| dc.description.abstract | Background
The rhomboid superfamily of proteins includes novel trans-membrane Serine-proteases that occur in all life-kingdoms, and are responsible for vital roles such as signaling in Drosophila, red blood cell invasion by Apicomplexa parasites, and quorum sensing in Providencia stuartii. Although they occur widely in prokaryotes, rhomboid proteins have not been characterized in most bacterial species. To address this gap, this study focused on bacterial rhomboids of an important genus Mycobacterium, under the following specific aims;
• To determine the nature and distribution of rhomboid proteins in mycobacteria
• To functionally characterize mycobacterial rhomboid genes using a rhomboid deficient strain of Providencia stuartii
• To genetically delete rhomboid genes from Mycobacterium smegmatis, a Mycobacterium tuberculosis surrogate/model, and gain insight into their roles in physiology.
Methods
General microbiological procedures, bioinformatics and classical molecular genetics approaches that included PCRs, nucleic acid hybridization, DNA cloning, DNA mutagenesis, genetic complementation assays, β-galactosidase assays, chemical and electro-transformation, gene knockout and knock-in and phenotype screening assays, to name a few, were employed.
Results
The genome of Mycobacterium tuberculosis reference strain, H37Rv, encodes two putative rhomboid proteases, Rv0110 and Rv1337, orthologues of which are widely represented in most Mycobacterium species. Bioinformatics analysis revealed that across the genus, there’s conservation and organization (i.e. synteny) of the genes encoding the orthologues of Rv1337, arranged in proximity with the glutamate racemaze (mur1) encoding gene. Conversely, the orthologues of Rv0110 were lost in Mycobacterium leprae and the Mycobacterium avium complex species. Additionally, mycobacterial rhomboids formed the six core transmembrane helices (TMHs) typical of rhomboid proteases, with the orthologues of Rv0110 forming seven TMHs (i.e. 6 TMHs core + 1), similar to most eukaryotic rhomboids, while the orthologues of Rv1337 formed only six TMH core, typical of some prokaryotic rhomboids. When transformed into Providencia stuartii null-rhomboid mutant (∆aarA), the genes encoding mycobacterial orthologues of Rv1337 fully complemented AarA activity (AarA is the rhomboid protein of Providencia stuartii). However, most genes encoding the mycobacterial orthologues of Rv0110 did not complement. Furthermore, upon gene deletion in Mycobacterium smegmatis, the ΔMSMEG_4904 single mutant (in which the MSMEG_4904 encoding gene orthologous to Rv1337 was deleted) formed the least biofilms, which was determined by the optical density measurement of the cultured cells stained by crystal-violet. The ΔMSMEG_4904 single mutant was also more susceptible to ciprofloxacin and novobiocin, antimicrobials that inhibit DNA gyrase. Conversely, the ΔMSMEG_5036 single mutant (in which the MSMEG_5036 encoding gene orthologous to Rv0110 was deleted) was not as susceptible. Surprisingly, the double rhomboid mutant ΔMSMEG_4904-ΔMSMEG_5036 (in which genes encoding both rhomboid homologues were deleted) was also not as susceptible, suggesting compensatory or epistasis mechanisms following deletion of the two rhomboid genes. Indeed, transforming the double rhomboid mutant with a plasmid encoding MSMEG_5036 produced phenotypes of the ΔMSMEG_4904 single mutant (i.e. susceptibility to ciprofloxacin and novobiocin).
Conclusion
Overall, the data in this thesis suggest that in some species of Mycobacterium, rhomboid proteases orthologous to Rv1337 appear to be physiologically more important in that they fully complement AarA, and gene deletion alludes to roles in DNA replication pathways. This bears significant implications regarding multi- and extensively- drug resistant pathogenic mycobacteria in that the rhomboid proteases may be mediating novel resistance mechanisms that are yet to be elucidated. Nevertheless, the ameliorated effect in the double mutant suggests that the rhomboid genes in Mycobacterium smegmatis exhibit compensatory or epistasis mechanisms. | en_US |
