A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine

Abstract

A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.

Figure 1. Compartment-specific expression profiling of the V. cholerae O1 tcp and ctx operons in ligated rabbit ileal loops.

DNA amplicon microarrays were used to monitor the expression of virulence genes by V. cholerae in two compartments of ligated rabbit ileal loops. Eight and 12 hours post inoculation, samples were obtained from fluid collecting in ileal loops during the infectious process. Four, eight and 12 hours post inoculation, samples were also obtained as a single fraction from epithelial surfaces and the overlying mucus gel. Each experiment was repeated 2–4 times and four microarrays were analyzed for each biological replicate. The expression of V. cholerae genes in each sample was compared with their expression during mid exponential phase growth in LB broth. Expression magnitudes are depicted by color in each cell of the heat map: shades of red indicate mRNA abundance in sample exceeds mRNA abundance in the mid exponential reference for the indicated gene; shades of green indicate lower mRNA abundance in sample than reference; and shades of black indicate nearly equal levels of mRNA in the experimental and reference samples for the indicated gene. The average fold difference values between experimental sample and reference are provided as numerical values in each cell. A 2-fold cut-off and a 0% false discovery rate was applied for the analysis of all samples. The complete data set is available in Tables S3, S4, S5, S6 and S7.

Figure 2. Single cell expression profiling and confocal microscopy of tcpA expression in ligated ileal loops.

(A) Structure and location of the tcpA-gfp(ASV) reporter on the V. cholerae large chromosome. The tcpA promoter was cloned in front of gfp(ASV), which encodes a destabilized GFP derivative, and the tcpA-gfp(ASV) fusion inserted as a single copy betweenVC0487 and VC0488 on the large chromosome of V. cholerae using the mTn7 transposon system. The structure and location of native tcpA remains intact within Vibrio Pathogenicity Island I. The native tcpA promoter reads through to toxT, thereby creating a positive feedback loop (indicated with red arrows). The paired ToxT molecules in the figure indicate that the promoters of tcpA-gfp(ASV), tcpA-F and ctxAB are activated by dimeric ToxT. (B–J) Confocal images of tcpA-gfp(ASV) expression by individual bacteria in ligated rabbit ileal loops. Bacteria harboring tcpA-gfp(ASV) were visualized using scanning laser confocal microscopy 4 hours (B, C, D), 8 hours (E, F, G) and 12 hours (H, I, J) post inoculation. The actin-rich epithelial surfaces were stained with phalloidin and are pseudo-colored blue; all V. cholerae were visualized using an O1-specific antibody and are pseudo-colored red; and, GFP-expressing bacteria are pseudo-colored green. Three identical images are shown for each time point: D, G and J visualize the epithelial surface and all V. cholerae four, eight and 12 hours post inoculation; C, F and I visualize the epithelial surface and the subset of bacteria that are expressing tcpA-gfp(ASV); and B, E and H superimpose images for the same time point to provide a composite portrait of tcpA-gfp(ASV)-expressing and non-expressing bacteria in the same visual field. Arrows on (E) indicate aggregates of tcpA-gfp(ASV)-expressing bacteria located away from the nearest epithelial surface 8 hours post inoculation. Main images are reconstructed Z-projections and show horizontal sections of the villi, while side panels show vertical sections at the positions indicated by white lines. Scale bars correspond to 50 µm.

Figure 3. Properties of the rrnBP1-gfp(ASV) reporter; expression of rrnBP1-gfp(ASV) and tcpA-gfp(ASV) as a function of distance from the epithelial surface.

The growth rate regulated promoter rrnBP1 was cloned in front of gfp(ASV) and the rrnBP1-gfp(ASV) fusion inserted as a single copy into the large chromosome of V. cholerae at the same site used for tcpA-gfp(ASV) (see Fig. 2). (A) Correlation between growth of V. cholerae in LB medium (blue) and average fluorescence from the rrnBP1-gfp(ASV) reporter (red) quantified using flow cytometry as the cells enter stationary phase. Error bars indicate the standard deviation of fluorescence from the individual bacteria in the culture. (B) Growth of V. cholerae during infection of the rabbit ileal loop measured as colony forming units per centimeter of ileum as determined from the analysis of ileal loop fluid samples obtained 4, 8 and 12 hours post inoculation. (C) Expression of rrnBP1-gfp(ASV) as a function of distance from the epithelial surface. Quantitative image analysis was used to analyze the expression of rrnBP1-gfp(ASV) as a function of distance to the nearest epithelial surface four hours post inoculation of rabbit ileal loops. Growth of V. cholerae as a function of location was estimated by computing the ratio between fluorescence from the V. cholerae O1 specific antibody (red) and fluorescence from the rrnBP1-gfp(ASV)-expressing bacteria (green). Ratios, depicted on the vertical axis, were determined from the average fluorescence intensity values from 14 different images obtained from different locations in the ileal loop. (D) Expression of tcpA-gfp(ASV) as a function of distance from the epithelial surface. Quantitative image analysis was used to analyze the expression of tcpA-gfp(ASV) as a function of distance to the nearest epithelial surface four hours post inoculation of rabbit ligated ileal loops. The values depicted on the vertical axis are the average ratios of fluorescence from the V. cholerae O1 specific antibody (red) and fluorescence from the tcpA-gfp(ASV) reporter (green) for 14 different images at different locations in the rabbit ileal loop.

Figure 4. Single cell expression profiling and confocal microscopy of the growth-regulated rrnBP1 promoter in ligated ileal loops.

Bacteria harboring the rrnBP1-gfp(ASV) growth reporter were visualized during infection of ligated rabbit ileal loops by scanning confocal microscopy 4 hours (A, B, C), 8 hours (D, E, F) and 12 hours (G, H, I) post inoculation. Actin-rich epithelial surfaces were stained with phalloidin (pseudo-colored blue); all V. cholerae were visualized using an O1-specific antibody (pseudo-colored red); and, GFP-expressing bacteria are pseudo-colored green. Three identical images are shown for each time point: C, F and I visualize the epithelial surface and all V. cholerae four, eight and 12 hours post inoculation; B, E and H visualize the epithelial surface and the subset of bacteria that are expressing rrnBP1-gfp(ASV); and A, D and G superimpose images for the same time point to provide a composite portrait of rrnBP1-gfp(ASV)-expressing and non-expressing bacteria in the same visual field. Arrows on (A) indicate aggregates of rrnBP1-gfp(ASV)-expressing bacteria typically associated with extruded epithelial cells located away from the nearest epithelial surface. Main images are reconstructed Z-projections and show horizontal sections of the villi, while side panels show vertical sections at the positions indicated by white lines. Scale bar corresponds to 50 µm.

Figure 5. Heterogeneous expression of tcpA-gfp(ASV) in rabbit ileal loops and during in vitro conditions of growth that induce the expression of V. cholerae virulence genes.

(A–B) Scanning confocal fluorescence microscopy was used to visualize V. cholerae harboring the tcpA-gfp(ASV) transcriptional reporter 12 hours post inoculation of ligated ileal loops. (A) The actin-rich epithelial surfaces were stained with phalloidin (colored blue); all V. cholerae were visualized using a V. cholerae O1-specific antibody (red); bacteria expressing tcpA-gfp(ASV) (green) are shown as a composite image in (A) and in isolation in (B). Arrows indicate examples of adjacent bacteria near the epithelial surface that exhibit different levels of tcpA-gfp(ASV) fluorescence. (C) Heterogeneity of tcpA-gfp(ASV) expression after growth of the reporter strain in AKI medium. (D) Heterogeneity of tcpA-gfp(ASV) expression during early stationary phase in LB medium containing 100 mM NaHCO₃. Scale bars corresponds to 15 µm.

Figure 6. The abundance of tcpA, ctxA and toxT transcripts within individual bacteria bifurcates into two populations after induction with bicarbonate and progression into stationary phase.

(A) Expression of the tcpA-gfp(ASV) reporter as a function of bicarbonate concentration. Average fluorescence from the tcpA-gfp(ASV) reporter construct was quantified using flow cytometry and plotted relative to the background fluorescence of an uninduced control. A linear response in fluorescence (vertical axis) was observed as a function of bicarbonate concentration (horizontal axis) 30 minutes after induction during exponential growth. (B) Growth of V. cholerae harboring tcpA-gfp(ASV) in LB medium, with and without bicarbonate. At time 0, NaHCO₃ was added to LB medium to a final concentration of 100 mM (blue curve). Growth was unaffected by bicarbonate addition when compared to the control, where a similar volume of water was added (red curve). Error bars indicate standard deviation calculated from 4 biological replicates. (C) Bifurcation of tcpA-gfp(ASV) expression as a function of time after induction with bicarbonate. Fluorescence intensity as a function of time was determined by flow cytometry after the addition of NaHCO₃ to an exponential phase culture. Unimodal induction of tcpA(gfp)ASV expression was observed 30 minutes after induction with NaHCO₃. Bifurcation of the population was observed during entry into stationary phase (∼90 minutes after addition of NaHCO₃). (D) Bifurcation of tcpA-gfp(ASV) expression as a function of NaHCO₃ concentration 3 hours post addition of bicarbonate to an exponential culture in LB medium as measured by flow cytometry. (E) Expression of ctxA and toxT also exhibit the bifurcation phenotype. Fluorescence activated cell sorting was used to collect populations of bacteria that had undergone bifurcation in tcpA-gfp(ASV) expression after induction with 100 mM NaHCO₃ and entry into stationary phase approximately 3 hours after induction. Two populations of bacteria were collected: GFP-positive; and, GFP-negative. A quantitative multiplex RT-PCR assay was used to measure, in each of the two populations, the abundance of transcripts corresponding to genes encoding major virulence determinants (ctxA and tcpA) and regulators of virulence gene expression (horizontal axis). Three chemotaxis genes localized at different positions in the genome were included as controls. The logarithm (base 2) of the ratio of expression levels for each gene in the two sorted populations was determined and plotted on the vertical axis. (F) Bifurcation in tcpA expression exhibits hysteresis after removal of inducer. V. cholerae harboring the tcpA-gfp(ASV) reporter was induced with 100 mM NaHCO₃ and GFP emission monitored by flow cytometry. During entry into stationary phase two hours after induction, bifurcation in the expression of GFP was established. The cells were then harvested (time zero), washed and resuspended in spent media from parallel cultures grown with and without bicarbonate. The fraction of induced bacteria over a 250 minute time course was calculated from the flow cytometry data as the ratio between GFP-positive and GFP-negative cells.

Figure 7. The tcpA promoter and CRP are required for the tcpA bistable phenotype.

Flow cytometry was used to analyze the effect of different mutations of the tcpA-gfp(ASV) reporter strain on the tcpA bistable phenotype. Samples were analyzed before induction (red curve) and 30 minutes (blue curve) and 3 hours (green curve) after induction with 100 mM NaHCO₃. (A) V. cholerae wild type strain harboring the tcpA-gfp(ASV) reporter. (B) tcpA-promoter deletion mutant. (C) crp deletion mutant.

Figure 8. Heterogeneity of tcpA expression in luminal fluid and after dilution into artificial seawater.

Fluorescence microscopy was used to analyze fluorescence from the tcpA-gfp(ASV) transcriptional reporter by bacteria in luminal fluid obtained from freshly incised rabbit ileal loops 12 hours post inoculation. All bacteria were stained with a V. cholerae specific antibody (shown in red) while GFP fluorescence from the tcpA-gfp(ASV) reporter is shown in green. An overlay of both colors is also shown. Most bacteria were present as planktonic cells; these showed heterogeneous distribution of tcpA-gfp(ASV) expression levels (A–C). Aggregates of V. cholerae expressing high levels of tcpA were also observed in the luminal fluid (D–F). The luminal fluid was diluted 1∶10 into artificial seawater and incubated for up to four hours. V. cholerae within aggregates continued to express high levels of the tcpA-gfp(ASV) reporter (G–I). Scale bar corresponds to 15 µm.

Figure 9. Model of the regulation of the tcpA bistable phenotype.

Regulation of the bistable switch before and after entry into stationary phase. Transcription factors that induce virulence gene expression are denoted by solid green arrows. The tcpA-promoter → ToxT autocatalytic feedback loop is labeled. Transcription factors that negatively regulate virulence gene expression during entry into stationary phase are denoted by solid red arrows; the proposed repression of the tcpA promoter by CRP-cAMP is denoted by a dashed red arrow. Based on RT-PCR data from Fig. 6E, genes in the regulatory cascade that exhibit homogeneous expression during entry into stationary phase are highlighted in grey, while genes showing bistable expression (tcpA-F, toxT and ctxAB) are highlighted in green. See text for additional details of this model.