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Mycobacterium tuberculosis pellicles express unique proteins recognized by the host humoral response.

ABSTRACT: Mycobacterium tuberculosis (MTB) causes both acute and chronic infections in humans characterized by tolerance to antibiotics and reactivation to cause secondary tuberculosis. These characteristics have led to renewed interest in the in vitro pellicle, or biofilm mode of growth, where bacteria grow to produce a thick aggregate at the air-liquid interface and exhibit increased phenotypic resistance to antibiotics. We infected guinea pigs with the virulent H37Rv strain of MTB for 60 days at which point we collected blood. To identify antigenic proteins, membrane protein extracts of MTB H37Ra pellicles and shaken cultures grown for 3, 5, or 7 weeks were probed with the infected animals' sera after the proteins were separated by two-dimensional gel electrophoresis (2DGE). Antigenic proteins were then identified using MALDI-TOF/TOF mass spectrometry peptide mass fingerprinting. Antigenic pellicle proteins were compared across the three timepoints to identify those that were produced consistently during pellicle growth. They were also compared to those membrane proteins identified from harvested shaken cultures to determine pellicle-specific vs. universally expressed proteins. Using this technique, we identified 44 distinct antigenic proteins, nine of which were pellicle-specific. The sequence of antigenic pellicle-specific proteins was checked for sequence conservation across 15 sequenced MTB clinical isolates, three other members of the MTB complex, as well as M. avium and M. smegmatis. The antigenic pellicle-specific protein Rv0097 was found to have very high sequence conservation within the MTB complex but not with related mycobacteria, while FabG4 was highly conserved in all mycobacteria analyzed. These conserved pellicle-specific proteins represent targets for the development of future diagnostic tests and vaccines.

SUBMITTER: Kerns PW

PROVIDER: S-EPMC4363115 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Mycobacterium tuberculosis pellicles express unique proteins recognized by the host humoral response.

Kerns Patrick W PW Ackhart David F DF Basaraba Randall J RJ Leid Jeff G JG Shirtliff Mark E ME

Pathogens and disease 20140226 3

Mycobacterium tuberculosis (MTB) causes both acute and chronic infections in humans characterized by tolerance to antibiotics and reactivation to cause secondary tuberculosis. These characteristics have led to renewed interest in the in vitro pellicle, or biofilm mode of growth, where bacteria grow to produce a thick aggregate at the air-liquid interface and exhibit increased phenotypic resistance to antibiotics. We infected guinea pigs with the virulent H37Rv strain of MTB for 60 days at which ...[more]

PMID: 24453174

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Project description:Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4(+) T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can be explored as antigen candidates for prime-boost vaccination strategies or extrapolated as markers for disease detection and progression.

Dataset Information

Mycobacterium tuberculosis pellicles express unique proteins recognized by the host humoral response.

Publications

Mycobacterium tuberculosis pellicles express unique proteins recognized by the host humoral response.

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