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.
Highlights
Tuberculosis, caused by Mycobacterium tuberculosis, is related to high morbidity and mortality rates and is a critical health concern worldwide (World Health Organization, 2016)
Because EspC is a highly immunodominant Region of difference 1 (RD1)-dependent secreted antigen specific for mycobacteria infection, we evaluated the release of cytokines from RAW264.7 macrophages stimulated with various concentrations of EspC
The results showed that EspC induced significant increases in Tumor necrosis factor (TNF)-α, IL-6, and monocyte chemoattractant protein (MCP)-1 levels in THP-1 cells (Figure 1C)
Summary
Tuberculosis, caused by Mycobacterium tuberculosis, is related to high morbidity and mortality rates and is a critical health concern worldwide (World Health Organization, 2016). Once infected with M. tuberculosis, hosts will initiate innate and adaptive immune responses against mycobacteria to restrict bacterial survival and eliminate the bacterium (Akira et al, 2001). M. tuberculosis can escape host immune defense and replicate within permissive macrophages through multiple strategies, including prevention of phagolysosome maturation, tolerance to the acidic environment of phagolysosomes, and inhibition of apoptosis and autophagy (Lee et al, 2009; Levitte et al, 2016; Saini et al, 2016). EsxA ( named 6-kDa-early-secreted antigenic target [ESAT-6]) is a well-known virulence factor of ESX-1 or RD1 in pathogenic mycobacteria and participates in host-pathogen interactions (Van Pinxteren et al, 2000; Brodin et al, 2005). Research on whether there are other ESX-1 secreted proteins that play an important role in the interaction between the host and mycobacteria is limited
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