The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo

Abstract

SummaryThe ability of Mycobacterium tuberculosis to grow in macrophages is central to its pathogenicity. We found previously that the widespread 210 strain of M. tuberculosis grew more rapidly than other strains in human macrophages. Because principal sigma factors influence virulence in some bacteria, we analysed mRNA expression of the principal sigma factor, sigA , in M. tuberculosis isolates during growth in human macrophages. Isolates of the 210 strain had higher sigA mRNA levels and higher intracellular growth rates, compared with other clinical strains and the laboratory strain H37Rv. SigA was also upregulated in the 210 isolate TB294 during growth in macrophages, compared with growth in broth. In contrast, H37Rv sigA mRNA levels did not change under these conditions. Overexpression of sigA enhanced growth of recombinant M. tuberculosis in macrophages and in lungs of mice after aerosol infection, whereas recombinant strains expressing antisense transcripts to sigA showed decreased growth in both models. In the presence of superoxide, sense sigA transformants showed greater resistance than vector controls, and the antisense sigA transformant did not grow. We conclude that M. tuberculosis sigA modulates the expression of genes that contribute to virulence, enhancing growth in human macrophages and during the early phases of pulmonary infection in vivo . This effect may be mediated in part by increased resistance to reactive oxygen intermediates.

Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth

“…M. tuberculosis strain 210 is widely distributed in the south-west and south-central USA (Barnes et al, 1997;Weis et al, 2002;Yang et al, 1998), and isolates of this strain grow more rapidly in human macrophages than other strains (Theus et al, 2005;Zhang et al, 1999), suggesting an enhanced capacity to avoid host defences. The ability of M. tuberculosis to grow in human mononuclear phagocytes is a central feature of its pathogenic potential.Strain 210 is a member of the widespread W-Beijing family of strains, and growing evidence indicates that the altered expression patterns of some genes contribute to the success of these strains (Reed et al, 2007;Sinsimer et al, 2008;Tsenova et al, 2005;Wu et al, 2004). We have previously shown that increased expression of the principal sigma factor SigA mediates the capacity of isolates of M. tuberculosis strain 210 to grow more rapidly in human monocytes and in mice (Wu et al, 2004).…”

“…TB294 (Table 1) is a member of M. tuberculosis strain 210 and was isolated from a patient with tuberculosis (Zhang et al, 1999). Transformants of TB294 bearing a sense sigA (TB294-pSigA) and vector control plasmid (TB294-pCV) were generated, as previously described (Wu et al, 2004). H37Rv was obtained from the Sanger Center.…”

“…For mycobacteria cultured in broth, RNA was extracted as described previously (Wu et al, 2004). To isolate RNA from intracellular mycobacteria, bacteria were harvested from MonoMac6 cells, as described by Wu et al (2004), and then RNA was extracted using the method described for broth-grown cultures, except that following extraction, the bacterial RNA was further purified with the MICROBEnrich kit (Ambion).Relative quantification of mRNA by real-time PCR. A mixture of 37 genome-directed primers designed to prime all ORFs in the M. tuberculosis genome was used for reverse transcription (Talaat et al, 2000).…”

“…PCR conditions were identical for all reactions, which were performed in triplicate. Assays were performed using the ABI Prism 7700 (Applied Biosystems) and were normalized for 16S rRNA, as described previously (Wu et al, 2004).DNA microarray analysis. The M. tuberculosis DNA microarray consisted of 4295 70-mer oligonucleotides representing the 3924 predicted ORFs of the H37Rv strain (http://www.sanger.ac.uk) and 371 probes designed to detect sequences in the CDC1551 strain (http://www.tigr.org).…”

Avaliação da virulência micobacteriana e modulação da resposta imune durante a infecção por isolados clínicos de Mycobacterium bovis e Mycobacterium tuberculosis.

“…Tais fatores envolvidos em mecanismos de reprogramação transcricional, baixo metabolismo catalítico e capacidade imunomodulatora são responsáveis pelo sucesso do M. tuberculosis em adaptar-se ao hospedeiro (Sachdeva et al, 2010). O sequenciamento completo do cromossomo circular da cepa H37Rv de M. tuberculosis em 1998 por foi de suma importância para auxiliar a compreensão acerca da patogenia induzida pelo complexo M. tuberculosis (Cole e Barrell, 1998; (Steyn et al, 2002;Wu et al, 2004).…”