GLOBAL REGULATION OF VIRULENCE FACTOR IN BORDETELLA PERTUSSIS

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The vir locus of Bordetella pertussis regulates the expression of multiple virulence-associated factors in response to environmental stimuli. Our studies are ultimately aimed at a molecular characterization of the mechanism by which environmental signals result in changes in expression of vir-regulated genes. The published DNA sequence of the vir locus predicts that three proteins, BvgA, BvgB, and BvgC are encoded, two of which show relatedness to a family of environmentally responsive regulatory proteins in bacteria termed "two component" systems. Gene fusions have been created through the use of PCR which have enabled us to express the vir-encoded proteins in E. coli in the form of fusion proteins to alkaline phosphatase. These have been used to develop polyclonal and monoclonal antibody reagents directed against the Bvg proteins. These antibodies allow us to detect their corresponding proteins in crude B. pertussis extracts. The use of these antibody tools has led us to the realization that BvgB and BvgC are in fact one protein which we are now calling BvgS (for sensor). The reason for the previous misunderstanding was an error in the DNA sequence which has been located and confirmed. We now have antibody reagents against both components of the vir regulatory system and are using those reagents to purify these proteins and test their functions biochemically. Preliminary experiments show that the monoclonal antibody against BvgS will be useful in immunoaffinity purification. Earlier genetic data has suggested that the sensor protein BvgS is functional as a multimer, probably a dimer. Our antibody tools have allowed us to begin investigating this hypothesis biochemically by using chemical cross-linking reagents on membrane preparations of B. pertussis. Preliminary evidence from these experiments supports our hypothesis that BvgS is active as a dimer.