IDENTIFYING M TUBERCULOSIS GENES

Project: Research project

Description

The disease tuberculosis has become a major health problem nationally and
internationally. More people die of infections with M. tuberculosis
worldwide than any other infectious agent. Central to the disease
process in tuberculosis are the interactions of the bacilli with host
macrophages. The infection of macrophages by M. tuberculosis can be
divided into three steps: adherence, entry, and intracellular survival.
The role virulence factors of M. tuberculosis play in these complex
interactions is virtually unknown. The goal of this proposed fellowship
is to identify, clone and characterize genes of M. tuberculosis which are
required for uptake, intracellular survival, and multiplication within
human macrophages. Mycobacterium smegmatis will be used to clone these
potential virulence factor genes of M tuberculosis. M. smegmatis is an
ideal strain to use in these studies because it grows rapidly in the
laboratory (in days instead of weeks as for M. tuberculosis), readily
expresses genes from other mycobacteria, can be genetically manipulated
by various techniques, is nonpathogenic, and is internalized and killed
by macrophages. The specific aims are to: 1. Prepare a genomic library from a virulent strain of M. tuberculosis
by using a plasmid vector system. Mycobacteria smegmatis will be
transformed with the genomic library by electroporation. Antibiotic
resistant transformations will be selected and pooled. 2. M. smegmatis transformants will be used to infect monolayers of human
macrophages and those clones which invade and/or survive intracellularly
within macrophages will be isolated. 3. Plasmids from M. smegmatis clones recovered from macrophage
monolayers will be isolated, restriction mapped, and sequenced to
characterize the cloned gene(s). 4. The role of these cloned genes in uptake and intracellular survival
of M. tuberculosis within macrophages will be verified by construction
of mutations in these gene(s). The mutations will be introduced into the
chromosome of virulent Mycobacterium tuberculosis by gene replacement
techniques. These mutant strains will then be tested for their abilities
to enter and survive within macrophages as compared to the virulent wild-
type strain.
StatusActive
Effective start/end date7/5/95 → …

Funding

  • National Institutes of Health

Fingerprint

Mycobacterium smegmatis
Tuberculosis
Macrophages
Genes
Clone Cells
Genomic Library
Virulence Factors
Plasmids
Mutation
Electroporation
Mycobacterium
Infection
Mycobacterium tuberculosis
Bacillus
Health

ASJC

  • Medicine(all)