M TUBERCULOSIS SURVIVAL IN MACROPHAGES

Project: Research project

Description

DESCRIPTION The infection of macrophages by Mycobacterium tuberculosis can be divided into four steps: adherence, entry, intracellular survival, and multiplication. The aim of the proposed research is to identify, clone, and characterize genes of M. tuberculosis which are required for intracellular survival within macrophages. Potential virulence factor genes of M. tuberculosis will be cloned by first constructing a recombinant library and incorporating it into the nonpathogenic Mycobacterium smegmatis, a rapidly-growing mycobacterium that is otherwise killed by macrophages. Clones with enhanced survival in macrophages will be identified and examined for the presence of M. tuberculosis genes involved in survival. In initial experiments, the applicant has demonstrated the feasibility of the proposed studies by isolating a plasmid clone from an M. tuberculosis library that reproducibly confers enhanced survival in U937 cells, and has isolated several cosmid clones, using the same strategy. The specific aims are: 1) To screen M. smegmatis clones containing an M. tuberculosis H37Rv recombinant library (either a plasmid or cosmid library) for survival in monolayers of the human macrophage-like cell line, U937. 2) To isolate M. smegmatis clones that survive within the U937 macrophage monolayers for further study. Plasmids or cosmids from these clones will be recovered and the M. tuberculosis genes contained will be restriction-mapped, sequenced, and compared to the published genome sequence of M. tuberculosis H37Rv as well as to other databases, and analyzed with other molecular techniques to characterize the cloned genes. 3) Genes identified and characterized in Specific Aim 2 which enhance M. smegmatis survival within macrophages will be selected and used to evaluate the role of these genes in virulent M. tuberculosis. Mutations will be constructed in these genes and introduced into the chromosome of virulent M. tuberculosis H37Rv by allelic exchange. The ability of the mutants to survive, persist, and replicate in the U937 macrophage survival assay will be tested and compared to the virulent wild-type parental strain. 4) Genes identified from the screening of the plasmid and cosmid libraries will be used in gene expression studies. Levels of gene expression will be monitored in vitro and within macrophages using integrating reporter gene vectors.
StatusFinished
Effective start/end date9/1/993/31/05

Funding

  • National Institutes of Health: $303,000.00
  • National Institutes of Health: $303,000.00
  • National Institutes of Health: $254,551.00
  • National Institutes of Health: $340,875.00

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Tuberculosis
Macrophages
Survival
Mycobacterium tuberculosis
Genes
Mycobacterium smegmatis
Clone Cells
Cosmids
Gene Knockout Techniques
Plasmids
Virulence Factors
Mycobacterium
Interferon-gamma
Reporter Genes
Gene Expression
Gene Library
Monocytes

ASJC

  • Medicine(all)
  • Immunology and Microbiology(all)