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Infection and Immunity, February 2000, p. 767-778, Vol. 68, No. 2
Department of Pathology and Laboratory
Medicine, University of Texas at Houston Medical School, Houston,
Texas
Received 15 October 1999/Accepted 9 November 1999
The mechanism of pathogenesis of Mycobacterium
tuberculosis is thought to be multifactorial. Among the putative
virulence factors is the antigen 85 (Ag85) complex. This family of
exported fibronectin-binding proteins consists of members
Ag85A, Ag85B, and Ag85C and is most prominently represented by 85A and
85B. These proteins have recently been shown to possess mycolyl
transferase activity and likely play a role in cell wall synthesis. The
purpose of this study was to generate strains of M. tuberculosis deficient in expression of the principal members of
this complex in order to determine their role in the pathogenesis of
M. tuberculosis. Constructs of fbpA and
fbpB disrupted with the kanamycin resistance marker
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Disruption of the Genes Encoding Antigen 85A and Antigen 85B of
Mycobacterium tuberculosis H37Rv: Effect on Growth in
Culture and in Macrophages
Km
and containing varying amounts of flanking gene and plasmid vector
sequences were then introduced as linear fragments into H37Rv by
electroporation. Southern blot and PCR analyses revealed disruption of
the homologous gene locus in one fbpA::
Km transformant and one fbpB::
Km transformant.
The fbpA::
Km mutant, LAa1, resulted from a
double-crossover integration event, whereas the
fbpB::
Km variant, LAb1, was the product of a
single-crossover type event that resulted in insertion of both
Km and plasmid sequences. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and Western blot analysis confirmed that expression
of the disrupted gene was not detectable in the fbpA and
fbpB mutants. Analysis of growth rates demonstrated that
the fbpB mutant LAb1 grew at a rate similar to that of the
wild-type parent in enriched and nutrient-poor laboratory media as well
as in human (THP-1) and mouse (J774.1A) macrophage-like cell lines. The
fbpA mutant LAa1 grew similarly to the parent H37Rv in
enriched laboratory media but exhibited little or no growth in
nutrient-poor media and macrophage-like cell lines. The targeted
disruption of two genes encoding mycolyl transferase and
fibronectin-binding activities in M. tuberculosis will
permit the systematic determination of their roles in the physiology and pathogenesis of this organism.
*
Corresponding author. Mailing address: Department of
Pathology and Laboratory Medicine, 6431 Fannin St., Houston, TX
77030. Phone: (713) 500-5272. Fax: (713) 500-0730. E-mail:
armitige{at}casper.med.uth.tmc.edu.
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