This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Mycobacterium tuberculosis ?H (Sigma Factor H or SigH) is a key transcriptional regulator that is involved in resistance to oxidative stress, and plays a role in the pathogenesis and immunopathology of tuberculosis. We determined that the M. tuberculosis ?H-deficient mutant (???? mutant) exhibits a very unique phenotype in a mouse model of infection via the aerosol route: it is able to reach the same bacterial load as the wild-type (WT) strain in mouse organs, but is unable to induce the typical lung granulomatous histopathology and pulmonary inflammatory response associated with the infection. Comparative microarray analysis of the transcriptome of broth-grown WT and ???? mutant strains indicates that the thioredoxin system (thioredoxin and thioredoxin reductase) is regulated by ?H. The ability of the thioredoxin system to act as a general protein disulfide reductant and reduce reactive oxygen species in a NADH/NADPH dependent manner has been established in several systems. It is likely that the ???? mutant is unable to protect itself from some of the effects of oxidative stress, as it is unable to induce the thioredoxin regulon;this would explain its inability to cause the tissue damage and immunopathology that is observed with the WT strain. In order to determine whether the """"""""reduced immunopathology"""""""" phenotype of the ???? mutant observed in an in-vivo mouse aerosol model of infection, is manifested in a more accurate, human-like, primate model of tuberculosis, and to employ this mutant as a model to further study the molecular and immunological determinants of TB pathogenesis we have proposed the following specific aim: Comparison of the in-vivo infection phenotype of the WT M. tuberculosis and the ???? mutant strain in a macaque model. Note: Dr. Kaushal had a one year pilot entitled """"""""The crucial role of M. tuberculosis ?H in the immunopathology of TB that ran from 7/1/06 to 6/30/07. After a competitive review process he was then selected for a full COBRE project upon the graduation of Dr. MacLean. This full project entitled """"""""Mycobacterium tuberculosis ?? and its regulon in the immunopathology of Tuberculosis"""""""" started October 2007. A.
SPECIFIC AIMS. SA1 In-vivo studies using pilot study samples, to identify molecular determinants of immunopathology: SA1a: Gene-expression and cytometry in the granulomatous lesions of animals infected with M. tuberculosis (Mtb) and the ???? mutant. We will compare the transcriptome of lesions from the two groups of animals as well as lesion gene-expression as a function of size, location and type. SA1b: In-vivo bacillary transcriptome during macaque infection. We will compare the transcriptome of BAL cells from the two groups of macaques. The underlying hypothesis is to conclusively determine if the composition of the in-vivo ?H regulon is similar to in-vitro data and whether Trx, Clp and mce regulons are specifically induced in-vivo, in Mtb. SA2 In-vivo studies involving a new experiment with rhesus macaques: SA2a: Confirmation of the """"""""imp"""""""" phenotype of the ???? mutant in macaques. We propose a comprehensive experiment (5 animals per group), to be infected with a lower dose of Mtb and the ???? mutant. It will allow a statistically valid comparison of immunopathology in macaque lungs in response to a physiologically relevant dose. SA2b: Role of GranB+ cytolytic T cells in immunopathology of TB. Our preliminary data showed a higher accumulation of CD8+ GranB+ T cells and higher expression of GranB in BAL cells from the animals infected with the ???? mutant, compared to Mtb. We hypothesize that one or more ??-dependent Mtb component(s) specifically interacts with the host immune system to suppress the cytolytic function mediated by GranB. We will study the recruitment kinetics of CD8+GranB+ T cells in peripheral blood, BAL and lungs of both groups of animals by flow cytometry. SA3 Ex-vivo Studies: We standardized the infection of rhesus lung-derived macrophages with Mtb. Using this model, we propose SA3a: Effect of ?? on host response to infection with tubercle bacilli. We will compare changes in the host proteome following infection with the two strains, extending our transcriptome findings. We expect to find differences in the expression of pro-inflammatory cytokines, other mediators and, crucially, of chemokines involved in immune cell recruitment to the lungs. We will focus on IL-6-STAT3, NFkB and IL-8 pathways that show enhanced expression during infection with Mtb but not the mutant, pro-inflammatory agents (TNF-a, g-IFN) and cytokines/chemokines with a defined role in TB. SA3b: Analysis of the role of IL6-STAT3, IL-8 and NFkB pathways in mediating immunopathology of TB. The role of these pathways will be examined in the context of ?? by silencing the expression of key genes from these pathways, in rhesus primary cells, and assess their influence on the infectivity of Mtb and the mutant.
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