Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.815612
Title: The role of LytR-CpsA-Psr proteins in cell envelope biogenesis of Mycobacterium smegmatis
Author: Sahu, Abhipsa
ISNI:       0000 0004 9358 5457
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2020
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Abstract:
Tuberculosis infection is one of the leading causes of mortality worldwide and is caused by Mycobacterium tuberculosis (Mtb). With an upsurge of multidrug-resistant tuberculosis, it is a global threat. Therefore, development of new drugs need immediate attention, and this needs identification of potential drug targets. The cell envelope of mycobacteria is one such attractive drug target owing to its role in maintaining the structural integrity and pathogenicity of the bacterium. The LytR-CpsA-Psr (LCP) family of proteins in Mycobacterium spp. have been shown to catalyze the coupling of arabinogalactan and peptidoglycan and possess pyrophosphatase activity. The four LCP protein homologues present in Mycobacterium smegmatis (Msmeg), MSMEG_0107, MSMEG_1824, MSMEG_5775 and MSMEG_6421, have not been extensively investigated with the focus on the existence and interplay of multiple LCP proteins. In this study with this non-pathogenic model organism, all four LCP homologues were shown to possess pyrophosphatase activity, with a significant higher activity displayed by MSMEG_0107 and MSMEG_5775. In order to further study the role of the LCP proteins on the physiology of the bacterium, single and double deletion strains lacking of the three non-essential lcp genes were created along with the respective complemented strains. All the generated mutants showed different phenotypes in the different assays, but usually not very severe. However, the double-deletion lcp mutant, ΔΔ(0107+5775) was the most affected mutant strain and displayed a disrupted cell envelope as evident from deprived growth rate, slower cellular aggregation, diminished biofilm formation on air-liquid interface, altered morphology, as well as an increased susceptibility to surface detergent, lysozyme and a wide range of antibiotics. Thus, the loss of both MSMEG_0107 and MSMEG_5775 exhibited profound effects on the mycobacterial cell envelope, and therefore could be further investigated as a possible combined drug target by extending these studies in Mtb. A novel approach in this study is the detection of exposed mycobacterial Galf moieties of arabinogalactan by EB-A2 monoclonal antibody, in the double lcp deletion mutant ΔΔ(0107+5775). Transcription profiling of all the lcp genes in the wild type strain and the mutants exhibited differential expression of these genes under both standard and stress conditions. A loss of MSMEG_5775 resulted in an upregulation of the other three lcp genes in comparison to the wild type strain under standard conditions. Under both acid and lysozyme stress, the loss of MSMEG_5775 downregulated all other lcp genes while loss of MSMEG_6421 upregulated these genes. Lastly, an in silico approach led to the identification of putative transcriptional factors in mycobacteria and related species which could be further investigated and experimentally confirmed. This study helped to understand the role of the lcp homologues in Msmeg better. From the differential expression studies, role of regulator(s) might be a significant approach to understand this family of proteins much better.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.815612  DOI:
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