ESX Secretion-Associated Protein C From Mycobacterium tuberculosis Induces Macrophage Activation Through the Toll-Like Receptor-4/Mitogen-Activated Protein Kinase Signaling Pathway

Abstract

Mycobacterium tuberculosis , as a facultative intracellular pathogen, can interact with host macrophages and modulate macrophage function to influence innate and adaptive immunity. Proteins secreted by the ESX-1 secretion system are involved in this relationship. Although the importance of ESX-1 in host-pathogen interactions and virulence is well-known, the primary role is ascribed to EsxA (EAST-6) in mycobacterial pathogenesis and the functions of individual components in the interactions between pathogens and macrophages are still unclear. Here, we investigated the effects of EspC on macrophage activation. The EspC protein is encoded by an espA/C/D cluster, which is not linked to the esx-1 locus, but is essential for the secretion of the major virulence factors of ESX-1, EsxA and EsxB. Our results showed that both EspC protein and EspC overexpression in M. smegmatis induced pro-inflammatory cytokines and enhanced surface marker expression. This mechanism was dependent on Toll-like receptor 4 (TLR4), as demonstrated using EspC-treated macrophages from TLR4 −/− mice, leading to decreased pro-inflammatory cytokine secretion and surface marker expression compared with those from wild-type mice. Immunoprecipitation and immunofluorescence assays showed that EspC interacted with TLR4 directly. Moreover, EspC could activate macrophages and promote antigen presentation by inducing mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor-κB activation. The EspC-induced cytokine expression, surface marker upregulation, and MAPK signaling activation were inhibited when macrophages were blocked with anti-TLR4 antibodies or pretreated with MAPK inhibitors. Furthermore, our results showed that EspC overexpression enhanced the survival of M. smegmatis within macrophages and under stress conditions. Taken together, our results indicated that EspC may be another ESX-1 virulence factor that not only modulates the host innate immune response by activating macrophages through TLR4-dependent MAPK signaling but also plays an important role in the survival of pathogenic mycobacteria in host cells.

Mycobacterium tuberculosisESX-1-secreted substrate protein EspC promotes mycobacterial survival through endoplasmic reticulum stress-mediated apoptosis

“…(Extracellular signal-regulated protein kinases (ERK) inhibitor) [21]. We further found that SB203580 treatment prominently increased EspC-induced apoptosis (Fig 2D).…”

“…Our previous studies have indicated that EspC interacts with TLR4 directly and initiates the production of pro-inflammatory cytokines by triggering the MAPK signaling pathway [ 21 ]. To further understand how EspC regulates the host immune response, RAW26.7 cells were incubated with EspC protein or solvent control for 24 h, and the proteins associated with EspC functions in macrophages were then investigated by iTRAQ.…”

“…The espC gene was cloned, expressed, and purified, and Ms::espC was constructed as described in our previous study [ 21 ]. Briefly, EspC expression was induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 12 h at 37 °C after the bacteria were grown to OD 600 = 0.6–0.8.…”

“…RAW264.7 cells were infected with Ms:: espC and Ms::PSQ as previously described [ 21 ]. At the indicated times after infection, the cells were collected and subjected to flow cytometry to assess apoptosis and ΔΨm or western blotting with specific antibodies for each target protein.…”

“…We have previously found that EspC activated macrophages and induced the secretion of pro-inflammatory cytokines through the TLR4-dependent mitogen-activated protein kinase (MAPK) signaling pathway [ 21 ]. In the present study, we demonstrate that EspC is another critical virulent factor from the Mtb ESX-1 system that mediates Mtb –macrophage interactions by triggering endoplasmic reticulum (ER) stress-mediated apoptosis and promoting mycobacterial infection.…”