scholarly journals Second-Generation Recombination-Based In Vivo Expression Technology for Large-Scale Screening for Vibrio cholerae Genes Induced during Infection of the Mouse Small Intestine

2005 ◽  
Vol 73 (2) ◽  
pp. 972-980 ◽  
Author(s):  
C. G. Osorio ◽  
J. A. Crawford ◽  
J. Michalski ◽  
H. Martinez-Wilson ◽  
J. B. Kaper ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Large Scale ◽  
Screening Method ◽  
Mouse Small Intestine ◽  
In Vivo Expression ◽  
Induced Genes ◽  
El Tor ◽  
Selection Of

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.

scholarly journals Characterization of Vibrio cholerae O1 El TorgalU and galE Mutants: Influence on Lipopolysaccharide Structure, Colonization, and Biofilm Formation

2001 ◽  
Vol 69 (1) ◽  
pp. 435-445 ◽  
Author(s):  
Jutta Nesper ◽  
Crystal M. Lauriano ◽  
Karl E. Klose ◽  
Dagmar Kapfhammer ◽  
Anita Kraiß ◽  
...  
Keyword(s):  
Organic Acids ◽  
Vibrio Cholerae ◽  
Bile Salts ◽  
Short Chain ◽  
Core Oligosaccharide ◽  
O Antigen ◽  
Mouse Small Intestine ◽  
Vibrio Cholerae O1 ◽  
El Tor

ABSTRACT Recently we described the isolation of spontaneous bacteriophage K139-resistant Vibrio cholerae O1 El Tor mutants. In this study, we identified phage-resistant isolates with intact O antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the galU gene. We inactivated another gal gene, galE, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both gal mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The galU and R-LPS mutants, but not thegalE mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vitro, we could show that galU and R-LPS mutants were more sensitive to short-chain organic acids, cationic antimicrobial peptides, the complement system, and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen-negative strains were found to be sensitive to complement and cationic peptides, but they displayed significant resistance to bile salts and short-chain organic acids. Furthermore, we found thatgalU and galE are essential for the formation of a biofilm in a spontaneous phage-resistant rugose variant, suggesting that the synthesis of UDP-galactose via UDP-glucose is necessary for biosynthesis of the exopolysaccharide. In addition, we provide evidence that the production of exopolysaccharide limits the access of phage K139 to its receptor, the O antigen. In conclusion, our results indicate involvement of galU in V. cholerae virulence, correlated with the observed change in LPS structure, and a role for galU and galE in environmental survival of V. cholerae.

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

2004 ◽  
Vol 186 (5) ◽  
pp. 1355-1361 ◽  
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.

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

1990 ◽  
Vol 7 (2-3) ◽  
pp. 221-228 ◽  
Author(s):  
Gunhild Jonson ◽  
Ann-Man Svennerholm ◽  
Jan Holmgren
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factors ◽  
El Tor

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

1985 ◽  
Vol 21 (6) ◽  
pp. 884-890 ◽  
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

scholarly journals Role of DnaK in In Vitro and In Vivo Expression of Virulence Factors of Vibrio cholerae

1999 ◽  
Vol 67 (3) ◽  
pp. 1025-1033 ◽  
Author(s):  
Sabyasachi Chakrabarti ◽  
Nilanjan Sengupta ◽  
Rukhsana Chowdhury
Keyword(s):  
Heat Shock ◽  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Heat Shock Gene ◽  
Insertion Mutant ◽  
In Vivo Expression ◽  
Shock Gene

ABSTRACT The dnaK gene of Vibrio cholerae was cloned, sequenced, and used to construct a dnaK insertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen. The central regulator of several virulence genes of V. choleraeis ToxR, a transmembrane DNA binding protein. The V. cholerae dnaK mutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity. Using Northern blot analysis and toxR transcriptional fusions, we demonstrated a reduction in expression of the toxR gene in the dnaK mutant strain together with a concomitant increase in expression of a htpG-like heat shock gene that is located immediately upstream and is divergently transcribed fromtoxR. This may be due to increased heat shock induction in the dnaK mutant. In vivo, however, although expression from heat shock promoters in the dnaK mutant was similar to that observed in vitro, expression of both toxR andhtpG was comparable to that by the parental strain. In both strains, in vivo expression of toxR was significantly higher than that observed in vitro, but no reciprocal decrease inhtpG expression was observed. These results suggest that the modulation of toxR expression in vivo may be different from that observed in vitro.

scholarly journals Differential Requirement for PBP1a and PBP1b inIn VivoandIn VitroFitness of Vibrio cholerae

2014 ◽  
Vol 82 (5) ◽  
pp. 2115-2124 ◽  
Author(s):  
Tobias Dörr ◽  
Andrea Möll ◽  
Michael C. Chao ◽  
Felipe Cava ◽  
Hubert Lam ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Vibrio Cholerae ◽  
Synthetic Lethality ◽  
Wild Type ◽  
Content Type ◽  
Infant Mouse ◽  
Mouse Small Intestine ◽  
Membrane Lipoproteins

ABSTRACTWe investigated the roles of theVibrio choleraehigh-molecular-weight bifunctional penicillin binding proteins, PBP1a and PBP1b, in the fitness of this enteric pathogen. Using a screen for synthetic lethality, we found that theV. choleraePBP1a and PBP1b proteins, like theirEscherichia colihomologues, are each essential in the absence of the other and in the absence of the other's putative activator, the outer membrane lipoproteins LpoA and LpoB, respectively. Comparative analyses ofV. choleraemutants suggest that PBP1a/LpoA ofV. choleraeplay a more prominent role in generating and/or maintaining the pathogen's cell wall than PBP1b/LpoB.V. choleraelacking PBP1b or LpoB exhibited wild-type growth under all conditions tested. In contrast,V. choleraelacking PBP1a or LpoA exhibited growth deficiencies in minimal medium, in the presence of deoxycholate and bile, and in competition assays with wild-type cells bothin vitroand in the infant mouse small intestine. PBP1a pathway mutants are particularly impaired in stationary phase, which renders them sensitive to a product(s) present in supernatants from stationary-phase wild-type cells. The marked competitive defect of the PBP1a pathway mutantsin vivowas largely absent when exponential-phase cells rather than stationary-phase cells were used to inoculate suckling mice. Thus, at least forV. choleraePBP1a pathway mutants, the growth phase of the inoculum is a key modulator of infectivity.

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

2015 ◽  
pp. 65-69 ◽  
Author(s):  
S. P. Zadnova ◽  
T. A. Kul’Shan’ ◽  
N. B. Cheldyshova ◽  
A. A. Kritsky ◽  
N. A. Plekhanov ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Vibrio Cholerae ◽  
Survival Capacity ◽  
El Tor

scholarly journals In Vivo Expression and Immunoadjuvancy of a Mutant of Heat-Labile Enterotoxin of Escherichia coli in Vaccine and Vector Strains of Vibrio cholerae

1999 ◽  
Vol 67 (4) ◽  
pp. 1694-1701 ◽  
Author(s):  
Edward T. Ryan ◽  
Thomas I. Crean ◽  
Manohar John ◽  
Joan R. Butterton ◽  
John D. Clements ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Vibrio Cholerae ◽  
Supernatant Fraction ◽  
E Coli ◽  
Heat Labile ◽  
In Vivo Expression ◽  
Heterologous Antigens

ABSTRACT Vibrio cholerae secretes cholera toxin (CT) and the closely related heat-labile enterotoxin (LT) of Escherichia coli, the latter when expressed in V. cholerae. Both toxins are also potent immunoadjuvants. Mutant LT molecules that retain immunoadjuvant properties while possessing markedly diminished enterotoxic activities when expressed by E. coli have been developed. One such mutant LT molecule has the substitution of a glycine residue for arginine-192 [LT(R192G)]. Live attenuated strains of V. cholerae that have been used both as V. cholerae vaccines and as vectors for inducing mucosal and systemic immune responses directed against expressed heterologous antigens have been developed. In order to ascertain whether LT(R192G) can act as an immunoadjuvant when expressed in vivo by V. cholerae, we introduced a plasmid (pCS95) expressing this molecule into three vaccine strains ofV. cholerae, Peru2, ETR3, and JRB14; the latter two strains contain genes encoding different heterologous antigens in the chromosome of the vaccine vectors. We found that LT(R192G)was expressed from pCS95 in vitro by both E. coli andV. cholerae strains but that LT(R192G) was detectable in the supernatant fraction of V. choleraecultures only. In order to assess potential immunoadjuvanticity, groups of germfree mice were inoculated with the three V. cholerae vaccine strains alone and compared to groups inoculated with the V. cholerae vaccine strains supplemented with purified CT as an oral immunoadjuvant or V. choleraevaccine strains expressing LT(R192G) from pCS95. We found that mice continued to pass stool containing V. cholerae strains with pCS95 for at least 4 days after oral inoculation, the last day evaluated. We found that inoculation withV. cholerae vaccine strains containing pCS95 resulted in anti-LT(R192G) immune responses, confirming in vivo expression. We were unable to detect immune responses directed against the heterologous antigens expressed at low levels in any group of animals, including animals that received purified CT as an immunoadjuvant. We were, however, able to measure increased vibriocidal immune responses against vaccine strains in animals that receivedV. cholerae vaccine strains expressing LT(R192G) from pCS95 compared to the responses in animals that received V. cholerae vaccine strains alone. These results demonstrate that mutant LT molecules can be expressed in vivo by attenuated vaccine strains of V. cholerae and that such expression can result in an immunoadjuvant effect.

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

2007 ◽  
Vol 75 (10) ◽  
pp. 5035-5042 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document