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Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression.

ABSTRACT: The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence analysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Comparison with the Salmonella typhimurium OmpB protein sequences revealed 99.5% homology. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD genes. The presence of the deletions within ompR was confirmed by Southern hybridization and sequencing of the DNA fragments surrounding the deleted regions by PCR. The S. typhi ompR mutants displayed a marked decrease in OmpC and OmpF porin expression as demonstrated by examination of outer membrane preparations. It was also found that S. typhi strains harboring the defined ompR deletions no longer agglutinated with Vi antiserum. However, when a functional ompB operon was introduced back into the S. typhi ompR mutants, either on a multicopy plasmid or as a single-copy chromosomal replacement, the Vi+ phenotype was restored. The levels of Vi synthesis were also found to be sensitive to different concentrations of sodium chloride present in the growth medium, although the levels of sensitivity varied between different isolates of S. typhi. It is therefore concluded that the ompR-envZ two component regulatory system plays an important role in the regulation of Vi polysaccharide synthesis in S. typhi and that one of the environmental signals for this regulation may be osmolarity.

SUBMITTER: Pickard D

PROVIDER: S-EPMC303057 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression.

Pickard D D Li J J Roberts M M Maskell D D Hone D D Levine M M Dougan G G Chatfield S S

Infection and immunity 19940901 9

The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence analysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Comparison with the Salmonella typhimurium OmpB protein sequences revealed 99.5% homology. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open read ...[more]

PMID: 8063417

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Project description:BackgroundSerotype-specific antibody responses to unconjugated pneumococcal polysaccharide vaccine (PPV) evaluated by a World Health Organization (WHO)-standardized enzyme-linked immunosorbent assay (ELISA) are the gold standard for diagnosis of specific polysaccharide antibody deficiency (SAD). The American Academy of Allergy, Asthma and Immunology (AAAAI) has proposed guidelines to interpret the PPV response measured by ELISA, but these are based on limited evidence. Additionally, ELISA is costly and labor-intensive. Measurement of antibody response to Salmonella typhi (S. typhi) Vi vaccine and serum allohemagglutinins (AHA) have been suggested as alternatives. However, there are no large cohort studies and cutoff values are lacking.ObjectiveTo establish cutoff values for antipneumococcal polysaccharide antibody response, anti-S. typhi Vi antibody, and AHA.MethodsOne hundred healthy subjects (10-55 years) were vaccinated with PPV and S. typhi Vi vaccine. Blood samples were obtained prior to and 3-4 weeks after vaccination. Polysaccharide responses to 3 serotypes were measured by WHO ELISA and to 12 serotypes by an in-house bead-based multiplex assay. Anti-S. typhi Vi IgG were measured with a commercial ELISA kit. AHA were measured by agglutination method.ResultsApplying AAAAI criteria, 30% of healthy subjects had a SAD. Using serotype-specific fifth percentile (p5) cutoff values for postvaccination IgG and fold increase pre- over postvaccination, only 4% of subjects had SAD. One-sided 95% prediction intervals for anti-S. typhi Vi postvaccination IgG (≥11.2 U/ml) and fold increase (≥2) were established. Eight percent had a response to S. typhi Vi vaccine below these cutoffs. AHA titer p5 cutoffs were ½ for anti-B and ¼ for anti-A.ConclusionWe establish reference cutoff values for interpretation of PPV response measured by bead-based assay, cutoff values for S. typhi Vi vaccine responses, and normal values for AHA. For the first time, the intraindividual consistency of all three methods is studied in a large cohort.

Project description:BackgroundThe two typhoid vaccines, the parenteral Vi capsular polysaccharide and the oral live whole-cell Salmonella Typhi Ty21a vaccine, provide similar levels of protection in field trials. Sharing no antigens, they are thought to confer protection by different mechanisms. This is the first head-to-head study to compare the humoral immune responses to these two vaccines.Methods50 age- and gender-matched volunteers were immunized, 25 with the Vi and 25 with the Ty21a vaccine. Circulating plasmablasts reactive with whole-cell Salmonella Typhi or one of the typhoidal antigenic structures, Vi, O-9,12, and H-d antigens, were identified as antibody-secreting cells (ASC) with ELISPOT. Homing receptor (HR) expressions were determined. These results were compared with ASC in four patients with typhoid fever. Antibodies to S. Typhi lipopolysaccharides were assessed in cultures of ALS (antibodies in lymphocyte supernatants) and in serum with ELISA.ResultsIn 49 out of 50 vaccinees, no typhoid-specific plasmablasts were seen before vaccination. On day 7, response to Vi antigen was mounted in 24/25 volunteers in the Vi, and none in the Ty21a group; response to S. Typhi and O-9,12 was mounted in 49/50 vaccinees; and to H-d in 3/50. The numbers of typhoid-specific plasmablasts (total of ASC to Vi, O-9,12 and H-d antigens) proved equal in the vaccination groups. The HR expressions indicated a mainly systemic homing in the Vi and intestinal in the Ty21a group, the latter resembling that in natural infection. Plasmablasts proved more sensitive than serum and ALS in assessing the immune response.ConclusionsThe typhoid-specific humoral responses to Vi and Ty21a vaccines are similar in magnitude, but differ in expected localization and antigen-specificity. The unforeseen O antigen-specific response in the Vi group is probably due to lipopolysaccharide contaminating the vaccine preparation. Only the response to Ty21a vaccine was found to imitate that in natural infection.Trial registrationCurrent Controlled Trials Ltd. c/o BioMed Central ISRCTN68125331.

Dataset Information

Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression.

Publications

Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression.

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