Comparative Proteomic Analysis of Cell Wall Proteins of Aminoglycosides Resistant and Sensitive Mycobacterium tuberculosis Clinical Isolates.

Introduction: The rising prevalence of Mycobacterium tuberculosis (M.tb) strains resistant to aminoglycosides (amikacin and kanamycin) challenges effective TB control and treatment. Understanding the mechanisms behind this resistance is crucial since aminoglycosides are a mainstay of TB therapy.

Aim: The study aimed to analyze the cell wall proteins overexpressed in aminoglycoside-resistant isolates of Mycobacterium tuberculosis using proteomics approaches.

Methods: We used two-dimensional electrophoresis and mass spectrometry to compare the cell wall proteomes of aminoglycosides-resistant and susceptible clinical isolates. The overexpressed protein spots were excised and identified using liquid chromatography-mass spectrometry (LC/MS). The identified proteins were subsequently analyzed for molecular docking, pupylation site identification, and STRING analysis.

Results: We found a total of nine significantly upregulated proteins in aminoglycosides-resistant isolates. Three of these proteins were the same (isoform), resulting in the identification of seven unique proteins. Specifically, Rv3841 and Rv1308 belonged to intermediary metabolism and respiration; Rv2115c to the cell wall and cell processes; Rv2501c, Rv2247 and Rv0295c to lipid metabolism; and Rv2416c to virulence, detoxification/adaptation. Notably, variations in these proteins support cell wall integrity, aiding mycobacteria's establishment and proliferation. Molecular docking study revealed that both drugs bind strongly to the proteins' active site regions. Additionally, the GPS-PUP algorithm successfully identified possible pupylation sites within these proteins, except Rv0295c. Based on interactome analysis using the STRING 12.0 database, we have identified potential interactive partners suggesting their role in aminoglycosides resistance.

Conclusion: Overexpressed proteins not only act to counteract or regulate drug effects but also have a role in protein dynamics that allow for resistance. Some of these identified proteins may serve as innovative drug targets and biomarkers for the early detection of drug-specific resistance in M.tb. Further research is needed to elucidate the mechanisms by which these potential protein targets contribute to resistance in AK and KM M.tb isolates.