Mechanism and functional analysis of chaperonins of Mycobacterium marinum

Rafique Butt, Shahida (2023). Mechanism and functional analysis of chaperonins of Mycobacterium marinum. University of Birmingham. Ph.D.

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Mycobacterial chaperonins have been demonstrated to play an important role in immunomodulation. Both the chaperonins of M. tuberculosis have been revealed to be involved directly or indirectly in pathology of tuberculosis. In a study, MtbCpn60.1 was found to be essential for granulomatous inflammation of infected mice and guinea pigs. We used M. marinum as a model organism to study the mechanism and functions of mycobacterial chaperonins. The genome comparison of M. marinum and M. tuberculosis and the phylogenetic analysis of 50 mycobacterial species and 12 other species of actinobacteria showed that both the organisms are close genetic relatives and share ∼50% homologous coding sequences in their genomes. To investigate the promoter regions of the chaperonins of M. marinum different lengths of the upstream regions of the chaperonin genes of M. marinum were cloned in pSD5B which is a promoter probe shuttle vector. The strongest promoter activity was observed in the fragment upstream to Mmcpn60.2 followed by that of Mmcpn10. No promoter activity was found in the intergenic region of Mmcpn60.1.

RNA-seq analysis of M. marinum WT, Dcpn60.1, and complemented strains was performed to study the transcriptome profile and the effect of deletion of cpn60.1 on the expression profile of M. marinum. The TPM values of the read counts of the triplicate samples of WT M. marinum showed that the expression level of cpn60.2 was slightly higher than 2-fold than that of cpn10. The expression of cpn10 was nearly doubled the expression of cpn60.1 indicating the differential expression of bicistronic cpn10-cpn60.1. In addition, DESeq2 analysis of the RNA-seq data demonstrated that the deletion of Mmcpn60.1 significantly differentially expressed (upregulated in KO) the sets of genes involved in three major sets of pathways. First, the highest fold enrichment pathways were related to stress response, chaperone activity and protein folding. The second highest fold enrichment pathways were related to cell wall, cell division, and peptidoglycan biosynthesis whereas the third highest fold enrichment pathways were related to polyketide synthase pathways. The results indicated that although not essential for cell survival but Mmcpn60.1 is directly or indirectly involved in regulation of key mechanisms of the cell.

The complementation analysis of the chaperonins of M. marinum demonstrated that MmCpn60.2 was able to complement the loss of GroEL in E. coli in either combination with MmCpn10 or GroES whereas MmCpn60.1 failed show complementation phenotype in all attempts. The results supported the previous findings that the Cpn60.2 plays the role of housekeeping chaperonin. The “sequence optimized”, RBS distance optimized, and double phosphomimetic mutant of MmCpn60.1 also failed to demonstrate the chaperone function in E. coli. MmCpn60.1 might perform chaperone function in its natural cell environment as indicated in DESeq2 analysis by significant upregulation of chaperone genes in KO mutant strain. Further investigation of the role of Cpn60.1 in peptidoglycan and cell wall lipids biogenesis would be advantageous in understanding its role in the pathogenesis of tuberculosis.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Life & Environmental Sciences
School or Department: School of Biosciences
Funders: Other
Other Funders: Darwin Trust of Edinburgh
Subjects: Q Science > QR Microbiology


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