Validation of LysX2 detection in Mycobacterium tuberculosis by Western blot

Mycobacterium tuberculosis (Mtb) remains a major global health challenge due to its capacity to survive host immune pressures and evade antimicrobial clearance, causing more than 1,2 million deaths every year. This persistence can be attributed to the ability of the mycobacterial cell wall to remodel and modify its surface characteristics, allowing the bacterium to adapt to environmental stressors such as phagosomal acidity. Centrally involved in this surface modification is LysX2, a membrane protein belonging to the aminoacyl-phosphatidylglycerol synthase family, that reduces the net negative charge of the mycobacterial cell surface enhancing bacterial survival to several host associated stresses. Unlike typical orthologues that show a cytoplasmic catalytic domain, LysX2 is characterized by a unique morphology, since its catalytic domain is predicted to be exposed to the outer side of the plasma membrane. This study aimed to establish an experimental framework for the validation and characterization of a newly developed antibody targeting the hydrophilic catalytic domain of LysX2. Mycobacterium smegmatis expressing LysX2 was first used as a non-pathogenic surrogate model to optimize the Western blot protocol and assess antibody performance. The validated protocol was subsequently applied to Mycobacterium tuberculosis protein extracts, where antibody specificity and reliability were further evaluated across multiple strains: a wild-type strain, a LysX2-deficient knockout control, and two alternative complemented strains. The latter were included to verify their capacity to restore LysX2 protein production to wild-type levels, thereby confirming both strain functionality and antibody sensitivity. The resulting optimized workflow provides a robust experimental foundation for future studies aimed at characterizing the morphology, function, and mechanism of action of LysX2 in mycobacterial biology, as well as its potential as a novel anti-tuberculosis therapeutic target.