Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature‐tagged transposon mutagenesis

Abstract

SummaryTuberculosis remains the greatest cause of death worldwide due to a single pathogen. In order to identify the genes required for the pathogenicity of Mycobacterium tuberculosis, a functional genomic approach was developed. A library of signature-tagged transposon mutants of this bacterium was constructed and screened for those affected in their multiplication within the lungs of mice. From 1927 mutants tested, 16 were attenuated for their virulence. The insertions harboured by the selected mutants were mapped on the M. tuberculosis genome and most of the mutated loci appeared to be involved in lipid metabolism or transport across the membrane. Four independent mutations identi®ed a cluster of virulence genes located on a 50 kb chromosomal region. These genes might be involved in the production of phthiocerol and phenolphthiocerol derivatives, a group of molecules restricted to eight mycobacterial species, seven of them being either strict or opportunistic pathogens. The interaction of ®ve mutant strains with mouse bone marrow macrophages was investigated. These ®ve mutants were still able to multiply in this cell type. However, in three cases, there was a growth defect in comparison with the wild-type strain. The other two strains exhibited no clear difference from the virulent strain, MT103, in this model. This study, which is the ®rst global research of virulence factors of M. tuberculosis, opens the way to a better understanding of the molecules that are key players in the interaction of this pathogen with its host.

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

“…A number of studies demonstrate a correlation between in vivo attenuation of mycobacterial mutants and impaired in vitro survival in macrophages (Camacho et al, 1999;Sassetti & Rubin, 2003). The in vivo phenotype of the lspA : : aph mutant is characterized by a pronounced reduction in c.f.u., which is already prominent soon after infection.…”

Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis

“…Signature tagged mutagenesis (STM) has been used successfully in several bacterial pathogens to identify virulence determinants in vivo [25][26][27][28][29][30][31][32][33]. Briefly, a pool of transposons is assembled, each harbouring a unique oligonucleotide tag.…”

Development of Signature-Tagged Mutagenesis in Burkholderia pseudomallei To Identify Genes Important in Survival and Pathogenesis

“…This locus was later found to code for a previously unknown type III secretion system critical for virulence and bacterial proliferation within macrophages (19,36). STM studies of other intracellular pathogens such as Mycobacterium tuberculosis (6), Listeria monocytogenes (3), Yersinia pestis (14), and Brucella suis (15) successfully identified a number of genes not previously linked to virulence, so we aimed to use a modified version of the technique on the genome-sequenced B. pseudomallei reference strain K96243 (21).…”