Mycobacterial Metallophosphatase MmpE Functions as a Nucleomodulin to Regulate Host Gene Expression and Promote Intracellular Survival
Nucleomodulins are bacterial effectors that target host cell nuclei and play crucial roles as virulence factors in mycobacterial pathogenesis. Mycobacterium tuberculosis, the causative agent of tuberculosis, presents a significant global health challenge; however, the mechanisms of nucleomodulins in Mtb remain unclear. In this study, we characterize the hypothetical protein Rv2577 (designated MmpE) as a critical nucleomodulin involved in immune evasion and intracellular persistence. We performed ChIP-seq in HEK293T cells transfected with EGFP-MmpE, identifying 2,903 potential MmpE-binding sites: 537 in intergenic regions and 1,144 in intragenic regions. Remarkably, 99.7% (2,894) of these sites were associated with protein-coding regions. Among them, 1,013 sites were located within 3 kb of transcription start sites (TSS), primarily targeting promoter regions (<1 kb: 63.18%; 1–2 kb: 22.9%; 2–3 kb: 13.92%), indicating MmpE's role in transcription regulation. Further analysis of the 1,013 potential target genes revealed significant enrichment in protein kinase-related genes, such as PRKCB, PLCG2, and PIK3CB, involved in immune activation and inflammatory responses via the PI3K–Akt and mTOR signaling pathways. Additionally, we identified four transcription factors (CREBBP, VDR, GREB1, and FOXP4) that MmpE directly targets in their promoters. This finding points to the critical role of MmpE in shaping host immune responses and highlights its potential as a therapeutic target in combating Mycobacterium tuberculosis infections. Overall design: This study used CUT&Tag to identify genome-wide binding sites of the MmpE protein in HEK293T cells. Cells were transfected with either MmpE-pEGFP or empty pEGFP-N1 vector as a control. After 36 hours, cells were harvested and subjected to CUT&Tag using an anti-GFP antibody to target MmpE-bound chromatin regions. Libraries were sequenced on the Illumina NovaSeq 6000 platform. Peaks were identified using MACS2, and downstream analyses including genomic annotation, GO enrichment, and motif discovery were performed using HOMER and TOMTOM.