Molecular epidemiology and fingerprinting of field strains of Mycobacterium tuberculosis

Tuberculosis is a major health problem for most developing countries and India holds the unfortunate distinction of having the largest TB population in the world. Kerala, a small picturesque state in the Southern tip of India also has its fair share of TB. One major focus of our research is to identify and fingerprint local field strains. Our initial studies on fingerprinting using IS6110 as a marker showed that a large number of strains have low (fewer than five) or no copy of this sequence making them untypable by using this polymorphism. We have investigated systems such as AFLP which scans whole genomes fro polymorphisms and found that while AFLP is powerful in discriminating between mycobacterial species, it has limited utility in typing isolates. Currently we are using methods such as MIRU, RD sequence polymorphism and Spoligotyping to to study the strains. Our studies using RD differences led to the identification of the presence of moaA3 in the field strains from Kerala which is absent in H37Rv and Ra. We have also identified a novel insertion of IS6110 in a field strain.

Identification and characterization of novel sequences of Mycobacterium tuberculosis from Kerala

Our studies on fingerprinting pointed out the possibility that the differences in the field strains extended beyond the fingerprint differences to the genome level. We have been employing AFLP and Subtractive Hybridisation for identifying novel sequences of the field strains. Using subtraction we have identified a novel region in the field strains from Kerala and this is being characterized.

Host Pathogen interaction

Macrophages are cells that phagocytose and kill invading pathogens and also present the antigens to the immune system for augmentation of the immune response. But pathogenic mycobacteria have developed systems to successfully survive in macrophages. We have designed a Host-Pathogen Interaction Analysis vector to identify the mycobacterial genes that are induced when the bacterium interacts with the macrophage. This vector has a promoterless cre gene which when induced by the mycobacterial promoter makes the system hygromycin resistant. We are using the system in Mycobacterium smegmatis and the vaccine strain M. bovis BCG to identify the genes that are induced in mycobacteria on interaction with THP1 cells, a monocyte-macrophage cell line. The characterization of some the genes that have been identified are in progress.