Mycobacterium tuberculosis manages to remain latent in the human body regardless of extensive chemotherapy. Complete eradication of tuberculosis (TB) requires treatment strategies targeted against latent form of infection, in addition to the current regimen of antimycobacterials. Many in vitro and in vivo models have been proposed to imitate latent TB infection, yet none of them is able to completely mimic latent infection state of M. tuberculosis. Highly infectious nature of the pathogen requiring BSL3 facilities and its long generation time further add to complications. M. aurum has been proposed as an important model organism for high throughput screening of drugs and exhibits high genomic similarity with that of M. tuberculosis. Thus, the present study was undertaken to explore if M. aurum could be used as a surrogate organism for studies related to M. tuberculosis latent infection. M. aurum was subjected to in vitro conditions of oxygen depletion, lack of nutrients and acidic stress encountered by latent M. tuberculosis bacteria. CFU count of M. aurum cells along with any change in cell shape and size was recorded at regular intervals during the stress conditions. M. aurum cells were unable to survive for extended periods under all three conditions used in the study. Thus, our studies suggest that M. aurum is not a suitable organism to mimic M. tuberculosis persistent infection under in vitro conditions, and further studies are required on different species for the establishment of a fast growing species as a suitable model for M. tuberculosis persistent infection.

We report a novel lacZ fusion vector and demonstrate its utility for expression analysis of genes associated with Mycobacterium tuberculosis latent infection. The vector contains E. coli (oriE) and mycobacterial (oriM) origins of replication, a kanamycin resistance gene (Km(r)) as selection marker, and a lacZ reporter gene in fusion with MCS for cloning of upstream regulatory sequence of the desired genes. β-galactosidase activity of the vector was standardized for expression analysis under latent mycobacterial conditions using Phsp60, a constitutive mycobacterial promoter, utilizing Mycobacterium smegmatis as model organism. Validation of the vector was done by cloning and expression analysis of PhspX (alpha crystalline) and Picl (isocitrate lyase), promoters from two of the genes shown to be involved in M. tuberculosis persistence. Both genes showed appreciable levels of β-galactosidase expression under hypoxia-induced persistent conditions in comparison to their actively replicating state. Expression analysis of a set of hypothetical genes was also done, of which Rv0628c showed increased expression under persistent conditions. The reported fusion vector and the strategy can be effectively used for short listing and validation of drug targets deduced from various non-conclusive approaches such as bioinformatics and microarray analysis against latent/persistent form of mycobacterial infection.

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