The Vibrio cholerae Pst2 Phosphate Transport System Is Upregulated in Biofilms and Contributes to Biofilm-Induced Hyperinfectivity

Abstract

Vibrio cholerae is the causative agent of the deadly diarrheal disease cholera. As part of its life cycle, V. cholerae persists in marine environments, where it forms surface-attached communities commonly described as biofilms. Evidence indicates that these biofilms constitute the infectious form of the pathogen during outbreaks. Previous work has shown that biofilm-derived V. cholerae cells, even when fully dispersed from the biofilm matrix, are vastly more infectious than planktonic (free-living) cells. Here, we sought to identify factors that contribute to biofilm-induced hyperinfectivity in V. cholerae, and we present evidence for one aspect of the molecular basis of this phenotype. We identified proteins upregulated during growth in biofilms and determined their contributions to the hyperinfectivity phenotype. We found that PstS2, the periplasmic component of the Pst2 phosphate uptake system, was enriched in biofilms. Another gene in the pst2 locus was transcriptionally upregulated in biofilms. Using the infant mouse model, we found that mutation of two pst2 components resulted in impaired colonization. Importantly, deletion of the Pst2 inner membrane complex caused a greater colonization defect after growth in a biofilm compared to shaking culture. Based on these data, we propose that V. cholerae cells in biofilms upregulate the Pst2 system and therefore gain an advantage upon entry into the host. Further characterization of factors contributing to biofilm-induced hyperinfectivity in V. cholerae will improve our understanding of the transmission of the bacteria from natural aquatic habitats to the human host.

INTRODUCTION

Vibrio cholerae, the Gram-negative facultative pathogen responsible for the deadly diarrheal disease cholera, causes significant morbidity and mortality worldwide (20). As part of its life cycle, V. cholerae survives in marine environments, where it forms surface-attached communities termed biofilms in association with copepods and other aquatic organisms (18). It is now understood that fragments derived from such environmental biofilms can provide the initial dose of V. cholerae during infection of a human host (8, 15).

For many bacterial species, cells contained within biofilms have increased recalcitrance to antimicrobial agents and stressful conditions relative to planktonic (free-living) counterparts (11, 26, 39). In the case of V. cholerae, biofilms are more resistant to bile salts and low pH (17, 49). Because only intact biofilms display full acid resistance, it was proposed that the biofilm structure aids in colonization of the host intestine, perhaps by protecting biofilm-encased cells during transit through the stomach (49). However, we recently showed that intact and fully dispersed V. cholerae biofilms grown on glass in the laboratory were equally infectious in the infant mouse model, and both were vastly more infectious than free-living cells (37). Therefore, the physiological state of individual biofilm-associated cells is responsible for the observed hyperinfectivity. We hypothesize that certain factors upregulated in biofilms contribute to this phenotype and that their expression in the biofilm primes V. cholerae for colonization of the host intestine. Importantly, such factors may not be identified as associated with virulence in studies relying on planktonic cell cultures and, therefore, may represent a novel set of virulence factors contributing to the transmission of V. cholerae from environmental reservoirs to humans.

Phosphate acquisition is important for species such as V. cholerae that survive in marine environments, where phosphate is limiting (32). Phosphate transport has been best studied in Escherichia coli, in which the two-component response regulator PhoB directly controls the transcription of numerous genes that are involved in phosphate metabolism and uptake (the Pho regulon) (3, 16). The Pst system, part of the Pho regulon, is an ATP-binding cassette transporter required for the uptake of phosphate into the cytoplasm. The Pst transporter consists of four proteins: PstS, a periplasmic binding protein, and PstC, PstA, and PstB, which form a permease complex that transports phosphate across the inner membrane (45).

Beyond well-established roles in phosphate homeostasis, members of the Pho regulon can also contribute to virulence (22). In pathogenic bacteria such as Proteus mirabilis, uropathogenic E. coli, and Salmonella enterica, dysregulation of the Pst system reduces virulence by impairing colonization and/or type III secretion (5, 6, 25). In V. cholerae, two pst loci are present in the genome, one of which (referred to as pst) has been shown to be important for colonization of the host based on studies in the infant mouse model (28, 32). PhoB, which directly regulates certain V. cholerae virulence factors in addition to its role in phosphate homeostasis, is also required for host colonization (32, 43). However, no studies have focused on the second pst locus in V. cholerae, which we have named pst2. There are currently no data comparing the regulation of the pst and pst2 loci, and the regulatory connections between PhoB and pst2 have not been determined.

In this study, we sought to identify factors that confer biofilm-induced hyperinfectivity. Because such factors are expected to be differentially regulated (transcriptionally or posttranscriptionally) during growth in a biofilm relative to planktonic cell culture, we compared the proteomes of V. cholerae cells grown under these two conditions. We identified several proteins that are more abundant in V. cholerae cells grown in a biofilm, including a phosphate binding protein (PstS2) encoded in the pst2 locus. We further determined the contribution of PstS2 and the PstCAB2 permease complex to biofilm production and intestinal colonization by V. cholerae. Together, our results indicate that the upregulation of the Pst2 phosphate uptake system during growth of V. cholerae in a biofilm enhances the ability of the bacterium to colonize the host. Thus, the Pst2 system contributes partially to the colonization advantage observed for biofilm-passaged cells, and our results with Pst2 lay the foundation for future studies aimed at finding additional factors that promote hyperinfectivity.

MATERIALS AND METHODS

Growth conditions and media.

V. cholerae C6706 and isogenic mutant strains were cultured in Luria-Bertani (LB) broth containing 100 μg/ml streptomycin (Sm). Morpholinepropanesulfonic acid (MOPS) medium (1× MOPS salts in distilled H₂O), supplemented with trace metals, was prepared as described previously (41).

Strain construction.

Strains and plasmids used in this study are listed in Table 1. Primer sequences are available upon request. In-frame deletions were generated using standard allelic exchange methods (10). All mutants were confirmed by PCR.

Table 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Genotype or description	Reference or source
V. cholerae strains
C6706	O1 El Tor Inaba; HapR+	40
C6706 ΔlacZ	In-frame deletion of lacZ	37
C6706 ΔpstS2	In-frame deletion of pstS2 (VCA0070)	This work
C6706 ΔpstCAB2	In-frame deletion of pstCAB2 (VCA0071 to -3)	This work
C6706 ΔvpsR	In-frame deletion of vpsR	This work
E. coli strains
DH5α λpir	F− Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 supE44 thi-1 gvrA96 relA1 λpir	21
SM10 λpir	thi recA thr leu tonA lacY supE RP4-2-Tc::Mu λpir	29
HB101(pRK2013)	Mating helper strain with IncP1 tra and ori(ColE1) plasmid	9
Plasmids
pCVD442::ΔpstS2	Allelic exchange vector for in-frame deletion of pstS2	This work
pCVD442::ΔpstCAB2	Allelic exchange vector for in-frame deletion of pstCAB2	This work
pCVD442::ΔvpsR	Allelic exchange vector for in-frame deletion of vpsR	38

Two-dimensional gel electrophoresis.

V. cholerae strain C6706 was grown overnight at 37°C in 1 ml of LB medium (planktonic culture) or diluted 1:1,000 from an overnight culture into 0.5 ml of LB medium in borosilicate glass tubes and incubated under static conditions at room temperature (approximately 23°C) for 27 h (biofilm culture). For protein extraction, 0.5 ml was pelleted from each of six independent planktonic cultures, and the cells were pooled and washed with LB. Total protein was extracted from biofilm cultures after removing the supernatants and washing the adherent biofilms twice with 0.85% NaCl. The attached cells were dispersed in 0.75 ml of LB medium by using an ultrasonic water bath for 1 min, pooled, and washed with LB. Dispersal in an ultrasonic water bath has been shown to be an effective means of disrupting biofilms without altering cell viability (37). Pooled planktonic or biofilm cells were pelleted once more and resuspended in 500 μl of lysis buffer (0.3% sodium dodecyl sulfate [SDS], 10 mM Tris [pH 7.4], 50 μg/ml RNase A, and 1 mM phenylmethylsulfonylfluoride). The suspensions were vortexed and placed on ice for 5 min. An equal volume of SDS boiling buffer (5% SDS, 10% glycerol, 60 mM Tris [pH 6.8]) was then added, and the protein concentration was determined using the bicinchoninic acid assay (Pierce). Afterwards, β-mercaptoethanol was added to a final concentration of 5% (vol/vol). Two-dimensional gel electrophoresis was carried out in duplicate by Kendrick Labs (Madison, WI) using the method of O'Farrell (30) with isoelectric focusing over a pH range of 4 to 8. Proteins differentially produced in planktonic and biofilm cells were identified visually. Selected spots were excised and identified by matrix-assisted laser desorption ionization mass spectrometry at the Columbia University Protein Chemistry Core Facility.

Isolation of RNA.

For most experiments, total RNA was extracted from cultures of V. cholerae grown with shaking to stationary phase (at an optical density at 600 nm [OD₆₀₀] of 1.2) or from dispersed biofilms grown as described above (pooled in groups of 3) using TRIsure (Bioline) and the RNeasy kit (Qiagen), according to the manufacturers' instructions (37). In some experiments, wild-type V. cholerae was grown in 2-ml cultures at 37°C with shaking to an OD₆₀₀ of 0.5, washed twice with MOPS medium without additives, and suspended in 2 ml of MOPS or MOPS supplemented with KH₂PO₄ to a final concentration of 50 μM. The suspended cultures were incubated at 37°C with shaking for 30 min before RNA extraction.

Quantitative real-time PCR.

For quantitative real-time PCR (qRT-PCR), 3 μg of RNA was treated using the TURBO DNA-free kit (Ambion) to remove genomic DNA contamination. Next, 500 ng of treated RNA was reverse transcribed using random hexamers with the Tetro cDNA synthesis kit (Bioline); as negative controls, reactions were run without reverse transcriptase. Reactions and controls were combined with 2× SYBR/fluorescein mix (SensiMix; Bioline) and 375 nM (each) forward and reverse primers and then run in a MyiQ thermocycler (Bio-Rad) using the following program: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 30 s. Melt curves were performed to verify the amplification of single products, and all values were adjusted for primer binding efficiency, as calculated from a standard curve of C6706 genomic DNA. Transcript levels were normalized to the levels of the housekeeping gene rpoB (34). The levels of rpoB under the experimental and reference conditions did not differ by more than approximately 1 cycle threshold in any experiment (data not shown).

Biofilm assays.

V. cholerae biofilms were cultured and stained with crystal violet as described previously (23, 37).

Competition experiments.

In vivo competition experiments were performed using 5-day-old CD-1 mice as described previously (37). Briefly, differentially marked samples were mixed 1:1 in 0.85% NaCl, and the inocula were plated on LB agar containing Sm and 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) to differentiate lacZ⁺ and lacZ-deficient strains and determine the input ratio. Mice were inoculated intragastrically with approximately 10⁵ CFU and sacrificed 21 h postinoculation. The small intestines were harvested, and tissue homogenates were plated on LB agar with Sm and X-Gal. The competition index was calculated as the ratio of blue versus white cells in the output normalized to the blue/white ratio of the initial inoculum. The mixtures used to inoculate mice for in vivo competition experiments were also diluted 1:1,000 into 2 ml of fresh LB medium and incubated overnight to determine the in vitro competition index. The animal protocol (09-259) was approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.

MOPS competition experiments.

Wild-type and ΔpstCAB2 mutant bacteria were grown as biofilms for 27 h, washed twice with 0.85% NaCl, and dispersed into 1 ml of MOPS medium with trace metals as described above. Both strains were mixed 1:1 into 2-ml cultures, and KH₂PO₄ was added to some cultures to a final concentration of 50 μM. Cultures were incubated at 37°C with shaking for 4 h or 24 h. Cells were then plated on LB agar supplemented with Sm and X-Gal, and the output blue/white ratios were normalized to the blue/white ratios of the initial mixture. Absolute CFU counts were also recorded for the mutant and wild-type at each time point.

RESULTS

The protein profiles of biofilm and planktonic V. cholerae cells are distinct.

To search for V. cholerae proteins responsible for the previously observed biofilm-induced hyperinfectivity, the protein contents of cells from biofilm and planktonic cultures were compared by two-dimensional gel electrophoresis. Because the phenotype was originally observed using stationary-phase shaking cultures and biofilms grown on glass in the laboratory (37), we analyzed cells grown under the same conditions in this study. Differences in the two protein profiles were readily apparent (Fig. 1), and in total, 152 spots reproducibly changed in intensity by ≥1.7-fold (data not shown). We selected four spots with dramatic, reproducible increases in intensity in the biofilm samples relative to the planktonic cell samples for identification by mass spectrometry (Fig. 1, bottom panels). Table 2 describes the proteins identified, including quantification of their relative levels in a biofilm compared to planktonic culture. One spot (Fig. 1, spot 36) contained peptides that matched two V. cholerae proteins: OppA, the periplasmic binding protein of the Opp oligopeptide transport system, and Lpd (dihydrolipoamide reductase), a component of the pyruvate dehydrogenase complex. Another spot (Fig. 1, spot 99) contained PstS2, the periplasmic binding protein of the Pst2 phosphate transport system. Finally, two small, basic protein spots (Fig. 1, spots 95 and 113) contained peptides matching a component of the large ribosomal subunit (Table 2).

Fig 1.

Proteomic profiles of V. cholerae cells grown as planktonic and biofilm cultures. The differential gel image shows spots that were more abundant (in red) or less abundant (in blue) in biofilm relative to planktonic culture. The four spots excised for peptide identification by mass spectrometry are labeled with their spot numbers (see text and Table 2 for details). The molecular weight markers are indicated on the right side of the gel; the direction of the isoelectric gradient is depicted above the gel. Montages of magnified images depicting spots 36 and 99 in the duplicate planktonic (PL) and biofilm (BF) gels are also shown.

Table 2.

Characteristics of several V. cholerae proteins enriched in biofilms

Spota	Locus	Name	Description	Mol wt	pI	Fold increaseb	Peptides identifiedc
36	VC1091	OppA	Periplasmic binding protein component of Opp oligopeptide transport system	55.1	6.5	4.1	23/73
36	VC2412	Lpd	Dihydrolipoamide dehydrogenase	55.1	6.5	4.1	12/58
99	VCA0070	PstS2	Periplasmic binding protein component of Pst2 phosphate transport system	36.7	6.3	3.2	15/36
95	VC0325	RplA	Ribosomal protein L1	29.7	7.1	7.2	7/32
113	VC2596	RplC	Ribosomal protein L3	25.3	7.1	9.7	11/30

^aProtein spots are those labeled by number in Fig. 1.

^bFold increase in average spot intensity in the biofilm samples relative to the planktonic samples.

^cNumber of peptides identified by mass spectrometry out of the total number of predicted trypsin digest products, including those with one missed cleavage event.

The identified proteins are not strongly regulated at the level of transcript abundance.

Because any of several types of regulation, ranging from changes in expression to altered protein stability, could explain the increased protein levels found in biofilms, we sought to determine whether the levels of the transcripts encoding these proteins changed along with protein abundance. We therefore measured the transcripts encoding the five identified proteins by qRT-PCR. When transcript levels in biofilms were compared to those in planktonic cultures, no large differences were noted (Fig. 2A). Transcript levels of pstS2, rplA, and rplC were effectively unchanged. In contrast, the levels of oppA and lpd declined in biofilms. Although these decreases were small, they were statistically significant (Fig. 2A). In a biofilm, V. cholerae cells are encased in a secreted exopolysaccharide matrix that is generated by the products of the vps genes. As a control, we measured the transcript level of vpsT, a transcription factor that is known to be upregulated in biofilms and to induce exopolysaccharide production (4). There were approximately 8-fold-higher levels of vpsT transcript in the biofilm samples, consistent with its role in biofilm formation (Fig. 2A).

Fig 2.

Changes in transcript levels of selected genes after growth in a biofilm. (A) The fold changes in transcript levels for the indicated genes in biofilm culture compared to planktonic (stationary-phase) culture were determined by qRT-PCR. The average fold changes from five biological replicates are shown (± standard deviations); data are from multiple independent experiments. The dashed line indicates no change in transcript. *, P < 0.05 compared to planktonic transcript levels, by the Mann-Whitney test. (B) Schematic of the V. cholerae pst and pst2 phosphate transporter loci. Genes depicted in black encode components of the Pst or Pst2 phosphate transporter system. Genes in gray are involved in phosphate or polyphosphate metabolism (ppk, polyphosphate kinase; ppx, exopolyphosphatase [31]). Genes in white are not predicted to be involved in phosphate metabolism. The illustration was made based on sequence information available at GenomeNet (www.genome.jp).

OppA has been associated with biofilm production in other Vibrio species (24, 47), and therefore we investigated an oppA deletion mutant. We determined that oppA is required for the growth of V. cholerae on peptides as the sole carbon source and that it contributes slightly to biofilm production, but the ΔoppA mutant displayed no colonization phenotype (data not shown). However, the importance of phosphate in V. cholerae pathogenesis has been previously demonstrated (32, 33, 43) and, therefore, PstS2 remained a promising candidate for further study.

The pst and pst2 loci are regulated differently in biofilms.

As mentioned above, pstS2 is part of pst2, one of two annotated phosphate transport loci in V. cholerae (Fig. 2B). The other locus, pst, has been the focus of prior studies, which have demonstrated its importance for survival in pond water, a nutrient-poor natural environment for V. cholerae (32). Dysregulation of PhoB in a pst mutant also leads to changes in levels of the second messenger cyclic-di-GMP and a severe defect in colonization of the infant mouse intestine (32, 33). At the pst locus, the periplasmic binding protein pstS1 is separated from the permease components by two genes in the opposite orientation. However, in the pst2 locus, pstS2 is found within the same operon as the genes encoding the three inner membrane permease components (pstCAB2). It is also worth noting that the phoU gene encoding the negative regulator of PhoB is found only at the pst locus, and the phoU gene was mutated along with pstCAB1 in the previous studies (32, 33) (Fig. 2B).

Because both systems are predicted to participate in phosphate uptake, we examined the transcriptional regulation of these phosphate transport loci to determine whether both are upregulated during growth in a biofilm. We used qRT-PCR to measure levels of the transcripts encoding the periplasmic binding protein pstS1 and the first permease component in each operon (pstC1 and pstC2). As shown in Fig. 3, the level of pstC2 transcript was significantly increased (approximately 40-fold) in biofilm compared to planktonic culture, but a slight decrease was observed for pstC1. Similar to the results shown above for pstS2 (Fig. 2A), pstS1 transcript levels remained unchanged. Whereas pstS1 and pstS2 had similar transcript levels under the planktonic reference condition, pstC2 was found at approximately 90-fold-lower levels than pstC1 (data not shown). Thus, pstC2 increases from a small amount of transcription in planktonic culture to a level much closer to that of pstC1 in biofilms. It was initially surprising that the transcript levels of pstS2 and pstC2, members of the same operon, showed different changes in biofilms. In E. coli, the multicistronic pst transcript is processed, and the separate fragments display differences in stability (2). A similar process may be at work in V. cholerae.

Fig 3.

Modulation of the pst and pst2 loci in biofilms. The fold changes in the transcript levels for the indicated genes in biofilm culture compared to planktonic (stationary phase) culture were measured by qRT-PCR. The average fold changes from five biological replicates are shown (± standard deviations); data are from multiple independent experiments. The dashed line indicates no change in transcript level. *, P < 0.05 compared to planktonic transcript levels by the Mann-Whitney test.

pst2 transcription is affected by phosphate availability.

We also investigated whether the upregulation of pstC2 expression is strictly biofilm induced or if the pst2 genes are expressed under low-phosphate conditions as well. We measured levels of the pstC2 transcript in a shaking culture of V. cholerae after a shift from rich medium (LB) to a nutrient-deficient MOPS medium lacking phosphate, using cells shifted to MOPS medium supplemented with 50 μM phosphate as a control. pstC2 levels were significantly higher in phosphate-deficient medium than in medium containing phosphate (5.5-fold ± 2.3-fold [mean ± standard deviation], n = 6; P = 0.0022 by the Mann-Whitney test), with no difference noted for pstC1 or pstS2 (data not shown). Levels of pstS1 and phoB transcripts were also higher (1.8-fold ± 0.1-fold and 1.7-fold ± 0.40-fold, respectively, n = 6; P < 0.05), perhaps as a reflection of the cellular response to phosphate starvation. Thus, phosphate starvation induces the expression of pstC2, but other aspects of growth in a biofilm likely affect its transcription (Fig. 3).

Evidence for a response to phosphate starvation by biofilm-associated cells.

As part of the qRT-PCR analysis of biofilm cultures, we also measured the amount of phoB transcript. As shown in Fig. 3, the transcript level of this master phosphate regulator increased significantly in biofilms compared to planktonic cells, indicating that the Pho regulon is activated during growth in a biofilm. This conclusion is supported by results showing that phoB and pstC2 transcript levels are elevated in surface-attached biofilm cells relative to unattached cells found in the liquid phase of the same culture (by approximately 25-fold and 15-fold, respectively [data not shown]). Because the latter experiments were performed using bacteria from the same tube and grown under the same conditions, we can surmise that the biofilm-associated bacteria experience phosphate limitation to a greater degree than the planktonic cells.

Biofilm production is reduced in a ΔpstS2 mutant, but the ΔpstCAB2 mutant produces wild-type levels of biofilm.

Because the components of the Pst2 system are upregulated at the translational or transcriptional level in biofilms, we sought to determine whether the system is required for biofilm formation. We generated in-frame deletions of pstS2 and pstCAB2 and tested both mutants for biofilm production. Both deletion strains grew equivalently to the wild type in LB medium under standard shaking culture conditions (data not shown). As determined by crystal violet staining, mutation of the genes encoding the permease (pstCAB2) did not affect biofilm production, while mutation of vpsR, which encodes a master regulator of exopolysaccharide production (48), nearly abrogated biofilm formation (Fig. 4A).

Fig 4.

Biofilm formation and fitness phenotypes of pst2 mutants. (A) Abilities of wild-type (WT) and mutant V. cholerae strains to form biofilms were assessed by crystal violet staining. The average values for at least nine independent cultures are shown (± standard deviation); data are from three independent experiments. **, P < 0.01 compared to WT; ***, P < 0.001 compared to each other strain by one-way analysis of variance with Tukey's post hoc test. (B) The ability of the ΔpstCAB2 mutant to survive in MOPS medium after dispersal from a biofilm was determined in a competition assay. Mutant and wild-type cells were competed in MOPS without phosphate (open symbols) or with 50 μM KH₂PO₄ (filled symbols). Each symbol represents the index for an individual culture; the horizontal line represents the median. Data from multiple independent experiments are shown (n ≥ 5). *, P < 0.05 compared to a hypothetical value of 1 (i.e., equal growth, marked by a dashed line) by the Wilcoxon signed rank test. (C) CFU were counted for the WT (filled symbols) and ΔpstCAB2 mutant (Δ, open symbols) in the input (0 h) and output (4 h and 24 h) samples from the competition experiments described for panel B. Data from the competition assays in MOPS medium without phosphate (left panel) and with 50 μM phosphate (right panel) are shown. Each symbol represents the count for the given strain in a single competition culture, and the horizontal line represents the median. Data from multiple independent experiments are shown (n ≥ 5). *, P < 0.05 by the Mann-Whitney test.

The ΔpstS2 mutant showed a slight but reproducible decrease in biofilm biomass in LB medium (Fig. 4A). However, the ΔpstS2 mutant forms biofilms containing wild-type numbers of CFU, and the transcript levels of vpsL and vpsT, two genes involved in the production of exopolysaccharide (48), are comparable to levels in a wild-type biofilm (data not shown). Thus, it appears that the ΔpstS2 mutant secretes less exopolysaccharide through the regulation of other vps genes and/or secretes less of other matrix material. PhoB has been shown to impair vpsR transcription and biofilm production in an El Tor strain of V. cholerae (36), and phoB expression is increased in ΔpstS2 biofilms (data not shown). It is possible that PhoB activity leads to the slight decrease in biofilm formation, but the exact cause remains unknown. Taken together, these data indicate that the pst2 locus, although it is induced during growth in a biofilm, does not considerably affect biofilm formation.

The Pst2 system contributes to the growth of V. cholerae in phosphate-deficient conditions after dispersal from a biofilm.

Previous work has shown that the Pst system is important for survival in pond water, a natural habitat for V. cholerae that has low levels of available phosphate (32). Because the pstC2 gene is strongly upregulated in biofilms and in media lacking phosphate, we hypothesized that the Pst2 system contributes to the fitness of V. cholerae in the context of growth in a biofilm and subsequent dispersal under low-phosphate conditions. We therefore tested the ΔpstCAB2 strain for growth in minimal medium after dispersal from a biofilm by using a competition assay. In this assay, the mutant and a differentially marked (lacZ-deficient) wild-type V. cholerae strain were grown as biofilms, dispersed, mixed at a 1:1 ratio, and incubated for 4 or 24 h in MOPS medium with or without phosphate. As shown in Fig. 4B, the ΔpstCAB2 mutant showed decreased fitness compared to the wild type at both time points after incubation in MOPS medium without added phosphate. However, when the medium was supplemented with high levels of phosphate (50 μM), the fitness defect disappeared (Fig. 4B). When examining the CFU in our input and output cultures, we observed that wild-type bacteria increased in number by approximately 5-fold after 4 h, regardless of the phosphate concentration (Fig. 4C). However, the ΔpstCAB2 mutant only increased by a similar amount in the high-phosphate medium; a significantly smaller increase (approximately 2-fold) was seen for the mutant in the phosphate-deficient medium (Fig. 4C, left panel). These data support a role for the Pst2 system in phosphate uptake during growth in a biofilm.

The Pst2 system contributes to the enhanced colonization of the small intestine by biofilm-derived V. cholerae cells.

Given the significant upregulation of pstC2 transcription in biofilms (Fig. 3) and the role of Pst2 in fitness under conditions of phosphate starvation after dispersal from a biofilm (Fig. 4), we wanted to assess the contribution of the second phosphate uptake system to V. cholerae pathogenesis. Specifically, we sought to determine whether the Pst2 system contributes to biofilm-induced hyperinfectivity. A role for the Pst2 system in this phenotype would be defined by greater attenuation of colonization by the mutant after growth in a biofilm compared to colonization after growth in planktonic cell culture.

We therefore tested the ΔpstS2 and ΔpstCAB2 mutants for colonization using the infant mouse model. These experiments involved coinfection of mice with a lacZ-deficient strain mixed 1:1 with the mutant strain before inoculation. We tested each mutant after growth in a biofilm as well as after growth to stationary phase in LB medium with shaking (planktonic culture). It should be noted that biofilms of V. cholerae were fully dispersed as individual cells immediately prior to inoculation, such that the structure of the biofilm was not relevant. In all cases, mutants were compared to the lacZ-deficient strain grown under the same conditions, and the inocula used for in vivo competition assays were also competed in vitro. We chose to compare wild-type and mutant cells grown the same way (i.e., biofilm or planktonic culture) to minimize differences in culture conditions that may have affected the results. Additionally, inoculating a mixture of biofilm-derived cells and planktonic cells of this strain leads to a relatively wide range of competition indices, reaching over 100 (37), which may have made differences between the wild-type and mutant harder to discern.

As shown in Fig. 5A, the mutant lacking the pstS2 gene was attenuated for colonization both after growth in a biofilm and in planktonic culture. This decrease in colonization efficiency was not due to an overall competitive disadvantage, because the same mixtures grew in vitro to levels equal to or greater than the wild type. In fact, when the ΔpstS2 mutant was grown in a biofilm and then dispersed, it outcompeted the wild-type strain in LB medium in vitro (median competition index, approximately 15) (Fig. 5A). There was considerable variability in the magnitude of the competition index from culture to culture, but this fitness increase was statistically different from the hypothetical value of 1 (i.e., equal growth; P = 0.001 by the Wilcoxon signed rank test). The ΔpstCAB2 permease mutant also displayed significantly reduced colonization efficiency after growth in a biofilm or in planktonic culture (Fig. 5B), and in both cases, the mutant grew at wild-type levels in vitro. Importantly, the in vivo colonization defect was significantly greater for the ΔpstCAB2 mutant after growth in a biofilm compared to planktonic culture (P = 0.0017, Mann-Whitney test). Therefore, the PstCAB2 permease can be defined as a factor contributing to the biofilm-induced hyperinfectivity of V. cholerae.

Fig 5.

Colonization phenotypes of the ΔpstS2 and ΔpstCAB2 mutants. The ΔpstS2 (A) and ΔpstCAB2 (B) mutants were assessed for their abilities to colonize the infant mouse small intestine in a competition assay. The competing wild-type and mutant strains were grown as planktonic cells in shaking culture (PL) or as biofilm (BF). The inocula consisted of 1:1 mixtures of wild-type and mutant strains grown under the same conditions. Each symbol represents the competition index for one mouse or one LB medium culture. The horizontal line represents the median. Median competition indices were compared to a hypothetical value of 1 (i.e., no difference in colonization) by the Wilcoxon signed rank test. The calculated P values are shown above each data set.

Phosphate starvation leads to a modest colonization advantage for planktonic V. cholerae cells.

Because the pst2 system is upregulated under conditions of phosphate starvation, and because the PstCAB2 permease plays a role in biofilm-induced hyperinfectivity, we examined whether phosphate starvation itself enhances the ability of V. cholerae to colonize the infant mouse intestine. For these experiments, wild-type V. cholerae and a differentially marked lacZ-deficient strain were grown to log phase in LB medium, then shifted to MOPS medium without phosphate (wild type) or MOPS medium supplemented with 50 μM phosphate (lacZ-deficient cells) prior to inoculation into infant mice and LB cultures. As shown in Fig. 6, the phosphate-starved cells demonstrated a slight, but significant, colonization advantage compared to the cells incubated in the presence of phosphate. A similar growth advantage was also seen in LB culture (Fig. 6, right), indicating that the response to phosphate starvation can be advantageous in multiple environments. The approximately 2-fold advantage observed for phosphate-starved cells was consistent with the 2- to 3-fold defect in colonization seen for the ΔpstCAB2 mutant biofilms.

Fig 6.

Phosphate starvation induces a slight colonization advantage. Phosphate-starved wild-type V. cholerae cells were assessed for the ability to colonize the infant mouse small intestine in a competition assay as described in Materials and Methods. Each symbol represents the competition index for one mouse (in vivo) or one LB medium culture (in vitro). The horizontal lines represent the medians. Median competition indices were compared to a hypothetical value of 1 (i.e., no difference in colonization or growth) by the Wilcoxon signed rank test, and the calculated P values are shown above each data set.

DISCUSSION

In this study, we searched for factors involved in the hyperinfectivity of V. cholerae biofilms by comparing the protein profiles of the pathogen during growth in planktonic versus biofilm cultures. Among the proteins upregulated in a biofilm was PstS2, a periplasmic protein predicted to bind phosphate. The Pst2 phosphate transport system, upregulated at the transcriptional level in biofilms, contributes to growth in medium lacking phosphate, and mutant strains lacking either pstS2 or pstCAB2 are attenuated for colonization in the infant mouse model. The attenuation for the permease mutant was greater after growth in a biofilm, consistent with a role for Pst2 in biofilm-induced hyperinfectivity.

Previously, we observed a strong biofilm-induced hyperinfectivity phenotype for C6706, the strain used in this study; biofilm-derived cells outcompeted cells grown in shaking culture by 10- to 100-fold (37). The current study revealed a 2-fold drop in colonization efficiency for the ΔpstCAB2 mutant grown as a biofilm, in comparison to the same mutant grown in planktonic culture. Therefore, the Pst2 system is only one of what is likely a large of number of factors contributing to hyperinfectivity and can, at best, be considered responsible for 2% of the phenotype. In addition, phosphate starvation increased colonization by a similar magnitude (approximately 2-fold), indicating that any phosphate starvation experienced within a biofilm is not a critical underlying cause for the observed hyperinfectivity. As yet, little is known about what other factors contribute to the phenotype, or even whether hyperinfectivity is VPS dependent. Recently, a V. cholerae mutant lacking two extracellular nucleases was shown to be impaired for colonization after growth in a biofilm (35). Those authors suggested that the nucleases may be required for dispersal from small clumps of biofilm. It would be interesting to see whether the nucleases are also required for normal colonization when cells are fully dispersed from biofilms before infection.

Our data indicate that V. cholerae upregulates the Pst2 phosphate transport system, both in response to conditions of phosphate starvation and during growth in a biofilm. Because the Pst2 system also contributes to proper colonization in the infant mouse model, the expression of the second system is likely to be induced in vivo. Unfortunately, we were unable to detect bacterial RNA in the mouse intestine at early time points after inoculation, when factors such as Pst2 may be most critical (data not shown). The Pst2 system appears to be dispensable for growth in planktonic culture in rich medium, when its transcript levels are normally low. However, the system is upregulated in response to phosphate starvation and contributes to survival under low-phosphate conditions. It is possible that the Pst2 system has a higher affinity for phosphate, making its activity advantageous under low-phosphate conditions, but no data are currently availably regarding the affinities of Pst and Pst2.

The increased expression of phoB in biofilms suggests that V. cholerae cells experience phosphate starvation during growth in a biofilm. Because phosphate is also an important resource inside the host, it is reasonable that growth in a biofilm, accompanied by phosphate stress and subsequent upregulation of the Pst2 system, provides a colonization advantage. Our data indicate that the Pst2 system assists in phosphate acquisition during growth in a biofilm and that this phosphate accumulation enhances fitness after dispersal. Specifically, our results support a model in which phosphate is taken up in a biofilm in a Pst2-dependent manner, providing V. cholerae with phosphate stores that allow the bacterium to grow even when no phosphate is available. In contrast, the ΔpstCAB2 mutant is unable to store additional phosphate and, therefore, grows less efficiently in phosphate-deficient medium. These results agree with previous work showing that polyphosphate storage in V. cholerae aids in survival under stress conditions in aquatic environments (19). In addition, the production of the system during biofilm formation may also prepare the bacteria to take up phosphate shortly after dispersal into a natural aquatic reservoir or within the host, or the Pst2 system may be important for sensing phosphate once the bacteria reach the small intestine.

Previous studies have shown that the Pst system is important for virulence in a number of pathogens. In the case of V. cholerae, deletion of the pst locus (including phoU) was shown to increase PhoB activity and thereby impair colonization (32). Similar dysregulation of PhoB was noted in other pathogens bearing pst operon deletions, and the altered expression of this critical master regulator was determined to be responsible for the virulence defects observed (7). In this study, deletion of the Pst2 permease (pstCAB2) did not cause increased expression of phoB or phoA, a known member of the Pho regulon (data not shown). Therefore, disruption of the V. cholerae Pst2 system may lead to reduced colonization in a manner different from disruption of the pst locus. Phosphate may be scarce in the host; thus, under the stressful conditions of host colonization, the Pst2 system may be needed to support the Pst system. The considerably greater colonization defect for a mutant lacking the pst locus (competition index, approximately 0.005 [32]) may support a secondary role for Pst2 in vivo, although the deletion of phoU and subsequent dysregulation of PhoB activity in the pst mutant make it difficult to directly compare those results with ours. Alternatively, the ΔpstCAB2 mutant may have acquired less phosphate than the wild-type strain during growth in a biofilm, leading to reduced growth in the host intestine.

We observed reduced phenotypes for the ΔpstS2 mutant compared to the ΔpstCAB2 mutant in terms of colonization and growth under low-phosphate conditions (data not shown). These differences may be due to redundancy among periplasmic phosphate binding proteins. In the archaeon Halobacterium salinarum, which possesses two pst loci, each PstS homolog has been shown to interact with both of the phosphate permeases (14). We have also identified an additional pstS homolog in V. cholerae (VCA0807) by using BLAST, and this gene is transcriptionally upregulated in biofilms (data not shown). Therefore, it is possible that a third protein also binds phosphate in the periplasm of V. cholerae, further reducing the relative importance of each periplasmic binding protein.

This study is a preliminary view of the biofilm proteome in V. cholerae. It is likely that components of other nutrient uptake systems and other factors are also upregulated in biofilm samples. One recent study used two-dimensional gel electrophoresis to uncover proteins enriched in a rugose variant of V. cholerae that overproduces the exopolysaccharide found in biofilms, compared with a smooth (nonrugose) variant. That study identified RbmA, a secreted protein that contributes to biofilm integrity (13). A more recent study identified several other matrix-associated proteins in V. cholerae biofilms (1). Undoubtedly, several structural proteins, such as RbmA and Bap1, are also enriched in biofilms, but were not identified here. Further analysis of the biofilm proteome will help to generate a clearer understanding of the physiological changes inherent to biofilm formation in V. cholerae. Whereas transcriptomic analyses can be informative, our data highlight the need for proteomic studies, because the proteins identified as being significantly increased in abundance in biofilms were not strongly regulated at the transcriptional level.

Previous studies using animal models to study V. cholerae have predominantly involved planktonically grown cells, but biofilm fragments may represent the true infectious particle during the natural course of cholera outbreaks. We and others speculate that factors upregulated in biofilms and involved in biofilm-induced hyperinfectivity may represent alternative molecular targets for the generation of V. cholerae vaccines and/or cholera therapeutics (44). Furthermore, other facultative bacterial pathogens become more infectious upon growth under conditions other than broth culture. For example, Yersinia pestis passaged through the flea vector assumes a state with increased antiphagocytic properties, which may preadapt it to resist host innate immune factors (42). Citrobacter rodentium, like V. cholerae, enters into a hyperinfectious state after passage through the host, which increases its ability to infect a subsequent host (12, 27, 46). Future studies aimed at identifying proteins enriched under conditions in which other bacterial pathogens are more infectious may uncover promising molecular targets in those organisms or even reveal conserved virulence pathways required for survival in the host.