Vibrio cholerae anaerobic induction of virulence gene expression is controlled by thiol-based switches of virulence regulator AphB

Abstract

Bacterial pathogens have evolved sophisticated signal transduction systems to coordinately control the expression of virulence determinants. For example, the human pathogen Vibrio cholerae is able to respond to host environmental signals by activating transcriptional regulatory cascades. The host signals that stimulate V. cholerae virulence gene expression, however, are still poorly understood. Previous proteomic studies indicated that the ambient oxygen concentration plays a role in V. cholerae virulence gene expression. In this study, we found that under oxygen-limiting conditions, an environment similar to the intestines, V. cholerae virulence genes are highly expressed. We show that anaerobiosis enhances dimerization and activity of AphB, a transcriptional activator that is required for the expression of the key virulence regulator TcpP, which leads to the activation of virulence factor production. We further show that one of the three cysteine residues in AphB, C₂₃₅, is critical for oxygen responsiveness, as the AphB^C235S mutant can activate virulence genes under aerobic conditions in vivo and can bind to tcpP promoters in the absence of reducing agents in vitro. Mass spectrometry analysis suggests that under aerobic conditions, AphB is modified at the C₂₃₅ residue. This modification is reversible between oxygen-rich aquatic environments and oxygen-limited human hosts, suggesting that V. cholerae may use a thiol-based switch mechanism to sense intestinal signals and activate virulence.

The Gram-negative bacterium Vibrio cholerae, the causative agent of the acute, dehydrating diarrheal disease cholera, has figured prominently in the history of infectious diseases as a cause of periodic, deadly pandemics. V. cholerae resides in aquatic environments between epidemics, and human infection normally starts with the ingestion of contaminated food or water. Vibrio cells surviving passage through the acidic gastric environment enter the small intestine, where they must produce an array of virulence factors including cholera toxin (CT) and the toxin co-regulated pilus (TCP) that are transcriptionally regulated by multiple systems (1). The primary, direct transcriptional activator of virulence genes is ToxT, whose transcription is regulated by the ToxRS and TcpPH proteins. Two additional activators encoded by unlinked genes, AphA and AphB, regulate the transcription of tcpPH.

The environmental cues within the host and their effect on the expression of virulence genes in V. cholerae in vivo remain poorly characterized. It has been shown that anaerobiosis serves as one of the host environmental factors that modulate virulence factor production (2). This is not surprising because it is generally presumed that the oxygen concentration in the intestine is low (3). A recent report showed that under anaerobic conditions, tcpP expression is higher and this effect depends on AphB (4). However, whether and how this AphB-mediated tcpP expression contributes to anaerobic virulence induction is unclear. In this study, we found that under anaerobic conditions, AphB proteins are more active than under aerobic conditions. This leads to higher expression of TcpP, which plays a key role in virulence factor production under anaerobic conditions. We further demonstrated that one of three cysteine residues (C₂₃₅) of AphB is critical for sensing O₂ concentration and modulating AphB activity.

Results

Virulence Gene Expression Requires Oxygen-Limiting Conditions.

When V. cholerae enters host intestines, genes related to V. cholerae pathogenesis, such as the tcp gene clusters, are strongly induced (5). In vitro, virulence genes can be induced using a set of artificial conditions, such as liquid AKI medium for El Tor strains or 30 °C and pH 6.5 for classical strains (6, 7). Culturing under AKI conditions requires an initial phase in the absence of shaking, suggesting that oxygen levels may have an effect on virulence gene induction in El Tor strains.

To further investigate the relationship between ambient oxygen levels and virulence factor expression, we examined the transcription and translation of the major virulence determinant TcpA of El Tor strain C6706 containing a P_tcpA-luxCDABE reporter plasmid in AKI under aerobic (shaking, without any stationary culturing), microaerophilic (stationary), and anaerobic (anaerobic chamber) conditions. We determined that O₂ levels did not affect Lux activity itself by comparing luciferase production in V. cholerae cells containing a P_BAD-luxCDABE plasmid grown under various conditions. We found that under microaerophilic and anaerobic conditions, tcpA was highly induced (Fig. 1A) and TcpA protein could readily be detected by Western blot (Fig. 1B). However, tcpA expression was dramatically reduced under aerobic conditions, and little TcpA was detected.

Fig. 1.

The effect of oxygen on virulence factor production. V. cholerae C6706 containing a P_tcpA-luxCDABE plasmid was grown under aerobic (shaking), microaerophilic (stationary), or anaerobic (anaerobic chambers) conditions in AKI medium (6) at 37 °C until OD₆₀₀ reached ≈0.2. (A) Transcription of tcpA. The units were calculated from Lux units (arbitrary light units/OD₆₀₀). Results are the mean of three experiments ± SD. (B) Whole-cell extracts (normalized by the total protein amounts) were subjected to Western blot analysis using anti-TcpA antiserum (Upper). The corresponding cell-free culture fluids were assayed for CT production (Lower). (C) GFP-labeled WT or tcpA mutants were inoculated on the surface of mouse small intestinal tissues and incubated at 37 °C aerobically or anaerobically for 4 h. Rinsed tissue samples were then visualized using a fluorescence confocal laser microscope. Representative micrographs are shown. (Scale bar: 10 μm.)

Oxygen had a similar effect on the production of CT, the other major virulence determinant, which is under the control of the same regulatory cascade as TCP (Fig. 1B). We also examined the effect of oxygen on virulence gene expression in O395, a classical strain, and MO10, an O139 strain, and found that TcpA was also greatly induced under oxygen-limiting conditions in these strains using the AKI medium (Fig. S1A), suggesting that anaerobiosis is important for virulence gene expression in various V. cholerae strains. Interestingly, when we used the previously described virulence-inducing conditions for classical strains, we found that TcpA production was higher under aerobic growth than under anaerobic growth (Fig. S1B). These data are consistent with a previous report (8). We do not know why classical strains behave differently in AKI medium grown at 37 °C compared with LB medium grown at 30 °C; however, 30 °C may not reflect the actual physiological conditions V. cholerae encounters during infection.

We then tested the ability of V. cholerae to adhere to intestinal tissues ex vivo. We dissected out fragments of mouse small intestines, loaded bacterial cultures (labeled with GFP) on top of the opened tissues, and incubated them aerobically or anaerobically for 4 h at 37 °C. After being rinsed with PBS, the samples were subjected to fluorescence confocal laser microscopy. We found that under aerobic conditions, very few bacterial cells were visible on the intestinal surface (Fig. 1C Left), whereas without oxygen, wild-type (WT) V. cholerae attached efficiently (Fig. 1C Center). The adherence required TCP, because a tcpA mutant failed to attach to intestines even under anaerobic conditions (Fig. 1C Right). Quantification of the attached bacteria confirmed the patterns observed by microscopy (Fig. S2). These observations imply that V. cholerae may detect oxygen-limiting conditions in the small intestine to induce gene expression required for colonization.

Anaerobiosis-Induced Virulence Gene Expression Acts at the Level of tcpP Expression Through AphB.

Signal transduction cascades coordinately regulate virulence in V. cholerae (1). Skorupski and colleagues recently showed that AphB responds to anaerobiosis to induce tcpP expression (4), but whether this induction contributes to the virulence induction that we observed above had not been investigated. To further confirm that the oxygen effect is mediated through TcpP, we compared transcriptional levels of genes encoding virulence regulators in bacteria grown aerobically and anaerobically using a luxCDABE transcriptional fusion plasmid (9). We found that transcription of both toxT and tcpP was induced under anaerobic conditions, whereas the expression levels of toxR were similar in the presence or in the absence of oxygen (Fig. 2A). Because toxR expression is independent of tcpP regulation, which is upstream of toxT, and ToxT can activate tcpA expression in Escherichia coli independently of oxygen concentration (Fig. S3), these results suggest that the effect of oxygen on virulence gene expression mainly occurs by modulation of tcpP transcription. To further confirm this finding, we measured tcpP transcript levels using an oligonucleotide-based S1 nuclease assay (10) and TcpP production using a Western blot. Consistent with the luminescence reporter data, both tcpP transcription and TcpP production were strongly induced under anaerobic conditions (Fig. 2 B and C).

Fig. 2.

The effect of oxygen on the expression of virulence regulators. (A) V. cholerae containing the indicated luxCDABE transcriptional reporter plasmids were grown under aerobic and anaerobic conditions in AKI medium at 37 °C until OD₆₀₀ reached ≈0.2, at which point luminescence was measured. Data are displayed as the fold induction in cultures grown anaerobically over those grown aerobically. The results are the mean of three experiments ± SD. (B) Total RNA was extracted from cultures grown under the conditions described above and subjected to oligo-based S1 nuclease assays (10) using ³²P-labeled probes complementary to tcpP. A probe for 16S rRNA was used as a control, and no difference between RNA harvested from O₂⁻ and from O₂⁺ cultures was detected (Fig. S4). (C) Whole-cell extracts from cultures above (normalized by the total protein amounts) were subjected to Western blot analysis using anti-TcpP antiserum.

It has been demonstrated that the expression of tcpP is activated by AphA and AphB (11). We thus wondered whether oxygen regulation of tcpP expression is mediated by AphA or AphB. Although O₂-limiting conditions did not alter either aphA or aphB expression (Fig. 2A), it remained possible that oxygen could affect the protein activity of AphA or AphB or of additional unknown modulators. We tested separately the effect of AphA and AphB on tcpP expression in the absence of or in the presence of oxygen. We introduced pBAD-aphA or pBAD-aphB plasmids into aphAB double mutants containing a P_tcpP-luxCDABE reporter plasmid and grew the resulting strains in the presence of 0.01% arabinose aerobically or anaerobically. We found that although expression of either AphA or AphB was able to induce tcpP expression, only AphB displayed oxygen-dependent activation of tcpP; i.e., under anaerobic conditions, tcpP was highly induced by AphB (Fig. S5A). A similar pattern of tcpP induction was also observed in an E. coli strain (Fig. S5B). Together, these data suggest that oxygen modulates AphB activity to regulate tcpP expression.

Oxygen Modulates AphB Activity Through a Cysteine Residue.

Located in the cytoplasm, AphB is a LysR-family protein that consists of an N-terminal DNA binding domain and a C-terminal substrate-binding domain. One of the LysR-family proteins, OxyR of E. coli, is a redox-sensing protein that activates expression of defense genes in response to oxidative stress (12). One of the six cysteine residues (C₁₉₉) in OxyR is crucial for sensing oxidation, as a cysteine 199 to serine (C199S) mutant loses function (13). To examine whether AphB may behave similarly to OxyR, we mutated each of its three cysteine residues to serine using site-directed mutagenesis and examined whether these mutations affect AphB function. Fig. 3A demonstrates that changing either or both C₇₆ and C₉₄ to serine residues has little, if any, effect on AphB activity. Both mutants could activate tcpP expression under anaerobic conditions, but not under aerobic growth. The C235S mutation, however, rendered AphB constitutively active and capable of inducing tcpP expression even during aerobic growth (Fig. 3A), suggesting that C₂₃₅ is critical for oxygen regulation of AphB activity. Moreover, we found that expression of AphB^C235S was sufficient to fully activate virulence gene expression under aerobic growth conditions (Fig. 3B). Similarly, expression of the C235S AphB mutant could enhance TcpP production in classical and O139 strains (Fig. S6). Finally, V. cholerae containing AphB^C235S could adhere to the intestinal surface efficiently, even under aerobic conditions (Fig. S7). These data indicate that AphB oxidative sensing is a major point of control in oxygen-related virulence induction, at least under our in vitro test conditions.

Fig. 3.

The importance of AphB cysteine residues in sensing oxygen levels. (A) aphB deletion mutants containing pP_tcpP-luxCDABE and either pBAD24 (vector control) or pBAD-aphB (WT and cysteine → serine mutants) were grown aerobically (filled bars) and anaerobically (open bars) in the presence of 0.01% arabinose as described above, and luminescence was measured. The tcpP induction was calculated by normalizing against Lux units of the vector control. The results are the mean of three experiments ± SD. (B) aphB deletion mutants containing pP_tcpA-luxCDABE and either pBAD-aphB^wt or pBAD-aphB^C235S were grown aerobically (filled bars) and anaerobically (open bars) in the presence of 0.01% arabinose as described above, and luminescence was measured (Upper). Results are the mean of three experiments ± SD. Whole-cell extracts (normalized by the total protein amounts) were subjected to Western blot analysis using anti-TcpA antiserum (Lower).

Oxygen-Dependent Modification of C₂₃₅ Prevents AphB Multimerization.

To understand why AphB is more active under anaerobic conditions, we applied in vivo cross-link assays to examine AphB multimerization. Fig. 4A shows that under anaerobic growth, significantly more WT AphB formed dimers than those grown under aerobic conditions, whereas similar amounts of dimers of AphB^C235S mutant proteins were formed under both aerobic and anaerobic conditions. In the absence of the cross-linking reagent, few AphB dimers were detected in all cells. These data indicate that the cellular oxidation state affects AphB multimerization and that this effect acts through the C₂₃₅ residue.

Fig. 4.

The effect of oxygen on AphB dimerization and activity in vitro. (A) In vivo cross-linking of AphB. aphB deletion mutants containing pBAD-aphB^wt or pBAD-aphB^C235S were grown aerobically and anaerobically to OD₆₀₀ ≈ 0.2. Resuspended cells were then treated with cross-linking reagent DSP and subjected to a Western blot using AphB antibodies. (B Upper) Gel shift assays using tcpP promoter DNA and 0.1 μM recombinant AphB^wt (lanes 2, 3, and 7) or AphB^C235S (lanes 4 and 5). The proteins were purified aerobically from shaking cultures (lanes 2–5) or purified in an anaerobic chamber from anaerobically grown cultures (lane 7). When indicated, 10 mM DTT was included in the reaction. (Lower) The dimer percentage of different purified AphB proteins was determined using size-exclusion chromatography.

We further tested the effect of oxygen on AphB biochemically. Recombinant AphB^wt and AphB^C235S containing a C-terminal His₆-tag were generated and purified. These AphB-His₆ fusion proteins were functional, as they could activate tcpP expression in E. coli. Purified AphB has been shown to bind tcpP promoter DNA in gel shift assays (11). We performed similar assays but omitted the reducing agent DTT from the binding reaction and found that WT AphB purified from cultures grown aerobically failed to bind tcpP promoter DNA (Fig. 4B, lane 2). Addition of DTT to the reaction restored the binding activity of AphB^wt (Fig. 4B, lane 3). As anticipated based on the data described above, AphB^C235S protein could shift the target DNA in the absence and in the presence of DTT (Fig. 4B, lanes 4 and 5). When AphB^wt was purified under anaerobic conditions from cultures grown anaerobically, the protein was active without addition of DTT even after it was exposed to the air (Fig. 4B, lane 7). In addition, we used size-exclusion chromatography to estimate the percent of dimers present in purified AphB protein samples. We found that AphB^wt purified from aerobically grown cultures contained few dimers (0.2%) and that DTT treatment promoted dimerization (Fig. 4B). Similar to AphB^wt purified from anaerobically grown conditions (9% dimers), AphB^C235S contained a significant percentage of dimers (6%), and DTT treatment did not further promote dimerization. The dimer percentage of purified AphB protein correlated well with activity and dimerization in vivo (Figs. 3A and 4A), as well as tcpP binding activity in vitro (Fig. 4B), implying that anaerobiosis-dependent dimerization of AphB is important for tcpP inducing activity.

To confirm that dimerization is important for AphB functionality, we randomly mutagenized the aphB coding sequence by error-prone PCR and screened for AphB missense mutants that could not activate tcpP expression. We found that an M133I mutation greatly reduced AphB dimerization under anaerobic conditions (Fig. S8A). The expression of tcpP, toxT, and tcpA in V. cholerae containing this mutation was significantly reduced, and this strain could not colonize well (Fig. S8B). Together, these data suggest that AphB may be modified at the C₂₃₅ residue under aerobic growth, rendering the protein inactive by preventing dimerization. Certain cellular enzymes may be involved in the AphB^C235 modification, as purified AphB is insensitive to oxygen. The modification also is cleavable by DTT.

In an effort to determine the possible modification of AphB at the C₂₃₅ residue during aerobic growth, AphB^wt and AphB^C235S purified aerobically from cultures grown aerobically were analyzed by nano flow-liquid chromatography coupled to tandem mass spectrometry. Samples were either trypsin-digested directly or reduced and alkylated using DTT/iodoacetamide (DTT/IAM) before digestion with trypsin. For each protein, multiple unique peptides were identified, and a high degree of peptide coverage was achieved. The overall data are summarized in Table S1. In the case of AphB^wt, without reduction/alkylation (DTT/IAM) treatment, the R.SAC₂₃₅SEGLGITLMPDVMLR.E peptide was identified either as disulfide-bonded to DC₇₆SPLLER or LASMTEEITDEC₉₄R peptides, but not in its unmodified form. A fragment corresponding to a cleavage product immediately after C₂₃₅ could also be detected. After DTT/IAM treatment all alkylated cysteine containing peptides were detected. With the AphB^C235S protein, the R.SAS₂₃₅SEGLGITLMPDVMLR.E peptide was readily detected independent of DTT/IAM treatment. These data indicate that before reduction, no free thiol group was available at position 235 in AphB^wt that was produced and purified under aerobic conditions, suggesting that in AphB^wt, the C₂₃₅ is modified.

To further evaluate the redox activity of C₂₃₅ in AphB, we purified recombinant AphB^C76S/C94S and AphB^{C76S/C96S/C235S} and reduced these proteins with DTT. We measured the free thiol content of these proteins using DTNB [5,5-dithiobis (2-nitrobenzoic acid)] assays (14) and found that reduced AphB^C76S/C94S contained approximately 1 thiol per monomer, whereas oxidized AphB^C76S/C94S and reduced AphB^{C76S/C96S/C235S} contained very little, if any, free thiol (Fig. S9A). Then, reduced AphB^C76S/C94S was incubated with cumene hydroperoxide (CHP), and we were able to detect the transient sulfenic acid intermediate (AphB^C235-SOH) by using NBD (4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole) assays (ref. 14; Fig. S9B). These data also suggest that the C₂₃₅ residue of AphB can be modified.

AphB Thiol-Based Switches Are Reversible.

The above results support a notion that AphB is more active during anaerobic growth and that oxidative modification at the C₂₃₅ residue leads to reduction of its activity. As the V. cholerae life cycle consists of transitions between oxygen-rich and -poor environments, we examined whether the effect of oxygen on AphB activity is reversible. We grew aphB deletion mutants containing P_tcpP-luxCDABE reporter plasmids and either pBAD-aphB^wt or pBAD-aphB^C235S aerobically and anaerobically in the presence of arabinose to induce the expression of aphB. We then removed arabinose and shifted the cultures to the opposite condition. As expected, AphB^C235S induced tcpP expression under all of the conditions (Fig. 5). Transferring of AphB^wt from anaerobic to aerobic growth diminished tcpP expression, whereas transfer from aerobic to anaerobic conditions restored tcpP expression (Fig. 5). These data suggest that the effects of oxygen on AphB activity are reversible.

Fig. 5.

The reversibility of the effect of oxygen on AphB. aphB deletion mutants containing pP_tcpP-luxCDABE and either pBAD-aphB^wt (wells 1, 3, 5, and 7) or pBAD-aphB^C235S (wells 2, 4, 6, and 8) were grown aerobically (wells 1–4) and anaerobically (wells 5–8) in the presence of 0.01% arabinose for 4 h. Arabinose was then removed, and cultures were shifted to anaerobic (wells 3 and 4) or aerobic (wells 7 and 8) conditions for an additional 4 h. Cultures were then transferred to 96-well plates, and photographs were taken in the dark using a LAS4010 Imager (GE Healthcare).

Discussion

For V. cholerae and other bacterial pathogens to successfully infect a host, they must readily alter their phenotypic properties to allow for successful colonization and growth in the host environment. V. cholerae displays complex patterns of transcriptional changes that accompany the adaptation to host environments. Extensive in vitro studies have demonstrated that the ability of V. cholerae to colonize and cause disease requires the intricately regulated expression of a number of virulence factors during infection. However, what and how host signals control V. cholerae gene expression after bacteria enter the small intestine and begin to colonize is largely unknown. Here we show that the low oxygen concentration in host small intestines may serve as one of the signals that activate virulence gene expression in V. cholerae. Oxygen availability has been shown to modulate virulence gene expression in a number of gastrointestinal pathogens, such as Shigella (15), Salmonella (16, 17), and enterohemorrhagic E. coli (18). We found that elevated virulence factor production under microaerophilic and anaerobic conditions is due to the increase in AphB transcriptional activation of the tcpP promoter, which leads to increased production of TcpP and thereby downstream virulence genes (Fig. 2). In addition, other global regulatory systems for anaerobic metabolism, such as Fnr and the ArcA/ArcB two-component system (19), may be involved in modulating expression of V. cholerae virulence genes. It has been shown that ArcA in V. cholerae is required to activate virulence gene expression (20), but the exact mechanism of this regulation is unclear. However, Fnr and ArcA/ArcB are not involved in AphB oxygen-dependent activity regulation (4).

AphB belongs to the LysR-family of transcriptional regulators (21). The LysR-family proteins have a conserved structure with an N-terminal DNA-binding helix–turn–helix motif and a C-terminal coinducer-binding domain (22). No coinducers of AphB have been identified. OxyR of E. coli is a well-characterized LysR-type protein that is activated in response to oxidation (23). The exact mechanism of OxyR action remains under debate: one group suggested that active forms of OxyR contain a single intramolecular disulfide bond (24), while another group showed that different stable chemical modifications at a single cysteine are responsible for OxyR activity (25). We found that thiol-mediated oxidative modification affects AphB activity. One of the three cysteine residues (C₂₃₅) in AphB is essential for sensing oxygen concentration. Our studies show that with regard to inducing tcpP expression, WT AphB is less active when cells are grown aerobically (Fig. 3) and cannot bind to tcpP promoter DNA in vitro in the absence of the reducing agent DTT (Fig. 4B). On the contrary, the C235S mutant becomes insensitive to oxidation (Figs. 3 and 4). AphB purified from anaerobically grown cultures is functional in the absence of reducing agents (Fig. 4B), similar to purified AphB^C235S, indicating that the AphB C₂₃₅ residue is reduced under low-oxygen conditions and the reduced form of AphB is active. Mass spectrometry analysis of aerobically purified AphB did not detect the peptides containing the C₂₃₅ thiol residue in the absence of a reducing agent (Table S1), suggesting that the C₂₃₅ residue is modified under aerobic conditions. Moreover, we found that more AphB dimers are formed under reducing conditions (Fig. 4), and dimerization may be important for AphB activity. Further studies are required to determine the exact modification and the mechanism by which the C₂₃₅ modification affects AphB activity.

Oxidative stress is the result of the production of reactive oxygen species during aerobic metabolism in bacteria, or a condition imposed on bacterial cells by the extracellular environment. Thiol-based regulatory modifications are often used by diverse bacterial species to respond to oxidative stress (26). Of note, many of the regulators involved in response to oxidative stress are basal repressors that lose their activity due to oxidative modification of cysteine residues, resulting in de-repression of oxidative stress resistance genes. Examples include OhrR in Bacillus subtilis (27) and MgrA and its homologs in Staphylococcus aureus (14, 28). Reduced AphB at C₂₃₅ is active and capable of activating virulence factor production under oxygen-limiting conditions, an environment similar to host intestines, while oxidative modification of AphB helps V. cholerae repress virulence that is no longer needed. This modification of AphB is reversible (Fig. 5), allowing V. cholerae to adapt to different environments rapidly. In addition, we found that although V. cholerae containing a nonmodifiable AphB mutant (AphB^C235S) colonized infant mice well, it had fitness disadvantages in aquatic environments with high salt concentrations (seawater) and oxidative stress (exposure to H₂O₂) (Fig. S10). This finding suggests that the ability to modify AphB at the C₂₃₅ residue may play important roles in oxidative environments, but how modifiable AphB confers a fitness advantage in aquatic environments is currently unclear. AphB in V. cholerae has been shown to regulate a number of genes other than tcpP (4), and in a closely related bacterium, V. vulnificus, an AphB homolog is believed to be a global regulator that plays an important role in environmental adaptation and survival (29, 30). Thus, V. cholerae may exploit the thiol-based regulatory switch to cope with its two lifestyles, survival in aquatic environments (aerobic) and colonization of the human intestine (anaerobic to microaerophilic).

Materials and Methods

Strains, Plasmids, and Culture Conditions.

All V. cholerae strains used in this study were derived from El Tor C6706 (31) unless otherwise noted and were propagated in Luria broth (LB) medium containing appropriate antibiotics at 37 °C unless otherwise noted. AKI medium was used to induce virulence gene expression (6). Cultures were grown aerobically (shaking at 250 rpm), microaerophilically (BBL CampyPak Microaerophilic System, or stationary growth), or anaerobically (MiniMACS anaerobic workstation, Microbiology International). Strain and plasmid constructs are described in the SI Materials and Methods.

Measuring Transcriptional Expression of Virulence Genes.

For Lux-based reporters, bacterial cultures were grown aerobically and anaerobically in AKI medium until OD₆₀₀ ≈ 0.2. The luminescence of cells was read using a BioTek Synergy HT spectrophotometer and normalized by the OD at 600 nm. Lux expression is reported as light units/OD₆₀₀. tcpP transcripts were measured using an oligonucleotide-based S1 nuclease assay (10). The oligo probe complementary to the first 50 nucleotides of tcpP mRNA with 4 noncomplementary nucleotides that are not complementary to the mRNA at the 3′ end was end-labeled with [γ-³²P]ATP.

TcpA, TcpP, AphB, and CT Production.

Whole-cell lysates were prepared from bacterial cultures as described above, and samples were normalized to the amount of total protein as assayed by the Bio-Rad protein assay. The samples were separated by SDS/PAGE on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane for Western blot analysis using polyclonal rabbit anti-TcpA, TcpP, or AphB antibody. CT production was measured by CT ELISA as described (32).

Gel Retardation Assays.

AphB-His₆ protein was expressed and purified on nickel columns according to the manufacturer's instructions (Qiagen) aerobically or anaerobically. PCR products of tcpP promoter regions were ³²P-labeled. Binding reactions contained 0.1 μM AphB proteins and 0.1 ng of DNA in a buffer consisting of 10 mM Tris·HCl (pH 7.9), 1 mM EDTA, 60 mM KCl, 10 mg/mL poly(dIdC), and when indicated, 10 mM DTT. After 20 min of incubation at 25 °C, samples were size-fractionated using 5% polyacrylamide gels in 1× TAE buffer (40 mM Tris·acetate/2 mM EDTA, pH 8.5). The radioactivity of free DNA and AphB-DNA complexes was visualized by using a Typhoon 9410 PhosphorImager (Molecular Dynamics).

Mass Spectrometry Analysis.

As indicated, purified proteins were treated with DTT and alkylated with iodoacetamide (IAM). Untreated and reduced/alkylated samples were then digested in-solution using trypsin, and peptides were analyzed with a Thermo LCQ Deca XP+ MS/MS spectrometer coupled to a LC Packings Ultimate Nano HPLC system controlled by Thermo Xcalibur 2.0 software. SEQUEST searches against both V. cholerae and E. coli protein databases used Thermo Bioworks 3.3 software, and results were filtered using standard values for Xcor and ΔC_N (33).

In Vivo Cross-Linking of AphB.

V. cholerae aphB⁻ deletion mutants containing pBAD-aphB (WT and C235S) were grown aerobically and anaerobically to OD₆₀₀ ≈ 0.2. Resuspended cells were then treated with 0.8 mM cross-linking reagent DSP [dithiobis(succinimidylpropionate), Thermo Scientific] as described (34). AphB multimers were detected by Western blot using AphB antibody.

Size-Exclusion Chromatography.

Purified AphB proteins from various sources were subjected to size-exclusion chromatography using a HiLoad 16/60 Superdex 200 column (GE Healthcare) and PBS as the running buffer. When indicated, proteins were treated with 0.1 mM DTT for 2 h. Chromatograms were normalized against absorbance values, and molecular mass was determined by running protein standards under the same conditions.

Ex Vivo Adherence Assays.

Five-week-old CD-1 mouse small intestines were cut open, trimmed to equal-sized pieces, and placed in 6-well dishes containing DMEM. Approximately 10⁷ GFP-labeled WT or tcpA mutants were inoculated on the surface of the mouse small intestinal tissues and incubated at 37 °C aerobically or anaerobically for 4 h. Tissue samples were rinsed with PBS three times and then visualized using a Zeiss Axiovert 200 M inverted fluorescence microscope. The images were analyzed by using Zeiss LSM510META (Version 3.2).