Deletion of ppe27 in Mycobacterium bovis BCG augments macrophage immune responses during infection

ABSTRACT Mycobacterial infection induces dynamic transcriptional changes in both the host and pathogen. In slow-growing mycobacteria, the ESX-5 secretion system plays a crucial role in virulence by exporting Pro–Glu (PE) and Pro–Pro–Glu (PPE) proteins, which modulate host immune responses and contribute to pathogenesis. Previous studies demonstrated that ppe27 overexpression in Mycobacterium smegmatis enhances bacterial survival and promotes host cell death, suggesting that PPE27 plays an important role in resistance to macrophage-induced cytotoxicity. However, its function during host–pathogen interactions remains poorly defined. To elucidate the function of PPE27, we generated a ppe27 deletion mutant of Mycobacterium bovis Bacillus Calmette–Guérin (BCG∆ ppe27 ) and performed RNA-seq to simultaneously profile host and bacterial transcriptional responses during macrophage infection. Host transcriptome analysis revealed that ppe27 deletion led to upregulated pro-inflammatory responses, including enhancement of the TNF-α, IFN-α, and IL-6 signaling pathways. In parallel, bacterial transcriptome analysis identified downregulation of JTY_3006 in BCG∆ ppe27 during infection compared with its expression in wild-type BCG. Amino acid sequence analysis indicated that JTY_3006 shares 100% identity to a S-adenosylmethionine (SAM)-dependent methyltransferase conserved among mycobacterial species. Taken together, these findings suggest that PPE27 may contribute to the negative modulation of host inflammatory responses and suggest that manipulation of PPE27 might reshape pathogenicity and inform future vaccine strategies. IMPORTANCE Tuberculosis remains the leading cause of death from a single infectious agent worldwide. Understanding the mechanisms that allow Mycobacterium tuberculosis to evade host defenses is essential for developing better vaccines, therapeutics, and prevention strategies. This study focuses on PPE27, a member of the PE/PPE protein family secreted through the ESX-5 secretion system. Although PE/PPE proteins are known to trigger strong immune responses, their precise role in pathogenesis has been unclear. Our study reveals that ppe27 deletion is associated with enhanced macrophage inflammatory responses. Understanding this mechanism provides valuable insight into host–pathogen interactions and offers new directions for tuberculosis vaccine and therapeutic development.