title: 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.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Alex T
authors.initials: AT
authors.lastname: Nielsen
authors.forename: Nadia A
authors.initials: NA
authors.lastname: Dolganov
authors.forename: Thomas
authors.initials: T
authors.lastname: Rasmussen
authors.forename: Glen
authors.initials: G
authors.lastname: Otto
authors.forename: Michael C
authors.initials: MC
authors.lastname: Miller
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Felt
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Torreilles
authors.forename: Gary K
authors.initials: GK
authors.lastname: Schoolnik
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002771:Cholera / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D002772:Cholera Toxin / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D054884:Host-Pathogen Interactions; D006801:Humans; D007422:Intestines / Q000382:microbiology* / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D011817:Rabbits; D014734:Vibrio cholerae / Q000302:isolation & purification / Q000502:physiology*; D014774:Virulence / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D015415:Biomarkers; C085807:TcpA protein, Vibrio cholerae; D036781:Fimbriae Proteins; D002772:Cholera Toxin