结核杆菌MTB引起的结核病仍威胁着人类健康，且其感染过程复杂机制不清。在前期自然基金资助下，我们发现乙酰化修饰在MTB感染中发挥重要作用，且蛋白乙酰化受去乙酰化酶NpdA调控。同时npdA敲除后感染小鼠毒力增强而高表达菌株毒力显著下降。究其原因，高表达株丧失ESX-1毒力蛋白EsxA分泌。有报道EsxA分泌及MTB毒力受核相关蛋白(类组蛋白)EspR调控。我们发现高表达NpdA菌株中受EspR调控EsxA分泌相关及其他基因转录水平显著下降，推测EspR是否有类似真核染色体结构紧疏来动态调控基因转录的表观遗传机制。本项目拟研究NpdA是否调控EspR去乙酰化改变其DNA结合能力，并影响染色体构象来动态调控下游基因转录和EsxA分泌及MTB毒力的机制。结果不仅从表观遗传角度揭示EXS-1蛋白分泌机制，且为研究感染过程毒力蛋白动态调控和MTB扩散及发现抗MTB药物另辟新径。

In 2018, the World Health Organization reported the “the global burden of tuberculosis (TB) remains enormous”. Mycobacterium tuberculosis (MTB), the etiological agent of TB, is one of most successful intracellular pathogens. MTB possesses a variety of mechanisms to survive with the host macrophages and escape from phagosomal compartment. In order to come up with new drug targets and treatment strategies, there is an urgent need to understand the molecular mechanisms supporting the success of this versatile pathogen. .Our previous studies have revealed lysine acetylation of ICL1 and DosR plays a critical role in the regulation of central carbon metabolism and virulence in MTB, and deacetylation of ICL1and DosR could be mediated by the only known protein deacetylase (NpdA) of MTB. The gene encoding deacetylase in MTB was further deleted to generate KO strain (∆npdA). Next, an integrated plasmid（pMV361）, expressing npdA under the control of native promoter or hsp60 promoter, generating complement strain (∆npdA:npdA)or overexpression strain (∆npdA:p60npdA). Expression of npdA was subsequently tested by qRT-PCR. In order to investigate whether NpdA regulates the virulence and pathogenesis of MTB, C57BL/6 mice were infected with H37Rv, ΔnpdA and ΔnpdA: p60npdA at dose of 5*105CFU. The results of bacterial loads in lung and spleen and mice mortality showed that the virulence of ΔnpdA was higher than that of WT, while survival ability of ΔnpdA:p60npdA strain in mice and mice mortality were dramatically decreased compared with KO and WT strain, indicating NpdA played an important role in MTB virulence and pathogenesis. Furthermore, we also found that the expression of EsxA (ESAT6), a secreted well-known virulence factor from ESX-1 secretion system, significantly reduced in ΔnpdA:p60npdA. Moreover, transcription of EsxA secretion-associated genes (espA and espC) was significantly reduced in ΔnpdA:p60npdA. On the contrary, the transcription of these genes was higher in ΔnpdA than in H37Rv wild type and KO strains. A key regulator of ESX-1 is EspR, a nucleotide-associated protein (NAP, also called histone-like protein), which regulates the expression of a wide range of virulence-associated transcripts in MTB. NAPs can modulate the bacterial chromosome through bending, wrapping and bridging of DNA in a sequence independent manner. To further investigate whether EspR play a vital role in secretion defect of the EsxA secretion and severe attenuation of ΔnpdA:p60npdA in mice, the several genes expression of top 10 EspR binding loci from ChIP-Seq was analyzed by qRT-PCR in H37Rv, ΔnpdA and ΔnpdA:p60npdA. 5 out of tested 10 genes was significantly down-regulated in transcriptional level in ΔnpdA:p60npdA compared to WT..Acetylation/deacetylation of histones is one of the most important tools in eukaryotic epigenetic machinery to attune transcription globally. Given that EspR is an acetylated protein at three lysine residues in our previous data, we hypothesized that whether EspR might be regulated by acetylation modifications akin to eukaryotic histones for maintaining the bacterial chromosome and global regulation of gene expression. Here, we will investigate the deacetylation mechanism of the EspR protein by NpdA in vitro and in vivo. Furthermore, whether acetylation of EspR might alter its affinity to DNA resulting in change in genome architecture, which further regulates transcription of espACD and other virulence-associated genes, which, in turn, controls the secretion of EsxA of ESX-1 and virulence of MTB. This research would help to gain a novel insight into secretion modulation of EXS-1 system which may contribute to a better understanding of virulence factors secretion as an intracellular Mtb strategy to rupture the phagosome and spread cell to cell, and also provide perspective targeting NAP-DNA interactions for the development of novel anti-TB drugs.