Comparative Analysis of Survival Capacity among Typical and Genovariant Strains of Vibrio cholerae, Biovar El Tor in vivo and in vitro

ABSTRACT We have constructed an improved recombination-based in vivo expression technology (RIVET) and used it as a screening method to identify Vibrio cholerae genes that are transcriptionally induced during infection of infant mice. The improvements include the introduction of modified substrate cassettes for resolvase that can be positively and negatively selected for, allowing selection of resolved strains from intestinal homogenates, and three different tnpR alleles that cover a range of translation initiation efficiencies, allowing identification of infection-induced genes that have low-to-moderate basal levels of transcription during growth in vitro. A transcriptional fusion library of 8,734 isolates of a V. cholerae El Tor strain that remain unresolved when the vibrios are grown in vitro was passed through infant mice, and 40 infection-induced genes were identified. Nine of these genes were inactivated by in-frame deletions, and their roles in growth in vitro and fitness during infection were measured by competition assays. Four mutant strains were attenuated >10-fold in vivo compared with the parental strain, demonstrating that infection-induced genes are enriched in genes essential for virulence.

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Expression of Cholera Toxin under Non-AKI Conditions in Vibrio cholerae El Tor Induced by Increasing the Exposed Surface of Cultures

Journal of Bacteriology ◽

10.1128/jb.186.5.1355-1361.2004 ◽

2004 ◽

Vol 186 (5) ◽

pp. 1355-1361 ◽

Cited By ~ 14

Author(s):

Joaquín Sánchez ◽

Gerardo Medina ◽

Thomas Buhse ◽

Jan Holmgren ◽

Gloria Soberón-Chavez

Keyword(s):

Cholera Toxin ◽

Vibrio Cholerae ◽

Expression Pattern ◽

Phase 1 ◽

Inert Atmosphere ◽

Surface Area Ratio ◽

Genes Encoding ◽

El Tor

ABSTRACT The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.

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Analysis of expression of toxin-coregulated pili in classical and El Tor Vibrio cholerae O1 in vitro and in vivo.

Infection and Immunity ◽

10.1128/iai.60.10.4278-4284.1992 ◽

1992 ◽

Vol 60 (10) ◽

pp. 4278-4284 ◽

Cited By ~ 27

Author(s):

G Jonson ◽

J Holmgren ◽

A M Svennerholm

Keyword(s):

Vibrio Cholerae ◽

Vibrio Cholerae O1 ◽

El Tor

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Expression of virulence factors by classical and El Tor Vibrio cholerae in vivo and in vitro

FEMS Microbiology Ecology ◽

10.1111/j.1574-6941.1990.tb01687.x ◽

1990 ◽

Vol 7 (2-3) ◽

pp. 221-228 ◽

Cited By ~ 1

Author(s):

Gunhild Jonson ◽

Ann-Man Svennerholm ◽

Jan Holmgren

Keyword(s):

Vibrio Cholerae ◽

Virulence Factors ◽

El Tor

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In vitro and in vivo cholera toxin production by classical and El Tor isolates of Vibrio cholerae.

Journal of Clinical Microbiology ◽

10.1128/jcm.21.6.884-890.1985 ◽

1985 ◽

Vol 21 (6) ◽

pp. 884-890 ◽

Cited By ~ 10

Author(s):

P C Turnbull ◽

J V Lee ◽

M D Miliotis ◽

C S Still ◽

M Isaäcson ◽

...

Keyword(s):

Cholera Toxin ◽

Vibrio Cholerae ◽

Toxin Production ◽

El Tor

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Hemolysin and the Multifunctional Autoprocessing RTX Toxin Are Virulence Factors during Intestinal Infection of Mice with Vibrio cholerae El Tor O1 Strains

Infection and Immunity ◽

10.1128/iai.00506-07 ◽

2007 ◽

Vol 75 (10) ◽

pp. 5035-5042 ◽

Cited By ~ 69

Author(s):

Verena Olivier ◽

G. Kenneth Haines ◽

Yanping Tan ◽

Karla J. Fullner Satchell

Keyword(s):

Vibrio Cholerae ◽

Intestinal Infection ◽

Lethal Challenge ◽

Adult Mice ◽

High Doses ◽

El Tor ◽

Vitro Data ◽

In Vitro Data

ABSTRACT The seventh cholera pandemic that started in 1961 was caused by Vibrio cholerae O1 strains of the El Tor biotype. These strains produce the pore-forming toxin hemolysin, a characteristic used clinically to distinguish classical and El Tor biotypes. Even though extensive in vitro data on the cytolytic activities of hemolysin exist, the connection of hemolysin to virulence in vivo is not well characterized. To study the contribution of hemolysin and other accessory toxins to pathogenesis, we utilized the model of intestinal infection in adult mice sensitive to the actions of accessory toxins. In this study, we showed that 4- to 6-week-old streptomycin-fed C57BL/6 mice were susceptible to intestinal infection with El Tor strains, which caused rapid death at high doses. Hemolysin had the predominant role in lethality, with a secondary contribution by the multifunctional autoprocessing RTX (MARTX) toxin. Cholera toxin and hemagglutinin/protease did not contribute to lethality in this model. Rapid death was not caused by increased dissemination due to a damaged epithelium since the numbers of CFU recovered from spleens and livers 6 h after infection did not differ between mice inoculated with hemolysin-expressing strains and those infected with non-hemolysin-expressing strains. Although accessory toxins were linked to virulence, a strain defective in the production of accessory toxins was still immunogenic since mice immunized with a multitoxin-deficient strain were protected from a subsequent lethal challenge with the wild type. These data suggest that hemolysin and MARTX toxin contribute to vaccine reactogenicity but that the genes for these toxins can be deleted from vaccine strains without affecting vaccine efficacy.

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A ToxR-dependent role for the putative phosphoporin VCA1008 in bile salt resistance in Vibrio cholerae El Tor N16961

Microbiology ◽

10.1099/mic.0.043117-0 ◽

2010 ◽

Vol 156 (10) ◽

pp. 3011-3020 ◽

Cited By ~ 10

Author(s):

Carolina Lage Goulart ◽

Guilherme Garcia dos Santos ◽

Livia Carvalho Barbosa ◽

Letícia Miranda Santos Lery ◽

Paulo Mascarello Bisch ◽

...

Keyword(s):

Vibrio Cholerae ◽

Sodium Deoxycholate ◽

Salt Resistance ◽

Growth Defect ◽

Dependent Manner ◽

Pandemic Strains ◽

El Tor ◽

First Time

The putative phosphoporin encoded by vca1008 of Vibrio cholerae O1 is expressed in vivo during infection and is essential for the intestinal colonization of infant mice. In vitro, its expression is induced under inorganic phosphate (Pi) limitation in a PhoB/R-dependent manner. In this work we demonstrated that VCA1008 has a strain-specific role in the physiology and pathogenicity of V. cholerae O1. Disruption of vca1008 led to a growth defect, an inability to colonize and a high susceptibility to sodium deoxycholate (DOC; the major bile compound) in the El Tor biotype strain N16961, but did not affect the classical strain O395 in the same way. Furthermore, vca1008 promoter activity was higher in N16961 cells grown under a low Pi supply in the presence of DOC than in the absence of the detergent. In the Pi-limited cells, vca1008 was positively regulated by PhoB, but when DOC was added to the medium, it negatively affected the PhoB-mediated activation of the gene, and enhanced vca1008 expression in a ToxR-dependent manner. These findings reveal for the first time a complex strain-specific interplay between ToxR and PhoB/R systems to control porin genes, as well as the influence of DOC on the expression of PhoB- and ToxR-regulated genes and pathogenesis in pandemic strains of V. cholerae.

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