Identification of the insertions, deletions and point mutations in rhomboid proteases from selected Mycobacterium species

Abstract

Background: Rhomboids, proteins that are conserved in all life kingdoms, are among the novel genes likely to encode roles in the pathogenesis of mycobacteria. While rhomboids have various specific functions across species, their roles in mycobacteria are not clearly defined. Whereas the insertions, deletions (inDels), point mutations and novel bifurcations have been identified among some mycobacterial rhomboids, their distribution and possible effect on mycobacterial rhomboid function has not been studied. The objective of this study was to functionally characterize additional rhomboid encoding genes from mycobacteria particularly those with InDels, point mutations and novel bifurcations, using bioinformatics and genetic approaches in Providencia stuartii. Methods: This was a laboratory-based experimental study with 56 mycobacterial isolates, from which rhomboid genes were PCR-amplified and cloned. Bioinformatics tools were applied to identify, retrieve and analyze rhomboid genes for evolutionary history, presence of signature. PCR-amplification of rhomboid genes from the isolates and some genes were cloned into pBCKS/ pBCSK plasmid vectors was done. Results: The Mycobacteria rhomboid proteases were acquired independently and they may have been acquired from the Acanthamoeba species (amoeba) through genetic exchange with these eukaryotes. Rhomboid protease 2 of M. abscessus is the ancestor of all the Rv1337 mycobacterial orthologues while Mycobacterium neaonurum is possibly the ancestor of all the Rv0110 mycobacterial orthologues. Multiple sequence alignment has revealed that mycobacterial rhomboids posses putative inDels and point mutations which may be authentic or due genome annotation errors (Kateete et al., 2010). One hundred and twelve (112) rhomboid amplicons were PCR-amplified from the MTBC and NTM species and are awaiting sequence confirmation of the inDels and point mutations. Furthermore, the rhomboids of 4 mycobacteria species were cloned and are being functionally characterized in P. stuartii. Conclusion: Rhomboid genetic exchange occurred between the Mycobacterium and the Acanthamoeba species. Given that the Acanthomoeba species effectively kill bacteria, yet mycobacteria resist this killing, it is postulated that the ability of the pathogenic mycobacteria, particularly the MTBC subspecies, to survive in macrophages (which are amoeba-like and are believed to be descendants of the amoeba) is an extension of this ancient lifestyle learned from the amoeba. For similar reasons, we hypothesize that the rhomboid proteases in the pathogenic Mycobacterium species evolved to be used in evasion mechanisms in macrophages