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.
Highlights
Clinical and pathological studies of diverse bacterial pathogens disclose a common theme: the infectious process evolves as a series of spatial and temporal patterns during migration of the pathogen through different tissues and cellular compartments of the host
During its residence in the host, the pathogen produces essential virulence determinants and often replicates rapidly, leading to a vast expansion of its biomass. This scenario is well established for Vibrio cholerae, the cause of a potentially fatal diarrheal illness, it has not previously been possible to identify precisely when or where virulence determinants are produced in the intestine. We addressed this question by investigating the expression of virulence genes by individual V. cholerae during infection of the small intestine
Confocal microscopy studies of green fluorescent protein (GFP)-labeled V. cholerae O1 El Tor in the rabbit ileal loop model of cholera 4, 8 and 12 hours after inoculation have previously shown that V. cholerae resides in at least three anatomically distinct sites in the ileal loop at the same time point: the epithelial surface; the mucus gel overlying the epithelial surface; and, in fluid that collects in the lumen of the loop [10]
Summary
Clinical and pathological studies of diverse bacterial pathogens disclose a common theme: the infectious process evolves as a series of spatial and temporal patterns during migration of the pathogen through different tissues and cellular compartments of the host. Spatial and temporal sources of microbial heterogeneity can be compounded by stochastic events that cause cell-to-cell transcriptional and phenotypic differences between genetically-identical individuals in the same microenvironment [1,2]. Together, these two sources of variation, one deterministic and the other probabilistic, pose significant experimental challenges that impede a deeper understanding of pathogenesis. These two sources of variation, one deterministic and the other probabilistic, pose significant experimental challenges that impede a deeper understanding of pathogenesis To address these challenges here we describe results from a study that employed a combination of site-specific and single cell gene expression methods to study Vibrio cholerae infecting the small intestine
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