Establishment and validation of Mycobacterium fortuitum model for Mycobacterium Tuberculosis Persistent Infection

Abstract

Persistent form of tuberculosis remains major hurdle in absolute and effective control of the disease As processes and mechanisms leading to development of persistent infection may take long time to manifest in host thus various in vitro models of Mycobacterium tuberculosis persistent infection have been developed Study using model systems employing M tuberculosis are associated with constrains in terms of cost establishment and maintenance of BSL 3 as well as screening period longer generation time Aim of the present study was to develop a rapidly growing mycobacteria based model mimicking M tuberculosis persistent infection which may offer advantages in terms of cost effectiveness and comparatively faster screening time for potential drugs and drug targets Mycobacterium fortuitum was selected on the basis of shorter generation time ease of handling nonrequirement of biosafety level3 and closer homology with M tuberculosis genome sequence On subjecting to physiological stress conditions encountered by persistent M tuberculosis M fortuitum showed constant CFU for extended periods of time the most important characteristic feature of a persistent infection Further validation of the organism also confirmed that it exhibited all physiological features of a persistent infection and upregulation of genes involved in persistence Identification of five novel gene sequences of M fortuitum was accomplished as homologs of M tuberculosis genes involved in persistence and submitted to GenBank database Upregulation of these M fortuitum homologs studied by realtime PCR predicted their involvement in persistence of M fortuitum An antisense knockout study of three of these genes was done which further confirmed their role in M fortuitum persistence Thus our studies suggest a conserved mechanism of persistence in M fortuitum and M tuberculosis Studies related to behavior of M fortuitum under acidinduced stress indicated that acidinduced stress may induce the conditions of persistent form of bacilli thus this condition may also be