The Regulation of ManLAM-Related Gene Expression in Mycobacterium tuberculosis with Different Drug Resistance Profiles Following Isoniazid Treatment

Abstract

Introduction: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) remains a global health concern because of the development of drug resistance. The adaptability of MTB in response to a variety of environmental stresses is a crucial strategy that supports their survival and evades host defense mechanisms. Stress regulates gene expression, particularly virulence genes, leading to the development of drug tolerance. Mannose-capped lipoarabinomannan (ManLAM) is a critical component of the cell wall, functions as a virulence factor and influences host defense mechanisms.Purpose: This study focuses on the effect of isoniazid (INH) stress on the regulation of ManLAM-related genes, to improve our understanding of virulence and drug resistance development in MTB. Materials and Methods: MTB with distinct drug resistance profiles were used for gene expression analysis. Multiplex-real time PCR assay was performed to monitor stress-related genes (hspX, tgs1, and sigE). The expression levels of ManLAM-related genes (pimB, mptA, mptC, dprE1, dprE2, and embC) were quantified by qRT-PCR. Sequence analysis of drug resistance-associated genes (inhA, katG, and rpoB) and ManLAM-related genes were performed to establish a correlation between genetic variation and gene expression. Results: INH treatment activates the stress response mechanism in MTB, resulting in a distinct gene expression pattern between drug resistance and drug-sensitive TB. In response to INH, hspX was up-regulated in RIF-R and MDR. tgs1 was strongly up-regulated in MDR, whereas sigE was dramatically up-regulated in the drug-sensitive TB. Interestingly, ManLAM-related genes were most upregulated in drug resistance, notably MDR (pimB, mptA, dprE1, and embC), implying a role for drug resistance and adaptability of MTB via ManLAM modulation. Conclusion:This study establishes a relationship between the antibiotic stress response mechanism and the expression of ManLAMrelated genes in MTB samples with diverse drug resistance profiles. The novel gene expression pattern in this work is valuable knowledge that can be applied for TB monitoring and treatment in the future.

Drug-resistant strains of Mycobacterium tuberculosis: cell envelope profiles and interactions with the host

“…In a separate study, treatment of M. bovis BCG with INH and EMB resulted in increased surface exposure of LAM, which could be linked to the reduction in MAc abundance by the action of INH, as well as an attempt from the bacteria to maintain cell envelope integrity after exposure to anti-TB drugs (Alsteens et al, 2008). This is supported by the upregulation of genes related to LAM biosynthesis following INH treatment (Yimcharoen et al, 2022). That said, this needs to be carefully considered as for this study, the authors used a MDR M.tb strain with a katG mutation, which should render it insensitive to INH.…”

“…Outside of the plasma membrane, a layer of peptidoglycan (PG) is covalently attached to the arabinogalactan (Carr et al, 2022;Nyang'wa et al, 2022, WHO, 2022b, WHO, 2022c. Wilson et al, 1995;Chen et al, 2002;Broussy et al, 2003;Schroeder et al, 2005; Vilchèze and Jacobs, 2007 PZA Pyrazinoic acid (active form of PZA) inhibits fatty acid synthesis by interfering with FAS I, blocks ATP production, and inhibits M.tb protein synthesis DS-TB Ngo et al, 2007;Zimhony et al, 2007 EMB Inhibits M.tb arabinosyltransferases to interfere with the biosynthesis of arabinose-containing cell envelope components and prevent bacterial division DS-TB Escuyer et al, 2001;Zhang et al, 2003;Amin et al, 2008;Goude et al, 2009;Zhang et al, 2020 (AG) polysaccharide. The non-reducing ends of AG are esterified to the long-chain (C 60 -C 90 ) mycolic acids (MAcs) that are unique in nature to Mycobacterium spp and complete the 'cell wall core' of the cell envelope (Jackson, 2014;Garcia-Vilanova et al, 2019).…”

“…Once activated, INH inhibits InhA, the M.tb enoyl reductase, by forming a covalent adduct with the NAD cofactor. This INH-NAD adduct acts as a slow, tight-binding competitive inhibitor of InhA to interfere with MAc biosynthesis (Wilson et al, 1995;Chen et al, 2002;Broussy et al, 2003;Schroeder et al, 2005;Vilchèze and Jacobs, 2007). RIF inhibits M.tb DNA-dependent RNA polymerase, leading to a suppression of RNA synthesis and bacterial death (Campbell et al, 2001;Tupin et al, 2010).…”

Drug resistantMycobacterium tuberculosisstrains have altered cell envelope hydrophobicity that influences infection outcomes in human macrophages

“…[23][24][25][26] There is also evidence that abolishment of LAM biosynthesis in M. abscessus is associated with increased susceptibility to multiple anti-TB drugs, 27 and that treatment of M.tb with isoniazid (INH) results in the upregulation of genes related to ManLAM biosynthesis. 28 Further, treatment of M.tb with ethambutol (EMB) results in truncated arabinan domains for both ManLAM and AG. 29,30 Initial studies investigated MDR-and XDR-M.tb strains using transmission electron and atomic force microscopy found increased cell envelope thickness of DRcompared to drug susceptible (DS)-M.tb strains.…”

Mycobacterium tuberculosis Adaptation in Response to Isoniazid Treatment in a Multi-Stress System That Mimics the Host Environment

“…Thus, studying the mechanism of mycobacterial adaptation in response to INH during an infection in a host is essential to improving our understanding of antibiotic resistance. INH treatment alters gene expression depending on the strains, growth phase, media, and culture conditions [18,20,21]. During the exponential phase, INH-treated Mtb regulates genes that encode enzymes in type II fatty acid synthesis, trehalose dimycolyl transferase, and other genes related to the isoniazid drug's action [18,22].…”