Investigating the role of the protein LysX2 of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, survives within the host through complex mechanisms of environmental adaptation and resistance to host killing, also thanks to the characteristics of its cell wall. Among the factors that modulate the bacterial cell wall, MprF-like proteins play a key role by modifying the surface charge through aminoacylation of phospholipids. M. tuberculosis possesses two proteins of this family, LysX and LysX2; while LysX has been well-characterized, the role and mechanism of LysX2 remain largely unknown. Preliminary studies conducted in Mycobacterium smegmatis suggested that LysX2 contributes to resistance to acidic pH, nitrosative stress, and antimicrobial peptides, modifying the glycopeptidolipid pool and reducing surface negative charge. In this work, we analyzed the role of LysX2 in its natural host, M. tuberculosis, by generating a knock-out strain using the ORBIT technique. Our results show that LysX2 is essential for rapid adaptation to mildly acidic pH; however, M. tuberculosis can still cope with the hostile environment through compensatory mechanisms, such as overexpression of the rv1169c gene, which encodes the PE11 protein involved in maintaining cell wall structure. RNAseq analysis also revealed upregulation of genes associated with oxidative stress (sigH, katG, rv2466) in the KO strain. Functional assays confirmed increased sensitivity of the KO strain to oxidative and nitrosative stress, as well as to vancomycin, suggesting increased cell wall permeability and altered surface properties. HPTLC lipid analysis showed accumulation of triglycerides in the KO at acidic pH, consistent with a stress adaptation response, but did not reveal the specific target of LysX2. Finally, the overall response of the KO strain to macrophage-mediated killing was evaluated by infecting the murine RAW264.7 macrophage cell line, again confirming that the KO strain is more sensitive than the wild-type. Our findings suggest that LysX2 is a crucial Mtb virulence factor, influencing resistance to key macrophage defense mechanisms. Future research will be essential to determine its molecular target and understand its mechanism of action, opening avenues for innovative therapeutic approaches.