Towards improved understanding of M. tuberculosis complex strain differences in The Gambia
23 October 2017
Promotiezaal Grauwzusters (UAntwerp, Stadscampus) - Lange St. Annastraat 7 - 2000 Antwerp (route: UAntwerpen, Stadscampus
3:00 PM - 5:00 PM
Nana Akua Boatema Ofori-Anyinam
Leen Rigouts, Bouke de Jong
PhD defence Nana Akua Boatema Ofori-Anyinam - Department of Biomedical Sciences
Knowledge of the physiology and metabolism of M. africanum lineages remains limited, relative to the rest of the M. tuberculosis complex (MTBC). Epidemiological studies showed that M. africanum L6 was more attenuated than M. tuberculosis, and less likely to cause disease in exposed contacts in the next two years. However, the underlying biological differences to explain the clinical phenotype were not yet uncovered. Furthermore, diagnostics for identification of the MTBC that are commonly used in The Gambia and other West African countries, were developed and evaluated mostly with the M. tuberculosis lineages. Therefore, studies conducted in this thesis explored biological reasons for M. africanum L6 attenuation and looked for differences that could affect the diagnostic performance of commonly used tools in West Africa.
We found significant underexpression, in sputum, of dormancy regulon genes in L6 compared to the more virulent L4, and confirmed that L6 favors hypoxic growth. As expression of the regulon increases intracellular survival and virulence, with impaired function associated with attenuation, our findings provide possible explanations for the attenuated clinical phenotype of L6.
Gene expression showed that L6 underexpressed an immunogenic protein detected by a widely used diagnostic assay (MPT64). We prospectively assessed the sensitivity of the MPT64 diagnostic for confirmation of M. africanum as MTBC in positive Mycobacterium Growth Indicator Tube (MGIT) cultures and found reduced sensitivity of this tool for detecting L6. To prevent misclassification, particularly in West Africa, MPT64 negative cultures should be confirmed with more sensitive molecular tools. In general, novel diagnostics should be evaluated against the entire MTBC before laboratory implementation.
A whole genome sequencing approach was used to investigate the mpt64 gene to determine the biological reasons for underexpression and reduced protein secretion in L6. This analysis showed how whole genome sequence analysis of the MTBC could be done in Africa and other low resource settings without vast bioinformatics infrastructural resources.
mpt64 and genes with predicted regulatory interactions had non-synonymous mutations with potential deleterious effects on protein function. Nutrient starvation was previously found to induce mpt64 underexpression and reduce antigen secretion. In L6, we found multiple mutations in carbon metabolism genes required for optimum use of nutrients in the MGIT medium, providing further reasons for MPT64 underexpression and -secretion. Our findings suggest that improved formulations of MGIT Growth media could increase isolation of L6 with the MGIT system and confirmation as MTBC by MPT64 rapid tests.