The influence of small RNAs on the physiology of Mycobacterium tuberculosis

[摘要] ENGLISH ABSTRACT: The role of bacterial small RNA (sRNA), i.e. RNA species between 50-500bp in size, in virulence, pathogenesis and drug resistance is gaining interest. In some bacterial species, it had been shown to play a crucial role in bacterial transcriptional and post-transcriptional regulation. sRNAs from various pathogenic bacteria were shown to modulate bacterial responses to the host and environment. In Mycobacterium tuberculosis, the causative agent of tuberculosis, more than 1000 sRNA species have been identified already; but the role of these sRNA in pathogenesis, virulence and stress responses is not well studied. Central dogma suggests that drug resistance in M. tuberculosis is associated with mutations in specific genes. However, a number of clinical drug resistant isolates do not harbour mutations in these genes, implicating other factors such as unknown mutations, as well as altered regulation of these resistance genes. Prediction of resistance, using molecular methods, can therefore be inaccurate in cases where known mutations are absent. In cases where known drug-resistance associated mutations are absent, mutations in other genes that regulate such resistance-associated genes might influence drug resistance. Growing evidence, in other bacteria and M. tuberculosis, hints at a role for mutations in intergenic regions and sRNAs species to play a role in bacterial growth and drug sensitivity. In light of this we hypothesised that mutations in sequences encoding sRNA or in sRNA target sequences influence the phenotype of M. tuberculosis clinical isolates. Using previously identified sRNA genes; we screened a genomic bank of clinical M. tuberculosis isolates for the presence of mutations in these sRNA encoding genes. A large number of isolates showed mutations in genes encoding for sRNAs. Furthermore, over-expression of sRNA using the plasmid pMV306 in Mycobacterium smegmatis showed differences in growth indicating that the presence of the extra copies of the three sRNA (mcr3, ASpks and mpr6) had a phenotypic effect on the bacterium. Overexpressed sRNAs did not affect the bacterial drug resistance phenotypes, although this requires further investigation before concluding the effect of sRNAs on drug resistance. We successfully modified a method to extract and purify sRNAs from Mycobacterium species, clean enough to perform Real Time Polymerase Chain Reaction even with small amounts. However challenges were faced in terms of quantification. Another challenge that still remains is obtaining reference genes specifically for sRNAs as we currently have none.

[发布机构] Stellenbosch University