Vibrio cholerae infection in the embryonated egg

Gardner, Earl W.; Lyles, Sanders T.; Lankford, C. E.; Hagens, Shirley J.

doi:10.1093/infdis/112.3.264

Cited by 15 publications

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“…Symbols: A, uninoculated animals (means offour to five mice); B, control animals given BHIplus dye (means ofat least five mice); C, test animals given BHI plus dye plus 6.5 X 106 CFU ofstrain CA401 (means ofat least 10 animals). human isolate (16), tested at the same dose, gives a comparable time-related FA response (results not shown).…”

Section: Resultsmentioning

confidence: 79%

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Intestinal fluid accumulation induced by oral challenge with Vibrio cholerae or cholera toxin in infant mice

Baselski¹,

Briggs²,

Parker³

1977

Infect Immun

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The diarrheal response of orally inoculated infant mice to viable Vibrio cholerae and purified cholera toxin was quantitated by means of a fluid accumulation (FA) ratio. The FA ratio is defined as the gut weight/remaining body weight. FA ratios were determined in relation to time of exposure and dose. Onset of fluid accumulation with viable cells of strains CA401 and 569B occurred 8 h postinoculation and reached a near maximum at 16 h. A dose of 4 x 106 colony-forming units of strain CA401 was required for a positive response 16 to 18 h postinoculation. Several other classical cholera strains demonstrated a similar dose-related response. Strain 569B, however, required a 100-fold higher dose to give a positive response. Several mutant cholera strains with decreased virulence in other model systems elicited FA ratios decreased from wild-type values. Onset of fluid accumulation with cholera toxin occurred 6 to 8 h postinoculation and reached a maximum by 10 h. A dose of 0.5 Ag was required for a positive response 10 to 12 h postinoculation. The positive response to toxin could be inhibited by preincubation with specific antitoxin.

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Section: Resultsmentioning

confidence: 79%

“…Infant mice were inoculated with a dose of the highly mouse virulent classical V. cholerae strain CA401, representing approximately 500 oral LD50. (CA4O1 is an isolate from a cholera patient [16].) Controls were given only Bil broth with 0.01% Evans blue dye.…”

Section: Resultsmentioning

confidence: 99%

Intestinal fluid accumulation induced by oral challenge with Vibrio cholerae or cholera toxin in infant mice

Baselski¹,

Briggs²,

Parker³

1977

Infect Immun

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“…MATERIALS AND METHODS Bacterial strains and media. V. cholerae CA401, originally obtained from C. Lankford (7) and characterized in earlier studies (2,3,9,18), was stored at -70°C in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) contain Lithium chloride-lithium acetate outer membrane preparation. Outer membrane was prepared by a modification of the lithium chloride-lithium acetate extraction method of Johnston et al (14).…”

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confidence: 99%

Identification and characterization of Vibrio cholerae surface proteins by radioiodination

Richardson¹,

Parker²

1985

Infect Immun

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Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or lodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride-lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides.Little is known about the surface or outer membrane structures of Vibrio cholerae. V. cholerae cells have two types of appendage: a single, polar flagellum enclosed in a sheath which appears contiguous with the outer membrane (5), and pili (1,36). The lipopolysaccharide of V. cholerae is chemically different from that of the family Enterobacteriaceae (13). V. cholerae cytoplasmic membrane and outer membrane are not differentially solubilized by Triton X-100 extraction (18), as are the membranes of Escherichia coli and Pseudomonas aeruginosa. Furthermore, the electrophoretic profile of V. cholerae outer membrane proteins in polyacrylamide gels is distinct from those of other gram-negative organisms. These studies and others (16,17,34) indicate that the outer membrane of V. cholerae is a complex structure containing many proteins.Proteins exposed on the bacterial surface are likely to contribute to colonization of the host, and improved knowledge of the surface proteins should further our understanding of their role in colonization. The surface proteins of several bacterial species have been identified by iodination with membrane-impermeable enzymes (15,20,29,31) or the water-insoluble reagent, 1,3,4,6-tetrachloro-3,6-diphenylglycouril (Iodogen) (33). We used lodogen-coated vessels and a covalently immobilized catalyst (lodo-beads) to radioiodinate whole cells and isolated outer membrane of V. cholerae. The major surface proteins on V. cholerae accessible to radioiodination were identified and shown to correspond, with a single exception, to the proteins in radiolabeled outer membrane obtained by lithium chloride-lithium acetate extraction (14). Outer membrane obtained by sucrose density centrifugation and Triton X-100 extraction (18) of labeled whole cells was shown to closely resemble the lithium-extracted outer membrane.MATERIALS AND METHODS Bacterial strains and media. V. cholerae CA401, originally obtained from C. Lankford (7) and characterized in earlier studies (2,3,9,18), was stored at -70°C in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) contain-* Corresponding author. t Present address: Center for Vaccine Development, University of Maryland Me...

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“…Rough cholera strains are avirulent (16), and recent mutant studies suggest that other cell surface changes may play a role in the virulence of V. cholerae (6). We studied the outer membrane proteins of V. cholerae strain CA401, a virulent wild-type strain.…”

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confidence: 99%

“…The samples were diluted 1:2 with the sample buffer (which contained 12.5% glycerol, 1.25% SDS, and 1.25% 2-mercaptoethanol in 0.25 M Trishydrochloride buffer [pH 6.8] with bromphenol blue added as a tracking dye), solubilized by boiling for 5 min, and applied to gels to obtain 20 to 30 jig of protein per lane. The gels were run with an ISCO model 493 power source, using constant voltage set at 60 V until the tracking dye had entered the separation gel and then increased to 160 V until the tracking dye reached the bottom of the gel (about 4 h total for the short gel and approximately- 16 h for the longer gel). The gels were stained for protein with Coomassie blue R250 (Sigma), destained according to Fairbanks et al (11), and dried onto Whatman no.…”

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Identification and preliminary characterization of Vibrio cholerae outer membrane proteins

Kelley¹,

Parker²

1981

J Bacteriol

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Outer membrane proteins of Vibrio cholerae were purified by sucrose density cenatifugation and Triton X-100 extraction at 10 mM Mg2e. The proteins were separated by polyacrylamide gel electrophoresis in the presence ofsodium dodecyl sulfate. V. cholerae outer membrane proteins presented a unique pattern when compared with the patterns of other gram-negative rods. There were 8 to 10 major bands (Mr 94,000 to 27,000), with most of the protein located in band 5 000), which thus appears to be the major structural protein of the outer membrane. Lipid and carbohydrate were associated with band 6.Outer membranes of gram-negative bacilli are composed primarily of proteins, lipids, and lipopolysaccharide. The outer membranes of members of the Enterobacteriaceae and Pseudomonadaceae (8,10,18,19,26,28,34,36) have been characterized, but little has been reported on the outer membrane of Vibrio cholerae (23, 37). V. cholerae differs from members of the Enterobacteriaceae and Pseudomonadaceae by having a lipopolysaccharide which lacks 2-keto-3-deoxyoctonate (29-31).Rough cholera strains are avirulent (16), and recent mutant studies suggest that other cell surface changes may play a role in the virulence of V. cholerae (6). We studied the outer membrane proteins of V. cholerae strain CA401, a virulent wild-type strain.Slightly modified methods, similar to those used for isolation of outer membranes of Enterobacteriaceae, were successful in obtaining outer membrane proteins of V. cholerae. Characterization of these proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed 8 to 10 major proteins, with more than 90% of the outer envelope protein appearing in a band having an Mr of -45,000.In this paper we present a useful method for isolating outer membrane proteins of V. cholerae, assign numbers to outer membrane proteins, and discuss some of the unique features of the V. cholerae outer envelope. MATERIALS AND MErHODS Bacterial strain. V. cholerae CA401 is a classical Inaba strain isolated in 1953 in Calcutta, India (22), which has been extensively characterized (3-6, 35). t Present address: MO 65212.Stock cultures were kept lyophilized or frozen at -70°C in brain heart infusion broth plus 15% glycerol. Working stocks were kept on meat extract agar slants (2) at 49C for no longer than 3 weeks.Cell culture and harvesting. V. cholerae was routinely grown in the semisynthetic medium (Syncase) of R. A. Finkelstein and C. E. Lankford (Bacteriol. Proc., p. 43, 1955) to mid-logarithmic growth phase and was occasionally grown in other media for comparison: complex medium, meat extract broth (2), and defined minimal A medium (27). Cultures were grown aerobically by inoculating approximately 20 ml of medium with 2 ml of an overnight culture in the same medium and incubating it at 35 to 370C with shaking (180 to 200 rpm) in a New Brunswick shaking waterbath until mid-logarithmic phase. Ten milliliters of this culture was used to inoculate 400 ml of the same medium in a 2-liter Erlenmeyer flask and incubated at 37°...

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Vibrio cholerae infection in the embryonated egg

Cited by 15 publications

References 0 publications

Intestinal fluid accumulation induced by oral challenge with Vibrio cholerae or cholera toxin in infant mice

Intestinal fluid accumulation induced by oral challenge with Vibrio cholerae or cholera toxin in infant mice

Identification and characterization of Vibrio cholerae surface proteins by radioiodination

Identification and preliminary characterization of Vibrio cholerae outer membrane proteins

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