RESUMEN
Bordetella pertussis dramatically alters its phenotype by sensing its environment via the BvgAS regulatory system. Increased concentrations of specific chemicals are used in vitro to induce modulation of the bacterium from the Bvg(+) virulent phenotype to a fully Bvg(-) phenotype. Varied expression of sets of Bvg(-)regulated molecules depends on the modulating capacity of the environment. We examined the effect of a number of chemicals on the modulating capacity of B. pertussis growth media, both alone and in combination with known modulators. It was demonstrated that under certain conditions the Bvg(-)intermediate protein, BipA, is coexpressed with the Bvg(-) antigen, VraA. This demonstrates that the patterns of molecules expressed in the different phenotypes of B. pertussis are more fluid than has previously been demonstrated. The in vitro modulator, sulfate, was found to be a relatively inefficient modulator of our Tohama I-derived B. pertussis strain. However, addition of nicotinic acid, MgCl2, or sucrose in combination with relatively low sulfate concentrations resulted in effective modulation. This suggests that multiple signals may affect modulation through the BvgAS system or possibly through other regulatory networks. In addition, the cooperative modulating effect of sucrose implicates osmolarity as an environmental stimulus that affects phenotypic modulation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bordetella pertussis/patogenicidad , Regulación Bacteriana de la Expresión Génica , Concentración Osmolar , Proteínas Bacterianas/genética , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Bordetella pertussis/fisiología , Medios de Cultivo , Humanos , Transducción de Señal , Factores de Transcripción , VirulenciaRESUMEN
Expression of virulence determinants by Bordetella pertussis, the primary etiological agent of whooping cough, is regulated by the BvgAS two-component regulatory system. The role of a second two-component regulatory system, encoded by risAS, in this process is not defined. Here, we show that mutation of B. pertussis risA does not affect Bvg-activated genes or proteins. However, mutation of risA resulted in greatly diminished expression of Bvg-repressed antigens and decreased transcription of Bvg-repressed genes. In contrast, mutation of risS had no effect on the expression of Bvg-regulated molecules. Mutation of risA also resulted in decreased bacterial invasion in a HeLa cell model. However, decreased invasion could not be attributed to the decreased expression of Bvg-repressed products, suggesting that mutation of risA may affect the expression of a variety of genes. Unlike the risAS operons in B. parapertussis and B. bronchiseptica, B. pertussis risS is a pseudogene that encodes a truncated RisS sensor. Deletion of the intact part of the B. pertussis risS gene does not affect the expression of risA-dependent, Bvg-repressed genes. These observations suggest that RisA activation occurs through cross-regulation by a heterologous system.
Asunto(s)
Proteínas Bacterianas/fisiología , Bordetella pertussis/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Operón , Receptores de Superficie Celular/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Bordetella pertussis/genética , Bordetella pertussis/patogenicidad , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Complejos Multienzimáticos/genética , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Análisis de Secuencia de ADNRESUMEN
Pulsed-field gel electrophoresis and gene typing were able to differentiate among 3,597 Bordetella pertussis isolates circulating in Alberta and Québec Provinces, Canada, from 1985 to 1994 and distinguish them from the strains used in vaccine production. This study provides a baseline for continued surveillance of prevalent and emerging strains of B. pertussis in Canada.
Asunto(s)
Bordetella pertussis/clasificación , Variación Genética , Alberta/epidemiología , Proteínas de la Membrana Bacteriana Externa/genética , Bordetella pertussis/genética , Bordetella pertussis/aislamiento & purificación , Electroforesis en Gel de Campo Pulsado , Humanos , Toxina del Pertussis/genética , Vacuna contra la Tos Ferina , Quebec/epidemiología , Factores de Virulencia de Bordetella/genética , Tos Ferina/epidemiología , Tos Ferina/microbiologíaRESUMEN
The intra- and interlaboratory variabilities of the molecular size measurements of each DNA fragment contributing to three pulsed-field gel electrophoresis (PFGE) profiles were assessed, as were the reproducibilities of the entire PFGE profiles for three Bordetella pertussis strains. The major source of variability within a laboratory occurred between subcultures rather than within gels or between gels. Each PFGE profile was generated reproducibly and was objectively defined by the molecular sizes of its composite fragments. A strain or profile most suitable for use as an internal reference standard was identified.
Asunto(s)
Bordetella pertussis/genética , ADN Bacteriano/análisis , Desoxirribonucleasas de Localización Especificada Tipo II , Electroforesis en Gel de Campo Pulsado , Reproducibilidad de los ResultadosRESUMEN
An LPSB-specific mAb was used to screen for ten Tn5 insertion mutants of Bordetella pertussis which have LPS which is phenotypically distinct from either wild-type LPSAB or LPSB. Silver-strained SDS-PAGE gels showed nine different LPS phenotypes, six of which contain two clinically undocumented LPS bands, designated IntA and IntB based on their proximity to the LPSA and LPSB bands, respectively. Binding assays with LPSA- and LPSB-specific mAbs established changes in epitope exposure for the various mutant LPS, both in cell-free form and as presented on the surface of whole cells. The possible involvement of a number of genes, both structural and regulatory, was indicated in production of the altered phenotypes. PFGE and Southern blotting showed that the Tn5 inserts of seven mutants mapped to a region of the B. pertussis chromosome shown previously to encode the bpl gene products of LPS biosynthesis. Mutants MLT3, MLT5 and MLT8, however, mapped to distinctly different parts of the chromosome. In addition, mutants MLT2 and MLT3 contributed to an accelerated frequency in the appearance of avirulent phase organisms despite their Tn5 inserts being over 1000 bp from the bvglASR locus. The alterations in LPS structure in the mutants changed their reactivity to strain-specific mAbs and their sensitivity to hydrophobic and hydrophilic antibiotics.