Abstract
To reveal functions of novel Mycobacterium tuberculosis (M. tb) proteins responsible for modulating host innate immunity is essential to elucidation of mycobacterial pathogenesis. In this study, we aimed to identify the role of a putative protein Rv0309 encoded within RD8 of M. tb genome in inhibiting the host inflammatory response and the underlying mechanism, using in-vitro and in-vivo experiments. A recombinant M. smegmatis strain Ms_rv0309 expressing Rv0309 and a mutant Bacillus Calmette-Guérin (BCG)ΔRS01790 strain with deletion of BCG_RS01790, 100% homologue of Rv0309 in BCG, were constructed. Rv0309 was found to localize in the cell wall and be able to decrease cell wall permeability. Purified recombinant rRv0309 protein inhibited lipopolysaccharide-induced IL-6 release in RAW264.7 cells. BCG_RS01790 in BCG or Rv0309 in Ms_rv0309 strain greatly inhibited production of IL-6, IL-1β, and TNF-α in RAW264.7 cells. Similarly, BCGΔRS01790 strongly induced expression of these cytokines compared with wild-type BCG and complement strain, cBCGΔRS01790::RS01790. Further BCG_RS01790 or Rv0309 suppressed cytokine production through NF-κB p65/IκBα and MAPK ERK/JNK signaling. Importantly, BCG_RS01790 in BCG and Rv0309 in Ms_rv0309 strain enhanced mycobacterial survival in macrophages. Mice infected with BCGΔRS01790 exhibited high levels of IFN-γ, TNF-α and IL-1β, and large numbers of neutrophils and lymphocytes in the early stage, and minimal lung bacterial load and inflammatory damage in late stage of the experiment. In conclusion, the cell wall protein Rv0309 or BCG_RS01790 enhanced mycobacterial intracellular survival after infection likely through inhibition of the pro-inflammatory response and decrease of bacterial cell wall permeability, thereby contributing to mycobacterial pathogenesis.
Highlights
Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (M. tb), is a leading cause of human death from a single infectious agent for a long time
A transmembrane structure was predicted in Rv0309 (TMHs = 1) (Figure 1B), indicating that this protein was a transmembrane protein, this result suggested that Rv0309 might interact with extracellular substances
Rv0309 and its homologs belonging to YkuD_like superfamily shared multiple conserved motifs with high homology in Mycobacteria (Figure 1D), indicating that this protein is vital to the physiological process of Mycobacteria and its potential function is worthy of deep investigation
Summary
Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (M. tb), is a leading cause of human death from a single infectious agent for a long time. M. tb is considered a highly successful intracellular bacterium that subverts host immune responses for long-term persistence [2]. Pathogenic mycobacteria have evolved sophisticated strategies to subvert signaling pathways that regulate innate immune responses in the hosts. Mycobacterial effector proteins, including secreted and cell surface-associated proteins, suppress the activation of the NF-kB and MAPK signaling pathways in macrophages, allowing mycobacteria to persist within the hostile macrophage environment [4, 5]. PtpA can inhibit the host’s nature immune response during M. tb infection [6]. Cell wallsurface proteins such as fibronectin-binding protein A (FbpA) and Rv0246c enhance intracellular mycobacterial survival by suppressing host inflammatory cytokine production [7, 8]
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