Introduction Mycobacterium leprae the causative agent
Mycobacterium leprae, the causative agent of leprosy, affects skin, peripheral nerves and upper respiratory tract . Although leprosy has been eradicated from most of the countries, it still affects approximately a quarter of a million people from India . Details of many bacteriological and pathological information including infection, parasitization and replication are still largely unknown because M. leprae is an unculturable pathogen . Genome sequencing revealed that the 3.3 mbp M. leprae genome encodes 1605 protein coding genes and 1115 pseudogenes [, , ]. Comparative genomic analysis of M. leprae genome with other mycobacterial genomes have established that its genome has gone through a reductive STF-118804 leaving behind a relatively short functional genome compared to other mycobacterial species such as Mycobacterium tuberculosis, Mycoplasma, Rickettsia and Chlamydia . The massive gene reduction has caused the elimination of a large number of functional genes, and some of the remaining genes have become nonfunctional gene or ‘pseudogenes’ [4,8]. Due to the slow rate of pseudogene elimination from the genome, the number of pseudogenes are exceptionally high in M. leprae . Most of these pseudogenes are linked to different metabolic and cellular pathways, including DNA repair. For example, genes associated with homologous recombination repair, such as recB, recC, recD, alkA, gyrB, mutT, dinG, dinF exist as a pseudogenes . However, M. leprae also harbors several protein coding genes that do not have an assigned function yet and commonly referred to as ‘hypothetical proteins’ . Therefore, it is possible that crucial DNA repair pathways essential for the survival of the bacteria could still be present among these hypothetical proteins. Thus, functional characterization of hypothetical proteins, especially that are involved in DNA repair, is necessary for a complete understanding of the mechanism of genome integrity of M. leprae.
In the present investigation, we have selected a hypothetical M. leprae protein ML0190 for functional characterization. We report that ML0190 belongs to the family of Fe(II)/2OG-dependent dioxygenase involved in DNA dealkylation repair [12,13]. The Escherichia coli AlkB is the most studied Fe(II)/2OG-dependent dioxygenase which is involved in the repair of methylated bases including N1-methyladenine (1meA) and N3-methylcytosine (3meC) [14,15]. The active site of these family of enzymes is located in a characteristic double-stranded β-helix (DSBH) fold. The conserved residues in the DSBH fold are involved in coordination of the essential Fe2+ ion and 2OG cofactors . Our initial sequence alignment and homology modelling study revealed that ML0190 belongs to AlkB family of Fe(II)/2OG-dependent dioxygenase with conserved DSBH domain and conserved catalytic residues. By qPCR analysis of skin biopsy samples from leprosy patients, we demonstrate that transcript of ML0190 is present during M. leprae infection. Using heterologous expression of ML0190 in DNA repair deficient strains of Saccharomyces cerevisiae and E. coli we prove that ML0190 provides resistance to DNA damaging alkylating agents.
Materials and methods
Results and discussion In E. coli, exposure to the DNA-alkylating agents induces an increase in expression of four DNA repair genes, namely, alkA, alkB, aidB and ada, which are regulated by the Ada protein and collectively called ‘ada regulon’ . In E. coli, ada and alkB genes are encoded in a single operon while alkA and aidB located separately in the genome. Characterization of ada regulon across different bacterial species revealed that there is considerable difference in the organization of ada regulon genes. In M. tuberculosis, the alkA and ogt genes constitute a single operon, whereas, putative alkB homologue, Rv1000c is located 370 kb away. In M. leprae, alkA and ogt are placed together akin to M. tuberculosis and hypothetical protein ML0190 is located approximately 1 mbp away. We present the results suggesting that M. leprae hypothetical protein ML0190 could be an AlkB homologue.