GENETIC SYSTEMS TO STUDY VIRULENCE IN BACTEROIDES

点击翻译按钮获取中文摘要

DESCRIPTION (Adapted from the applicant's abstract): The long term objectives of this proposal are to understand the factors that contribute to the virulence and pathogenicity of Bacteroides fragilis, the most important obligately anaerobic organism in suppurative intra- abdominal and pelvic infections in man. The investigators have recently isolated transposon insertion mutations with alterations in several functions which are candidates to be virulence factors; these include increased sensitivity to oxygen (loss of aero-tolerance); decreased binding to animal cells in tissue culture (loss of specific adherence); reduced ability to take up the essential heme molecule for enzyme and co-enzyme formation. Using a detection system for differential gene expression, the investigators have begun to isolate candidate promoter fragments for genes that are turned on, or turned on to a higher level of expression in the model systems of infection and/or after exposure of B. fragilis cells to oxygen or hydrogen peroxide. Their first objective is to analyze the new insertion mutants with demonstrated defects in growth in the tissue culture monolayer systems and in the rat granuloma pouch model. The moxR-region is defined by the aero-sensitive moxR-homolog mutants YT65.2.10 and YT120.2.20. They will determine if the genes in the mox-region comprise a common transcription group and establish how these genes are regulated. They will use a sensitive mutagenesis assay to follow early effects of oxygen exposure. They expect to see a significant difference in mutation frequencies in the moxR-homolog mutants compared with the parental strain. The rubredoxin region is particularly interesting since this protein is often involved in electron transfer reactions that may involve oxygen and some of its toxic products. They will determine the genes downstream of the rubredoxin structural gene and create specific deletion mutations in them to test for alterations in their in vivo growth potential. The B. fragilis binding deficient mutant, YT58.1.3 will be used to characterize genes and proteins required for binding to the tissue culture monolayer. Methods to detect surface proteins will be applied to these mutants to try and identify the protein(s) affected by the insertion. They will study the process of iron and heme uptake in B. fragilis by taking advantage of the heme permease gene newly identified in their laboratory, the newly identified angR-homolog discovered in transposon mutant MGD13.1, and the sequences of the hupA,B genes known to be involved in heme binding. Methods for detecting environmentally activated gene expression applied to bacterial cells during infection may reveal global and gene specific regulatory pathways involved in virulence. The investigators will continue to apply the cre/lox system that they have adapted for use in B. fragilis to obtain additional candidates for genes that are activated in vivo or after exposure to oxygen or hydrogen peroxide. To identify transposon mutants with defects in abscess formation, the hallmark of B. fragilis infections, they will adapt a sub-cutaneous abscess model from the mouse for use in the rat.