Ongoing evolution of PE/PPE genes in <i>Mycobacterium tuberculosis</i> associated with drug resistance and host immune response

The Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family comprises approximately 10% of the <i>Mycobacterium tuberculosis</i> (Mtb) genome and is characterized by GC-rich, highly repetitive sequences. As a result, these genes are usually excluded from short-read-based whole-genome sequencing analyses, leaving their sequence diversity and evolutionary dynamics poorly characterized. Recently, a genome masking approach demonstrated that roughly 54% of PE/PPE sequences are recoverable from short-read data, providing an opportunity to examine the evolution of this gene family at a population level. Here, we analyzed 51,229 Mtb genomes to characterize sequence diversity and selection pressures across the PE/PPE gene family. Overall, we observed that PE/PPE genes are under relaxed purifying selection compared to other gene categories, as evidenced by higher ratios of nonsynonymous to synonymous polymorphisms (<i>pNpS</i>) and greater mutation burdens. We identified 12 PE/PPE genes with signatures of positive selection and 7 with selective pressure associated with antibiotic resistance. Among these genes, <i>PPE51</i> exhibited selection favoring loss-of-function mutations, which occurred only in Mtb strains that were already multidrug-resistant (MDR). This pattern suggests either compensatory evolution or adaptation related to resistance against second-line or newly introduced drugs. Additionally, we identified T-cell epitopes in six PE/PPE genes that were subject to diversifying selection, suggesting immune-driven adaptation. Collectively, this work provides a baseline characterization of genetic diversity in PE/PPE genes and highlights specific genes that may be involved in adaptation to host immunity and antibiotic pressure and represent candidates for further investigation.IMPORTANCETuberculosis remains a significant global health challenge, partly due to <i>Mycobacterium tuberculosis</i> (Mtb)'s remarkable evolutionary adaptation to antibiotics and human immune responses. Around 10% of its genome comprises PE/PPE genes, whose functions and evolutionary dynamics are poorly understood due to their repetitive sequences and high GC content. In this study, we analyzed 51,229 global Mtb genomes using an advanced genome-masking method, revealing numerous PE/PPE genes under positive selection, potentially facilitating antibiotic resistance and immune evasion. Notably, <i>PPE51</i> often loses its function in strains resistant to multiple antibiotics, suggesting a role in bacterial survival during drug treatment. Additionally, we identified mutation-prone regions within six PE/PPE genes, highlighting potential targets for future vaccine development. Collectively, our findings underscore the crucial role of PE/PPE genes in Mtb evolution and drug resistance, providing valuable insights to inform novel therapeutic and vaccine strategies.