PHENOTYPIC CHARACTERIZATION OF HOST-PATHOGEN INTERACTION IN Mycobacterium africanum

Abstract Background: Mycobacterium africanum (MAF) and Mycobacterium tuberculosis sensu strico (MTBss) are two members of a closely related bacterial species of Mycobacterium tuberculosis complex (MTBC) that causes tuberculosis (TB) in humans. However MAF is known to cause up to 50% of human pulmonary TB in West Africa only. MAF has been subdivided into MAF West African 1 (MAF1) (Lineage 5) and MAF West African 2 (MAF2) (Lineage 6), as two distinct phylogenetical lineages within MTBC. Subsequently the absence of Mycobacterium tuberculosis deletion gene (TbD1) strains in MTBss has been referred to as modern lineage whilst ancient lineage (MAF1 and MAF2) have the presence of TbD1. Ghana represents one of the few countries within Central West Africa known to have this unique genetic diversity of MAF1, MAF2 and MTBss that causes TB cases in significant proportions. While previously it was thought MAF is genetically very closely related to MTBss such that there are no important phenotypic differences between the two species, current advance in molecular biology indicate that substantial genetic difference exit between the two that can translate into significant phenotypic differences including immunogenicity and virulence. Aim: The aim of the study was to analyze the phenotypic features of host-pathogen interaction in Mycobacterium africanum and compared to Mycobacterium tuberculosis sensu strico strains. Methodology: The study was embedded in 2 different projects. Retrospective archived cryopreserved peripheral blood mononuclear cells (PBMCs) of MAF-infected and MTBssinfected patients were stimulated with growth medium (negative control), Staphylococcus enterotoxin B (SEB, positive control) and recombinant early secreted antigenic protein 6 kiloDalton/culture filtrate protein 10 kiloDalton fusion protein (rESAT-6/CFP-10), surface stained for T-subsets (CD4 and CD8) and intracellular cytokine, interferon gamma (IFN-), and acquired with FACS Calibur flow cytometer. The second study used characterized large sequence polymorphism (LSP) clinical isolates identified as MAF1, MAF2 and MTBss to determine intracellular growth assay in human monocyte–derived macrophages (MDM), mean doubling time and pro-inflammatory tumour necrosis factor–alpha (TNF-α), interleukin 6 (IL-6) and 12p70 cytokines by enzyme-linked immunosorbent assay (ELISA). Results: The percentage frequencies of CD4+IFN-+ and CD8+ IFN-+ T cells of MAFinfected patients did not differ from the percentage frequencies CD4+ IFN-+ and CD8+ IFN-+ T cells of MTB-infected patients in response to rESAT-6/CFP-10 fusion protein (p>0.05). Uptake of MAF1, MAF2 and MTBss representing modern and ancient strains respectively at 4hours was not significant (p>0.05). Mean intracellular growth index from 24hours to 72hours was significantly rapid for MTBss (modern) lineage compared to MAF1 and MAF2 (ancient) lineages (p