The Mycobacterium tuberculosis SigD sigma factor controls the expression of ribosome-associated gene products in stationary phase and is required for full virulence

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Abstract
During infection Mycobacterium tuberculosis is exposed to several environmental conditions depending on the stage and severity of the disease. To survive, M. tuberculosis uses alternate sigma factors to regulate its gene expression in response to the changing host environment. In order to better understand the way in which stress response genes are regulated, the extracytoplasmic sigma factor gene sigD was deleted and subsequently complemented in the CDC1551 strain of M. tuberculosis. The ΔsigD mutant strain exhibited an in vitro growth rate in rich medium identical to that of both the sigD-complemented and wild-type CDC1551 strains. Additionally, no differences were observed in short-term intracellular growth between the mutant, complemented, and wild-type bacteria within the J774A.1 macrophage cell line. However, tumour necrosis factor (TNF)-α levels in macrophages infected with the ΔsigD mutant were decreased as compared to levels observed in macrophages infected with the wild-type bacteria. In time-to-death studies, C3H mice infected with the ΔsigD mutant exhibited a mortality delay compared to those infected with either the complemented or wild-type strains. Although mice infected with the ΔsigD mutant died at a reduced rate, the bacillary loads in the lungs and spleen of these mice were comparable to those seen in mice infected with either the complemented or wild-type strains. Microarray analysis of the ΔsigD mutant relative to wild type revealed that SigD directs the expression of a small set of ribosomal genes and adenosine triphosphate transporters whose expression is normally induced during stationary phase growth in vitro. Altered expression of a subset of these genes was confirmed by quantitative reverse transcription polymerase chain reaction analysis. Promoter-like elements resembling the consensus sequence AGAAAG-N16−20-CGTTAA were found upstream of 19 of the genes underexpressed in the ΔsigD mutant suggesting this may be the recognition sequence for the M. tuberculosis SigD-holoenzyme, EσD. These data indicate that the M. tuberculosis SigD sigma factor governs the expression of a small set of ribosomal genes typically expressed in stationary phase during in vitro growth and that loss of sigD reduces macrophage TNF-α secretion as well as the lethality of M. tuberculosis infection in mice.

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