Identification of new protein-coding open reading frames in Mycobacterium leprae and characterization of hypothetical protein ML0190

Leprosy is a public health problem and early diagnosis for the treatment is essential to evade the disabilities. However, annotation of M. leprae genome is still not complete and many genes and pseudogenes, hypothetical proteins, particularly genes encoding small proteins, remains to be discovered. Therefore, further analysis of the genome sequence of M. leprae is required to find potential genes, especially DNA repair genes, that can cause drug resistance. By using a set of 96 bacterial genes as a reference, 61 DNA repair pathway genes were identified in M. leprae based on sequence similarity and domain architecture. Expression of these genes was confirmed by qPCR. Sequencing of M. leprae genome has revealed massive gene decay with two-third of its genome encoding pseudogenes. The pseudogenization of M. leprae genome raises an obvious question, how could M. leprae remain a successful intracellular parasite with such rudimentary genome? It has been proposed that intergenic regions of M. leprae genome could harbor short protein-coding genes that might have been overlooked during gene annotation. Therefore, intergenic regions of M. leprae genome were scanned for small ORFs having conserved domains and canonical start and stop codons. The gene expression for intergenic ORFs was confirmed by qPCR with RNA extracted from skin biopsies of leprosy patients. Comparative bioinformatic analysis identified several potential M. leprae “hypothetical proteins” not yet annotated. Presently it is unclear whether these proteins are expressed or are metabolically relevant therefore one of the hypothetical M. leprae protein, HP ML0190 was characterized. Through mRNA expression and in vivo assays, it was observed that this 615-bp open reading frame encodes a protein with sequence, structural and functional similarity to Fe(II)/2OG-dependent dioxygenase (AlkB) family involved in DNA dealkylation repair