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.

核调节蛋白（Nucleomodulins）是一类靶向宿主细胞核的细菌效应因子，在分枝杆菌致病过程中作为毒力因子发挥关键作用。结核分枝杆菌（Mycobacterium tuberculosis，简称Mtb）是结核病的致病菌，给全球公共卫生带来了重大挑战，但Mtb中核调节蛋白的作用机制仍不明确。本研究对假定蛋白Rv2577（命名为MmpE）进行了功能表征，证实其为参与免疫逃逸与胞内存活的关键核调节蛋白。
我们在转染了EGFP-MmpE融合蛋白的HEK293T细胞中开展染色质免疫共沉淀测序（ChIP-seq），共鉴定得到2,903个MmpE潜在结合位点：其中537个位于基因间区、1,144个位于基因内区。值得注意的是，其中99.7%（2,894个）位点与蛋白编码区相关。在这些位点中，1,013个位于转录起始位点（TSS）上下游3 kb范围内，且主要靶向启动子区域（<1 kb：63.18%；1~2 kb：22.9%；2~3 kb：13.92%），表明MmpE参与转录调控过程。
对这1,013个潜在靶基因的进一步分析显示，其显著富集于蛋白激酶相关基因，例如参与PI3K-Akt及mTOR信号通路介导的免疫激活与炎症反应的PRKCB、PLCG2和PIK3CB。此外，我们还鉴定出MmpE在其启动子区域直接靶向的4种转录因子：CREBBP、VDR、GREB1及FOXP4。这一结果揭示了MmpE在调控宿主免疫应答中的关键作用，并提示其有望成为对抗结核分枝杆菌感染的治疗靶点。
实验整体设计：本研究采用CUT&Tag技术，在HEK293T细胞中鉴定MmpE蛋白的全基因组结合位点。将细胞分为两组，分别转染MmpE-pEGFP重组质粒与空pEGFP-N1载体作为对照。转染36小时后收集细胞，使用抗GFP抗体开展CUT&Tag实验，以富集MmpE结合的染色质区域。构建的测序文库在Illumina NovaSeq 6000平台上完成测序。使用MACS2软件进行峰区识别，并通过HOMER和TOMTOM软件完成基因组注释、基因本体（Gene Ontology，GO）富集分析与基序发现等下游分析。