noninfected cells were prepared as control

noninfected cells were prepared as control. or migration in humans. Introduction Tuberculosis (TB) remains a leading cause of death and disability worldwide. According to data from your World Health Business (WHO)1, ~10.4 million people were estimated to have fallen ill with TB and 1.4 million people died from TB in 2015. (Mtb), the etiological agent of the disease, survives inside the host macrophages either in Filgotinib an active or non-replicative state. The treatment of active TB requires at least 6 months, which often prospects to the emergence of multidrug-resistant Mtb strains due to inadequate treatment or poor individual compliance. WHO reported that about half of the patients with multidrug-resistant TB are not successfully treated, and the emergence of drug-resistant TB has become a Filgotinib major global threat1C4. Thus, it is urgent for us to better understand the molecular mechanisms of the interactions between Mtb and host immune system in order to identify new effective therapeutic targets. Mtb PtpA is usually a secreted, low-molecular-weight protein tyrosine phosphatase (PTP) that is important for Mtb pathogenicity in vivo but not essential for Mtb growth in vitro5. The crystal structure of Mtb PtpA revealed the PTP loop (residues 11C18) in its active site, along with three conserved active-site residues including Cys11, Arg17, and Asp126. Mutations of those three residues (C11A, R17A, and D126A) in Mtb PtpA cause loss of its phosphatase activity6. Mtb PtpA can prevent phagosome-lysosome fusion by dephosphorylating host protein VPS33B, and prevent phagosome acidification by binding to subunit H of the macrophage V-ATPase complex to block V-ATPase trafficking7, 8. Furthermore, binding of Mtb PtpA to ubiquitin via a ubiquitin-interacting motif-like region activates PtpA to dephosphorylate JNK, p38, and VPS33B, leading to suppression of innate immunity. Mtb PtpA can also suppress the activation of NF-B by competitively binding to the Npl4 zinc-finger domain of TAB3 independently of its phosphatase activity9. Those previous studies were mainly focused on the regulatory function of Mtb PtpA in the cytoplasm of host cells. Here, we show that Mtb PtpA is not only present in the cytoplasm but also in the nucleus of host cells. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis10, 11, we find that nuclear PtpA interacts with host DNA. PtpA appears to regulate the transcription of a variety of protein-coding genes, some of which are known to be involved in host innate immune signaling, cell proliferation, and migration. In addition, PtpA-expressing Bacillus Calmette-Guerin (BCG) promotes cell proliferation and migration of a human lung adenoma cell line in vitro and in Filgotinib a mouse xenograft model. Our findings reveal additional mechanisms by which Mtb PtpA inhibits host innate immunity. Further research is needed to test whether mycobacteria, via PtpA, might affect cell proliferation or migration in humans. Results Identification of PtpA in the nucleus of host cells Previous studies on the regulatory function of Mtb PtpA were mainly focused on how it interferes with the innate immune system as a phosphatase in the cytoplasm. The amino acid sequence of PtpA from BCG is identical to that of Mtb PtpA. With an aim to probe the subcellular location of PtpA in host cells, we performed confocal microscopy experiments using different BCG strains including wild-type (WT) BCG, PtpA-deleted BCG (BCG PtpA), BCG PtpA complemented with WT PtpA (PtpA?+?PtpA) and BCG PtpA complemented with phosphatase-inactive PtpA (PtpA?+?D126A). Our results indicated that some PtpA, but not PtpB (another mycobacterial secreted PTP12), entered the nucleus of human macrophage-like U937 cells (which were differentiated from U937 human monocytic cells) during BCG infection, and that this nuclear localization was independent of the phosphatase activity of PtpA (Fig.?1a). We also conducted a time course analysis for intracellular localization of PtpA, and observed that the nuclear localization of TIE1 PtpA increased over time within 6?h post-infection (Fig.?1b). Filgotinib Consistently, overexpressed PtpA was also largely co-localized with the nucleus of U937 cells and human lung adenoma A549 cells (Supplementary Fig.?1aCc). We then further performed immunoblot analysis of PtpA in U937 cells stably expressing Myc-tagged Mtb PtpA. PtpA was not only present in the cytoplasm but also in the nucleus of U937 cells (Fig.?1c). Consistent results were also obtained from the immunoblot analysis of U937 cells infected with different BCG strains including WT BCG, BCG PtpA, BCG (PtpA?+?PtpA), and BCG (PtpA?+?D126A) (Fig.?1d). Thus, PtpA is present.