-FLAG, and flgOP were created by PCR amplification using the oligonucleotides VC2206 Down NcoI and VC2206 Up XbaI (flgP), VC2206 Down NcoI and 2206 Flag Tag Up (flgP-FLAG), and VC2207 Down NcoI and {Delta}VC2206 Up XbaI (flgOP). The PCR fragments were digested with NcoI and XbaI and ligated into pBAD24 (15) digested similarly to form pKEK845 (flgP), pKEK944 (flgP-FLAG), and pKEK1081 (flgOP). The expression plasmid containing Campylobacter jejuni cj1026 was created by PCR amplification of C. jejuni 81-176 genomic DNA (kind gift of S. Blanke) with oligonucleotides CJ1026 Down NcoI and CJ1026 Up XbaI, followed by digestion with NcoI and HindIII and ligation into pBAD24 digested similarly to form pKEK1079. Plasmids expressing flgP from flgOp in a low-copy-number vector were constructed by PCR amplification with oligonucleotides VC2208p Up and VC2207 Up XbaI with the KKV2096 ({Delta}flgO) DNA template for flgOp {Delta}flgO flgP (pKEK1044). This latter fragment also contains a {Delta}flgO in-frame deletion. The PCR fragment was digested with EcoRI and XbaI and then ligated into pWSK30 digested with the same enzymes.

The plasmids containing the flgP-phoA translational fusion was created by PCR amplification using oligonucleotides VC2206 Down NcoI and VC2206 Up XmaI w/o Stop with strain O395 DNA; the fragment was digested with NcoI and XmaI and ligated into pBAD24 digested similarly to form pKEK1033 (flgP). Then, the phoA fragment was PCR amplified from Escherichia coli DH5{alpha} using oligonucleotides phoA Down XmaI and phoA Up HindIII, digested with XmaI and HindIII, and ligated into pKEK1033 digested similarly, to create pKEK1035, which expresses flgP-phoA.

Site-directed mutagenesis utilizing the QuickChange technique (Stratagene) and oligonucleotides VC2206C18GF and VC2206C18GR was performed to introduce the cysteine-to-glycine mutation (C18G) in FlgP in plasmids pKEK845, pKEK944, and pKEK1044, resulting in pKEK1037, pKEK1038, and pKEK1080, respectively.

Bacterial strains. Table 3 contains a list of bacterial strains used in this study. E. coli strain DH5{alpha} (16) was used for all cloning experiments, while the E. coli strain WM3046 (gift of William Metcalf, University of Illinois) was used to transfer plasmids to V. cholerae by conjugation. The V. cholerae {Delta}flgP and {Delta}flgO strains KKV1965 and KKV2096, respectively, were constructed as described previously (42) by mating pKEK802 and pKEK920, respectively, into V. cholerae strain KKV598. The correct construction of all strains was verified by PCR and sequencing.


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  		TABLE 3. Bacterial strains used in this study

 
β-Galactosidase and alkaline phosphatase assays. V. cholerae strains were transformed with plasmids containing the promoter-lacZ or protein-phoA fusions listed in Table 3, grown in LB medium supplemented with appropriate antibiotics (and 0.1% arabinose with pBAD plasmids) at 37°C, and harvested at an optical density of 600 nm of approximately 0.4 to 0.8. Bacterial cells were permeabilized with chloroform and sodium dodecyl sulfate (SDS) and assayed for β-galactosidase activity by the method described by Miller (33) or for alkaline phosphatase activity by the method described by Michaelis et al. (32).

Protein detection. Isolation of V. cholerae outer membrane (OM) proteins was performed using a method adapted from Lohia et al. (26). Fractionation of whole-cell lysates into OM, periplasmic, inner membrane, and cytoplasmic fractions was accomplished by the method of Bose and Taylor (2). Proteins were detected by separation on an SDS-15% polyacrylamide gel, followed by Western immunoblotting using anti-FLAG M2 (Sigma) monoclonal antibody, rabbit polyclonal anti-OmpU antiserum (gift of J. Peterson, University of Texas Medical Branch), or rabbit polyclonal anti-beta lactamase (Chemicon International) and ECL detection reagent (Amersham-Pharmacia).

For detection of lipid incorporation into FlgP, strains were grown in LB medium plus antibiotics at 37°C overnight and then normalized to an optical density of 0.4 at 600 nm. Arabinose was added to a final concentration of 0.1%, and the strains were grown for 5 min before the addition of [3H]palmitic acid to a final concentration of 25 µCi/ml. Cultures were grown at 37°C for 24 h and pelleted, and the supernatant was removed; cultures were then resuspended in 1x sample buffer and boiled. Samples were separated by SDS-15% polyacrylamide gel electrophoresis (PAGE) and then fixed with 5% glacial acetic acid, 5% isoproponal, and water. The gel was then treated with Autofluor (National Diagnostics) and imaged by autoradiography.

Electron microscopy. Strains were grown to mid-log phase in LB medium supplemented with 0.1% arabinose and then centrifuged and resuspended in 0.15 M NaCl. Samples were adhered to carbon-formvar-coated grids (Electron Microscopy Sciences) and stained with 7% uranyl acetate before microscopy with a JOEL 1230 microscope.

In vitro/in vivo virulence assays. CT in culture supernatants was measured using a ganglioside GM1 enzyme linked immunosorbent assay (ELISA) with rabbit polyclonal antiserum against the purified B subunit of CT (44). TCP was measured by CTX{phi}-Kan phage transduction (47). The in vivo colonization assays were performed as described by Gardel and Mekalanos (14) using 5- to 6-day-old CD-1 suckling mice. The inoculum consisted of ~105 CFU for both wild-type and mutant strains. Colonies recovered from intestinal homogenates were confirmed to carry plasmids by plating on appropriate antibiotics.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VC2206 and VC2207 are regulated by the flagellar regulatory protein FlrC. Immediately adjacent to one of the large flagellar gene clusters in the V. cholerae genome (cluster I) (36) lies an operon with two genes encoding conserved hypothetical proteins (VC2206 and VC2207) (Fig. 1A). This operon lies adjacent to and is convergently transcribed with the flgAMN operon. Microarray data examining transcription in an flrC strain suggested that this operon is positively regulated by FlrC, indicating that it represents a class III flagellar gene operon (K. Klose and F. Yildiz, unpublished results). The promoter region upstream of this operon (VC2207p) was fused to lacZ, and β-galactosidase activity was measured in V. cholerae strains with deletions in the four major flagellar regulatory genes, rpoN, flrA, flrC, and fliA. Transcription of VC2207p was dependent upon {sigma}54 (RpoN), FlrA, and FlrC but not {sigma}28 (Fig. 1B, FliA). This pattern of expression is characteristic of class III (FlrC dependent) promoters (6, 36). A reasonably good consensus {sigma}54 binding site was found located –112 to –97 with respect to the initiating methionine codon. This sequence (GGGTATAAATTTTGCT; –24 and –12 elements are underlined) maintains the invariant GG and GC elements with 10-bp spacing in between and shows an identical –12 sequence with two previously characterized FlrC-dependent promoters (6). Our data demonstrate that the VC2207-VC2206 operon is positively regulated by FlrC.


Figure 1
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  		FIG. 1. flgOp is a class III (FlrC-dependent) promoter. (A) Organization of the flgOP operon. (B) Expression of flgOp in V. cholerae flagellar regulatory mutant strains. V. cholerae strains KKV598 (wild-type), KKV56 ({Delta}rpoN), KKV59 ({Delta}flrA), KKV98 ({Delta}flrC), and KKV1113 ({Delta}fliA) carrying plasmid pKEK782 (flgOp-lacZ) were assayed for β-galactosidase activity during logarithmic growth in LB medium. Assays were performed in triplicate, and standard deviations are shown.

 
VC2206 (flgP) is required for V. cholerae motility. The VC2207 gene is predicted to encode a precursor protein of 222 amino acids with a molecular mass of 25 kDa. Homologues of VC2207 are found in other Vibrio species and Photobacterium profundum, which are all in the family Vibrionaceae. We have evidence that VC2207 is required for V. cholerae motility (data not shown) and, hence, named this gene flgO, but the focus of the current study is the second gene in this operon, VC2206.

The VC2206 gene is predicted to encode a precursor protein of 145 amino acids with a molecular mass of 16 kDa. A PSI-BLAST search indicated that the protein contains a conserved domain within amino acids 33 to 132, DUF400, a domain of unknown function that is also found in the pathogenic bacteria Helicobacter pylori and C. jejuni. When the protein was analyzed using the program LipoP, version 1.0, the results indicated that it contained a lipoprotein signal peptide and that the cleavage site was between amino acids 17 and 18, with the +2 amino acid position occupied by a glutamine, suggesting localization to the OM (18). The homologous proteins in members of the Vibrionaceae share 61 to 83% identity and 80 to 94% similarity with VC2206 in V. cholerae, while the homologues in H. pylori (HP0837) and C. jejuni (Cj1026) share 30 to 37% identity and 56 to 62% similarity.

To determine whether VC2206 is required for motility, an in-frame deletion was introduced into the chromosome of V. cholerae (see Materials and Methods). The motility of the resultant V. cholerae {Delta}VC2206 strain was measured in motility agar plates (Fig. 2). The {Delta}VC2206 strain had a nonmotile phenotype in motility agar and appeared nonmotile in wet mounts by light microscopy, although an occasional motile cell could be detected. The {Delta}VC2206 strain exhibited growth kinetics indistinguishable from the wild-type O395 strain (data not shown), indicating that this mutation does not affect the growth rate. Because this gene affects motility and shares homology with C. jejuni flgP (43), we have renamed it flgP and will refer to it exclusively by this name in this report.


Figure 2
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  		FIG. 2. Motility phenotype of a V. cholerae {Delta}flgP mutant strain. V. cholerae strains KKV598 (wild-type [WT]), KKV1965 ({Delta}flgP), and KKV1965 carrying plasmids pKEK1044 ({Delta}flgP/flgOp {Delta}flgO flgP; this is a low-copy-number vector expressing flgP from flgOp in the absence of flgO) and pKEK1080 [{Delta}flgP/flgOp {Delta}flgO flgP(C18G); this is a low-copy-number vector expressing flgP(C18G) from flgOp in the absence of flgO] were inoculated into motility agar and incubated at 30°C for 15 h.

 
Complementation of the {Delta}flgP mutant with various plasmids expressing FlgP restored motility to the {Delta}flgP mutant in all instances, including expression of FlgP from the pBAD promoter (data not shown) as well as expression of FlgP from the natural flgO promoter in the low-copy-number plasmid pWSK30 (Fig. 2, flgOp {Delta}flgO flgP), which restored wild-type motility. This construct was PCR amplified from a {Delta}flgO strain, to allow for flgP expression from the natural flgO promoter in the absence of flgO. The {Delta}flgP mutant strain was also complemented for motility by a pBAD24 plasmid expressing FlgP with a C-terminal FLAG tag (data not shown). This FlgP-FLAG construct was used in localization studies described below. Finally, we attempted to complement the {Delta}flgP mutant strain with a pBAD24 plasmid expressing the FlgP homologue cj1026 from C. jejuni strain 81-176, but this construct was unable to restore motility to the {Delta}flgP V. cholerae mutant (not shown).

FlgP is required for normal flagellar synthesis. Flagellar structure of {Delta}flgP cells was observed by transmission electron microscopy. When observed by electron microscopy, most of the wild-type V. cholerae cells possess a single attached flagellum that has smooth edges and exhibits a typical "sine wave" appearance (Fig. 3A). Most of the {Delta}flgP cells also possess a flagellum (field view shown in Fig. 3C), but the flagella have rough, uneven edges and lack the typical sine wave appearance (Fig. 3B shows two examples). Many of these flagella were found to possess atypical curvature, some even exhibited "right-angle" turns. The flagella of the {Delta}flgP cells appear to be fragile, as many appeared split or broken (Fig. 3C, arrows). Complementation of the {Delta}flgP mutant with FlgP expressed from flgOp (flgOp {Delta}flgO flgP, described above) (Fig. 3D) restored the presence of flagella that resembled the wild-type strain.


Figure 3
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  		FIG. 3. Electron micrographs of the V. cholerae {Delta}flgP mutant strain. Transmission electron micrographs of V. cholerae strains KKV598 (wild-type; A) KKV1965 ({Delta}flgP) (B and C), KKV1965(D) carrying plasmid pKEK1044 ({Delta}flgP/flgOp {Delta}flgO flgP; this is a low-copy-number vector expressing flgP from the flgOp in the absence of flgO), and KKV1965 (E) carrying plasmid pKEK1080 [{Delta}flgP/flgOp {Delta}flgO flgP(C18G); this is a low-copy-number vector expressing flgP(C18G) from flgOp in the absence of flgO]. Bar, 500 nm (A, B, D, and E) and 2 µm (C). The filled arrows in panel C point to two examples of apparently "split" flagella.

 
FlgP has little effect on class IV flagellar gene transcription. The V. cholerae flagellum is composed of five flagellins, and four of the flagellin genes (flaB, flaC, flaD, and flaE) are {sigma}28 dependent and thus class IV genes, while the flaA flagellin gene is FlrC dependent and a class III gene (6, 22, 36). We measured transcriptional activation of the five flagellin genes in the {Delta}flgP strain. Plasmids containing the five flagellin promoters fused to lacZ were transformed into the {Delta}flgP strain. Analysis of β-galactosidase activity of the flagellin promoters in the {Delta}flgP strain showed transcription levels of the class IV promoters only slightly reduced compared to the wild-type strain (>75% wild-type expression levels), indicating that FlgP is not critical for class IV gene expression (Fig. 4). Transcription of the class III flaA gene showed elevated expression in the {Delta}flgP strain.


Figure 4
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  		FIG. 4. Expression of flagellin promoters in the flgP V. cholerae strain. V. cholerae strains KKV598 (wild-type) and KKV1965 ({Delta}flgP) carrying plasmids pKEK80 (flaAp-lacZ), pKEK79 (flaBp-lacZ), pKEK76 (flaCp-lacZ), pKEK77 (flaDp-lacZ), and pKEK81 (flaEp-lacZ) were assayed for β-galactosidase activity during logarithmic growth in LB medium. Assays were performed in triplicate, and standard deviations are shown.

 
FlgP localizes to the OM. To determine whether FlgP is localized outside the bacterial cytoplasm, we constructed a plasmid that expressed a C-terminal fusion of FlgP to a signal-sequenceless alkaline phosphatase (PhoA). Because PhoA is not active within the bacterial cytoplasm (30), measurement of the PhoA activity of these fusions allowed us to determine whether FlgP is secreted outside the cytoplasm. The flgP-phoA fusion plasmid was transformed into the {Delta}flgP mutant and assayed for alkaline phosphatase activity. This revealed that FlgP-PhoA appeared to be secreted, since it exhibited alkaline phosphatase activity higher than our positive control (data not shown), a V. cholerae strain with a chromosomal acfA-phoA fusion, which is known to be secreted outside the bacterial cytoplasm (35).

OM localization of FlgP was tested using a C-terminal FLAG-tagged FlgP protein, since we do not have specific antiserum to FlgP (FlgP-FLAG complements the {Delta}flgP strain for motility, as mentioned above, indicating proper localization of this protein). OM, periplasmic, and inner membrane fractions were isolated (26) from the {Delta}flgP strain expressing FlgP-FLAG and then separated by SDS-PAGE. The FLAG-tagged protein was identified by Western immunoblotting utilizing anti-FLAG M2 antibody (Sigma) (Fig. 5). Whole-cell lysate samples of the same strain were also probed on the Western immunoblot to ensure that the FLAG-tagged protein was expressed. Whole-cell samples were matched to equivalent protein levels, and OM samples were matched to equivalent protein levels. FlgP-FLAG could be clearly detected in the OM sample, indicating that FlgP localizes to the OM. Further fractionation studies revealed a lack of FlgP-FLAG in the periplasm and detectable FlgP-FLAG in the inner membrane fraction as well. The OM samples were enriched for the known OM protein OmpU and lacked the periplasmic protein beta-lactamase (expressed from the plasmid), as determined by Western blotting (Fig. 5), while the periplasmic fractions contained beta-lactamase but lacked OmpU, indicating that these samples are enriched specifically for OM and periplasmic proteins, respectively.


Figure 5
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  		FIG. 5. OM localization of FlgP. Bacterial pellets (WC, whole cell) and enriched inner membrane (IM), periplasmic (Peri), and OM fractions were prepared from V. cholerae strain KKV1965 ({Delta}flgP) carrying either pKEK944 (expresses FlgP-FLAG) or pKEK1038 [expresses FlgP(C18G)-FLAG]. Samples were separated by PAGE and subjected to Western immunoblot analysis with anti-FLAG ({alpha}-FLAG), anti-beta lactamase ({alpha}-Bla), and anti-OmpU ({alpha}-OmpU) antibodies.

 
FlgP is a lipoprotein and lipidation is necessary for OM localization but not motility. As mentioned above, bioinformatic analysis indicated that FlgP may be a lipoprotein. If the predicted lipoprotein signal peptide, MMKSSLLLLVAALMMTG, is cleaved by signal peptidase II, it would liberate an N-terminal cysteine (C18) to be lipidated. The putative site of lipidation in FlgP-FLAG was altered by site-directed mutagenesis (C18G), yielding FlgP(C18G)-FLAG. To determine whether FlgP is a lipoprotein, the {Delta}flgP strain alone or expressing FlgP-FLAG or FlgP(C18G)-FLAG was grown in the presence of [3H]palmitic acid; the wild-type strain was also grown under similar conditions. Whole-cell extracts were separated by SDS-PAGE, and incorporation of [3H]palmitate was visualized by autoradiography (Fig. 6). The FlgP-FLAG protein incorporated [3H]palmitate, but the FlgP(C18G)-FLAG protein did not. Both proteins were confirmed to be present at equivalent amounts by Western immunoblotting (data not shown). No 3H-labeled FlgP could be detected in the {Delta}flgP strain carrying no plasmid, as expected. No 3H-labeled FlgP could be detected in the wild-type strain either; this is likely due to the low natural levels of FlgP found in the wild-type strain. These results demonstrate that FlgP is a lipoprotein that is lipidated at residue C18.


Figure 6
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  		FIG. 6. [3H]palmitate incorporation into FlgP. V. cholerae strains KKV598 (WT) carrying no plasmid (–) and KKV1965 ({Delta}flgP) carrying no plasmid (–) and plasmids pKEK944 (expresses FlgP-FLAG) and pKEK1038 [expresses FlgP(C18G)-FLAG] (all shown in respective order at the top of the gel) were grown in the presence of [3H]palmitate for 24 h with 0.1% arabinose. Whole-cell lysates were separated by 15% SDS-PAGE and imaged via autoradiography.

 
Lipidation of FlgP is necessary for its localization to the OM, as determined by Western blot analysis of fractions from the {Delta}flgP strain expressing FlgP(C18G)-FLAG (Fig. 5). FlgP(C18G)-FLAG was not detected in the OM fraction, and only low levels of FlgP(C18G)-FLAG were present in the inner membrane fraction, despite high levels of expression of FlgP(C18G)-FLAG in the whole-cell extracts. This was in contrast to the wild-type FlgP-FLAG protein, which was found at higher amounts in both the OM and inner membrane fractions. Notably, the FlgP(C18G)-FLAG protein in the inner membrane was of apparently higher molecular weight than FlgP-FLAG, consistent with the predicted lack of signal peptide processing in this mutant protein. No detectable FlgP(C18G)-FLAG was found within the periplasmic fraction.

Interestingly, FlgP(C18G) was able to complement the {Delta}flgP mutant strain for wild-type levels of motility (Fig. 2). This was true for flgP(C18G) expressed from its natural promoter in a low-copy-number vector [Fig. 2, flgOp {Delta}flgO flgP(C18G),] as well as for flgP(C18G) expressed from the high-copy-number vector pBAD24 and with or without a C-terminal FLAG tag (data not shown). The {Delta}flgP cells expressing FlgP(C18G) synthesize flagella that resemble the wild-type strain, i.e., of apparent wild-type length and curvature, with smooth edges (Fig. 3E). Our results show that lipidation of FlgP is necessary for OM localization but not for motility and normal flagellar synthesis.

Lipidation of FlgP is necessary for enhanced intestinal colonization. The {Delta}flgP strain was analyzed for virulence defects utilizing the infant mouse intestinal colonization assay (Fig. 7). The {Delta}flgP strain showed an approximately 20-fold defect in intestinal colonization, with a competitive index (CI) of 0.05 (± 0.04) versus the isogenic wild-type strain. Complementation of the {Delta}flgP strain with FlgP expressed from its natural promoter (flgOp {Delta}flg OflgP; pKEK1044) resulted in the ability of this strain to colonize the infant mouse small intestine similar to the wild-type strain (CI, 1.12 ± 0.8). Interestingly, the same plasmid construct that expresses the C18G mutant form of FlgP [flgOp {Delta}flgO flgP(C18G); pKEK1080] had little effect on the {Delta}flgP strain's ability to colonize the intestine, as this strain still demonstrated an approximately 10-fold defect in colonization (CI, 0.125 ± 0.12). The observed defects in colonization of the {Delta}flgP and the {Delta}flgP/flgOp {Delta}flgO flgP(C18G) strains were determined to be significantly different than the colonization of the {Delta}flgP/flgOp {Delta}flgO flgP strain by a Students' two-tailed t test [for {Delta}flgP versus {Delta}flgP-flgOp {Delta}flgO flgP, P is 0.0006; for {Delta}flgP/flgOp {Delta}flgO flgP(C18G) versus {Delta}flgP/flgOp {Delta}flgO flgP, P is 0.0009]. This indicates that lipidation of FlgP is necessary for enhanced intestinal colonization.


Figure 7
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  		FIG. 7. Intestinal colonization of a {Delta}flgP V. cholerae strain. Strains KKV1965 ({Delta}flgP) and KKV1965 carrying plasmids pKEK1044 ({Delta}flgP/flgOp {Delta}flgO flgP; this is a low-copy-number vector expressing flgP from flgOp in the absence of flgO) or pKEK1080 [{Delta}flgP/flgOp {Delta}flgO flgP(C18G); this is a low-copy-number vector expressing flgP(C18G) from flgOp in the absence of flgO] were coinoculated with the wild-type strain O395 perorally into infant mice at a ratio of ~1:1; intestinal homogenates were recovered at 24 h postinoculation and the numbers of CFU of wild-type and mutant strains were determined. The CI is given as the output ratio of mutant:wild-type divided by the input ratio of mutant:wild-type; each value shown is from an individual mouse.

 
In vitro competition experiments (performed in LB medium) showed no competitive defects of the {Delta}flgP mutant versus the wild-type strain, indicating no growth defect (not shown). Measurement of CT and TCP expression under in vitro virulence factor-inducing conditions revealed wild-type levels of CT and TCP expression by the {Delta}flgP strain as well as by the various complemented forms of this strain (not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
V. cholerae flagellar synthesis and motility contribute to cholera pathogenesis, but the exact role they play in virulence is still not completely understood. The contribution of motility to disease is likely to be complex, since some nonmotile strains have been shown to express higher levels of certain virulence factors, including CT and TCP, yet they also colonize poorly and cause less fluid accumulation in some animal models (14, 40). The flagellar motor regulates the polysaccharide expression involved in biofilm formation, and this in turn also affects the virulence of the organism (24, 50). Chemotaxis is dependent on flagellar-based motility, and chemotaxis has also been shown to regulate the virulence of V. cholerae (3). Up-regulation of motility gene expression has recently been correlated with egress of V. cholerae from the mucosal surface following colonization (34). Thus, the contribution of flagellar-based motility may involve a combination of opposing effects on virulence, and the net effect may vary in different strains, which might explain some of the previous strain-specific and biotype-specific observations regarding motility and virulence.

V. cholerae flagellar synthesis shares some common elements with the well-described systems in Salmonella enterica serovar Typhimurium and Caulobacter crescentus (28, 49), such as mostly the same structural subunits, and yet V. cholerae flagellar synthesis also has some unique aspects. For example, the Vibrio flagellar motor is energized by a sodium gradient across the membrane rather than a proton gradient, and this probably reflects the prevalence of sodium in the marine environment where vibrios are normally found (1). Another unique attribute of the flagellum specific to Vibrio spp. and a few other bacteria (including e.g., H. pylori) is the sheath covering the flagellar filament. The sheath appears to be an extension of the OM, since lipopolysaccharide is present in the flagellar sheath (12). Studies in H. pylori and various Vibrio species have identified the presence of specific proteins, including lipoproteins, in the flagellar sheath (13, 27). Yet very little is known about the flagellar sheath, and its function is not clear, although it has been hypothesized to both shield the antigenic flagellins from host recognition and facilitate interactions of the bacterium with surfaces (13).

The V. cholerae flagellar genes are transcribed in a four-tiered hierarchy that shares elements with, but is distinct from, the flagellar transcription hierarchies of S. enterica serovar Typhimurium and C. crescentus (36). The flagellar transcription hierarchy found in V. cholerae is probably similar, if not identical, in all Vibrio spp. and is also similar to the four-tiered hierarchy found in Pseudomonas aeruginosa (9). Since FlrC regulates class III gene transcription, which is embedded within the hierarchy, we suspected that the flgOP operon, which is activated by FlrC and immediately adjacent to the flgAMN genes, would likely contribute to flagellar synthesis and motility. In this report we demonstrated that FlgP is required for V. cholerae motility.

FlgP is an OM lipoprotein necessary for motility. In this report, we demonstrated that FlgP is a lipoprotein and that residue C18 is the site of lipidation, consistent with bioinformatics analysis that predicted the presence of a 17-amino-acid N-terminal lipoprotein signal sequence cleaved by signal peptidase II. Moreover, FlgP localizes to the OM, as predicted by the +2 rule (39), and lipidation is required for OM localization. Since the flagellar sheath is an extension of the OM, we suspect that FlgP is likely present in the V. cholerae flagellar sheath. Several lipoproteins have been identified in the flagellar sheath of other bacteria (13, 27).

Immunofluorescence studies to localize FlgP-FLAG within whole (nonpermeabilized) V. cholerae cells failed to detect the protein (data not shown), suggesting that the protein (or at least the FLAG-tagged C terminus) may be located on the periplasmic face of the OM rather than on the exposed OM surface. Interestingly, lipidation is not required for motility, and cells expressing nonlipidated FlgP(C18G) make apparently normal flagella. Fractionation studies failed to detect nonlipidated FlgP within the OM or periplasmic fractions and found very low amounts in the inner membrane fraction. The typical processing of bacterial lipoproteins proceeds in three basic steps on the periplasmic face of the inner membrane: transfer of a diacylglyceride to the cysteine sulfhydryl group of the unmodified prolipoprotein, cleavage of the signal peptide by signal peptidase II, and finally acylation of the N-terminal cysteine of the apolipoprotein (38). Because the cysteine residue has been replaced in FlgP(C18G), this protein cannot be processed by this pathway and would be predicted to remain at the inner membrane surface. The small amount of FlgP(C18G) in the inner membrane fraction had an apparently higher molecular weight than the native protein, consistent with a lack of signal peptide processing of the C18G mutant. The low levels of FlgP(C18G) recovered after fractionation, despite the high levels of expression in whole cells, may also indicate loss of protein during the fractionation procedure, perhaps due to periplasmic aggregates not recovered in the soluble periplasmic fraction.

The function of FlgP in flagellar synthesis is unclear. The {Delta}flgP cells synthesize flagella, but these flagella are clearly different from the flagella of the wild-type strain. The edges of the {Delta}flgP flagella appear rough, unlike the smooth edges of wild-type flagella, and it appears as if the sheath is not completely formed around the flagellin filament. The {Delta}flgP flagella also lack the smooth sine wave appearance of wild-type flagella, with the flagellin filament making irregular turns. This suggests that the flagellin subunits are not inserting and stacking correctly into the filament or are unstable once inserted into the filament. Transcription of class IV flagellar genes is relatively normal in the {Delta}flgP strain, indicating that FlgP does not affect the class III-class IV switch. The rough flagella produced by the {Delta}flgP strain do not function correctly, since this strain appears nonmotile both in motility agar and by visual inspection under the light microscope. We conclude that FlgP is important for the correct structure of the flagellum and suspect it may play some role in sheath deposition around the flagellum.

Homologues of FlgP are found in Vibrio, Helicobacter, and Campylobacter spp., as well as several other genera (Photobacterium, Pseudoalteromonas, Shewanella, and Idiomarina), all polarly flagellated organisms. The FlgP homologue (cj1026) was recently characterized in C. jejuni (43), and it was also shown to be required for motility. C. jejuni FlgP also localizes to the outer membrane, but it is not yet clear if this protein is also a lipoprotein. We showed here that the V. cholerae {Delta}flgP mutant failed to be complemented for motility with C. jejuni FlgP, but this may have been due to the instability of C. jejuni FlgP in the absence of a coexpressed protein, FlgQ (43), which is not present in V. cholerae. Alternatively, this could be due to a different role of C. jejuni FlgP in motility, since a C. jejuni flgP mutant makes apparently normal flagella, unlike the V. cholerae flgP mutant. Also, unlike the V. cholerae flgP gene, the C. jejuni flgP gene is not regulated by the flagellar transcription hierarchy. Still, the C. jejuni and V. cholerae FlgP proteins contain the domain of unknown function, DUF400 (accession number PF04164), suggesting that they share a conserved function which might relate to their roles in motility.

Lipidation of FlgP is required for normal intestinal colonization. The V. cholerae {Delta}flgP mutant was approximately 20-fold more defective for intestinal colonization than the wild-type strain, demonstrating that FlgP plays some role in this process. A number of genes have been identified that affect the colonization of V. cholerae in the infant mouse, but the role of many of these genes remains unclear. TCP is essential for intestinal colonization, and although it clearly mediates bacteria-bacteria interactions, it has not yet been shown to facilitate adhesion to the host cell surface (21). Recently, a chitin-binding protein, GbpA, has been shown to bind to GlcNac residues on both chitin surfaces and epithelial cells (20, 31). Disruption of GbpA leads to a colonization defect similar to that shown here in the {Delta}flgP strain. The colonization defect of the {Delta}flgP strain is not due to lack of motility alone, since many isogenic nonmotile strains have been shown in our laboratory to exhibit a very slight defect (approximately threefold) (5, 22, 36) in this assay. It is thus possible that FlgP represents an adhesin that facilitates binding to the epithelial surface, similar to GbpA.

Interestingly, lipidation of FlgP is required for enhancement of intestinal colonization even though it is not required for motility. Lipidation of FlgP localizes the protein to the OM; since unlipidated FlgP fails to facilitate intestinal colonization, FlgP may function as a surface-exposed adhesin. Alternatively, its apparent role in stabilization of the flagellar sheath may be required for some other surface-exposed intestinal adhesin present in this structure. This may be a conserved function of FlgP in other bacteria as well (e.g., H. pylori), although it has not yet been reported whether C. jejuni FlgP is also required for enhanced colonization within the chicken intestine (43). FlgP thus represents a novel colonization factor that is regulated by the V. cholerae flagellar hierarchy.


   ACKNOWLEDGMENTS
 
We thank Steve Blanke and William Metcalf for supplying materials used in this study.

This study was supported by NIH grant RO1 AI43486 to K.E.K.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249. Phone: (210) 458-6140. Fax: (210) 458-4468. E-mail: kklose{at}utsa.edu Back

{triangledown} Published ahead of print on 2 November 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Journal of Bacteriology, January 2008, p. 231-239, Vol. 190, No. 1
0021-9193/08/$08.00+0     doi:10.1128/JB.00924-07
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