Date of Award
tuberculosis, TB immune response
Tuberculosis is characterized by dynamic interactions between M. tuberculosis (Mtb) and the human immune response. The cytokine IFN-Î³ triggers macrophage production of bactericidal nitric oxide by inducible nitric oxide synthase (NOS2) and is essential for mammalian control of Mtb infection. Mice lacking NOS2 are unable to control replication of Mtb and rapidly succumb to infection. The persistent nature of TB infection suggests that Mtb has evolved counter-immune mechanisms to survive in the face of NOS2 and other pathways downstream of IFN-Î³. A differential signature-tagged transposon mutagenesis screen was conducted to identify mutants attenuated in NOS2-/- mice, but retaining virulence in IFN-Î³-/- mice. Such mutants may be deficient in counter-immune responses to IFN-Î³ -dependent, NOS2-independent immune pathways. Four mutants with the phenotype of interest were pulled from a screen of 96: pks6/Rv0405 - a polyketide synthase, Rv0072 - a membrane spanning domain of a putative glutamine transporter, Rv2958c - a glycosyl-transferase, and pstA1/Rv0930 - a membrane spanning domain of an inorganic phosphate transporter. Monotypic IV infections confirmed that these mutants replicate freely in the tissues of IFN-Î³-/- mice and kill these mice with similar kinetics to wild-type Mtb. In contrast, the mutants have little or no growth advantage in NOS2-/- mice and are highly attenuated in these mice. It was discovered that none of the four mutants were producing a key mycobacterial surface lipid: phthiocerol dimycocerate (PDIM). In vivo phenotypes of the mutants were reproduced in a PDIM deficient strain cloned from a subpopulation pre-existing in the parent stock. Hence it was discovered that PDIM deficiency causes IFN-Î³ dependent, NOS2 independent attenuation of H37Rv. Moreover, the PDIM deficient strains demonstrated NOS2 dependent attenuation as well as early IFN-Î³ independent attenuation. Autonomously from PDIM, the pstA1 mutant is impaired for survival during WT and NOS2-/- macrophage infection and during in vitro Pi starvation. It is also deficient for uptake of orthophosphate and is hyper-sensitive to H202, SDS, and acidified nitrite exposure. Phosphate starvation of H37Rv induces hypersensitivity to H202 and SDS suggesting that disruption of pstA1 may disrupt metabolism and/or gene regulation in a manner consistent with the effects of phosphate starvation.
Kirksey, Meghan Alida, "A Search for the Counter-Immune Mechanisms of Mycobacterium Tuberculosis" (2007). Student Theses and Dissertations. 7.