Mce1C and Mce1D facilitate N. farcinica invasion of host cells and suppress immune responses by inhibiting innate signaling pathways

The mammalian cell entry (Mce) family of proteins consists of invasin-like membrane-associated proteins. The roles of Mce1C and Mce1D proteins in host–pathogen interactions have not been investigated. In this study, we demonstrate that Mce1C and Mce1D protein is localized in the cell wall fraction of N. farcinica. Both N. farcinica Mce1C and Mce1D proteins are expressed at the level of protein and mRNA and elicit antibody responses during infection. Mce1C and Mce1D facilitate the internalization of Escherichia coli expressing Mce1C protein or latex beads coated with Mce1D protein by HeLa cells, respectively. We further demonstrate that Mce1C and Mce1D can suppress the secretion of the proinflammatory factors TNF-α and IL-6 in macrophages infected with Mycobacterium smegmatis expressing Mce1C or Mce1D and promote the survival of M. smegmatis expressing Mce1C or Mce1D in macrophages. In addition, Mce1C and Mce1D supress the activation of the NF-κB and MAPK signaling pathways by blocking the phosphorylation of AKT, P65, ERK1/2, JNK, or P38 in macrophages. These findings suggest that Mce1C and Mce1D proteins facilitate N. farcinica invasion of HeLa cells and suppress host innate immune responses by manipulating NF-κB and MAPK signaling pathways, which may provide a target for N. farcinica treatment.

, target proteins were isolated by cutting the gel slices, which were stained by 0.25 mol/l KCl and then grinded. Mice were immunized subcutaneously with the gel three times every 2 weeks, and serum was collected 7 days after the last immunization. Titers of serum were determined by ELISA.

Immunogenicity of Mce1C and Mce1D proteins.
Western blot was carried out with our earlier protocol with slight modifications 18  Thermo Fisher) were combined with 1 ml of PBS containing 60 μg of mce1D protein; latex beads without protein coating served as controls. The mixtures were incubated for 2 h at 37 °C. HeLa cells were seeded in a 24-well polystyrene tissue culture plate to continue incubation until a cell monolayer formed. Due to the difficulty of Mce1C protein purification, we used the recombinant bacteria to carry out the experiment. The recombinant E. coli expressing Mce1C proteins was cultured in LB medium and then induced with 0.2 mmol/l IPTG at 30 °C for 2 h. The induced E. coli was pelleted and resuspended in medium to prepare the inoculum. Recombinant E. coli cells were added to the monolayer at a multiplicity of infection (MOI) of 10:1 and incubated at 37 °C for 4 h, washed three times with PBS, and processed for electron microscopy as described previously 30 .
For Mce1D, a 200-μl aliquot of the latex beads coated with mce1D protein or latex beads alone was added to HeLa cells monolayers grown in a 24-well plate. The cells were incubated at 37 °C for 24 h and washed three times with PBS and then processed for electron microscopy.
The cells infected with recombinant E. coli or incubated with mce1D protein were fixed with 2% glutaraldehyde, postfixed in 1% osmium tetroxide, and dehydrated with an increasing concentration-graded series of ethanol solutions. Ultrathin sections of the cell samples were cut and then examined by transmission electron microscopy (TEM, HT7700, Japan) 18 .
Blocking assay. To examine the potentially neutralizing activities of anti-Mce1C and anti-Mce1D antibodies, the ability to inhibit bacterial invasion of epithelial cells was analyzed.
The logarithmic phase N. farcinica were incubated with anti-Mce1C, anti-Mce1D, or control serum for 1 h at room temperature. The HeLa cells was cultured and maintained in DMEM with 10% fetal bovine serum. For the assay, HeLa cells were seeded onto 24-well plates for 24 h before use. The assay was performed in triplicate. The cells were infected at an MOI of 10 with N. farcinica preincubated with antisera for 1 h at 37 °C. Extracellular bacteria was killed by amikacin (200 μg/ml). Then the HeLa cells were incubated with 1 ml of sterile water for 20 min at 37 °C to release the intracellular bacteria. Serial tenfold dilutions of the lysates were plated on BHI agar for determination of CFU numbers.
N. farcinica infection assay. The infection of HeLa cells was carried out following a protocol with slight modifications 31 . Cells was maintained in DMEM with 10% FCS. Two days before infection, an estimated 2 × 10 5 cells/well were incubated in a 6-well plate. Overnight cultures of N. farcinica was diluted 1:100 in fresh Real-time PCR. Real-time PCR was performed to analyze the relative levels of the mRNA transcripts, and secA was used as an internal control. cDNA syntheses were performed at 37 °C for 15 min and at 85 °C for 5 min using Prime Script reverse transcription reagents (TaKaRa, Japan). Real-time PCR was carried out by using the SYBR Premix Ex Taq II reagents (TaKaRa) and according to the manufacturer's instructions. The primers for targeted genes are listed in Table 2.
Construction recombinant M. smegmatis. M. smegmatis strain mc 2 155 was grown in LB supplied with 0.05% Tween 80 until mid-log phase. Cells were then harvested and washed three times with cold 10% glycerol. Next, 50 μl of the competent cells was mixed with 2 µg of pMV261-mce1C, pMV261-mce1D, or pMV261 vector and then electroporated following the standard setting of 2.5 kV, 1,000 Ω, and 25 μF using the Gene Pulser Xcell apparatus (BioRad). Cells were harvested with 1 ml of BHI and incubated for 2 h at 37 °C with gently shaking, and the culture were plated in BHI plates containing 50 μg/ml kanamycin. The expression of mce1C and mce1D in recombinant M. smegmatis was confirmed by using corresponding specific primers and Western blotting. Western blot analysis. After various treatments, RAW264.7 cells were lysed with RIPA buffer supplemented with phosphatase and protease inhibitor (CWBIO, China). Protein were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore). The membranes were incubated overnight at 4 °C with primary antibodies NF-κB p-P65, P65, p-P38 MAPK, P38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-AKT, AKT, and β-actin. Subsequently, membranes were incubated with HRP-conjugated anti-rabbit IgG (Beyotime Biotechnology, China) or anti-mouse IgG antibodies (Southern Biotech, USA) and developed using the Western Lightning Plus ECL kit (PerkinElmer, USA).

Statistical analysis.
Analyses were performed using the SPSS 22.0. Group means and standard deviations (SDs) were compared with the use of two-sided Student's t tests. P < 0.05 was considered to indicate statistical significance.          The results showed that the phosphorylation of ERK1/2, JNK, AKT, and P65 was inhibited to varying degrees in RAW264.7 cells for the indicated time periods (Fig. 9c), which further strengthens the preceding results. Interestingly, the phosphorylation of P38 was activated by Mce1C mainly at an early induction time, which consisted of the preceding assays, and the phosphorylation of P38 was obviously inhibited at 8 h after induction.

Results
These results indicate that both Mce1C and Mce1D inhibit the activation of AKT/NF-κB and MAPK signaling pathways and suppress the innate immune response. In addition, the roles of Mce1C and Mce1D in modulating P38 and JNK signaling pathways differed.

Discussion
Invading host cells is the initial step in the pathogenesis for intracellular pathogens 30 and survival in macrophages is also essential to the pathogenesis of N. farcinica. To facilitate the invasion function, mce proteins may be located on the cell surface, suggesting that these proteins play an important role in the host-pathogen interactions. In this study, we mainly studied the interaction between   www.nature.com/scientificreports/ In these assays, we mainly focused on the potential role in invasive function of Mce1C and Mce1D on mammalian cells, and we further studied the function of innate immune regulation involved in its interaction with macrophages after the pathogen entered macrophage cells. Macrophages are important innate immune cells that facilitate the clearance of intracellular bacteria. Besides, macrophages could establish various mechanisms to respond to the invasion of pathogens 39 .
In the present work, we found that Mce1C or Mce1D serves as a virulence factor contributing to the survival of recombinant M. smegmatis Mce1C or Mce1D in macrophages (Fig. 8c). Macrophages can secrete proinflammatory cytokines, such as TNF and IL-6, which can enhance the innate immune response and activate the corresponding T cells, thereby enhancing the host's ability to remove infected bacteria 25,40 . M. tuberculosis could subvert immune responses and avoid the elimination of the bacilli within macrophages by regulating innate immune signaling pathways 20 . We demonstrated that the M. smegmatis expressing Mce1C or Mce1D protein could decreased the expression of pro-inflammatory cytokines, such as TNF-α (Fig. 8a) and IL-6 ( Fig. 8b), in RAW264.7 cells, which in turn inhibit the development of efficient immune responses and contribute to the survival of M. smegmatis.
There are several reports that pathogens can produce effector molecules to suppresses inflammatory signal pathways. Yersinia effector YopJ targets and downregulates both the NF-κB and MAPK signal pathways 41 . Shigella flexneri effector OspG can suppress the activation NF-κB signal pathway by affecting the degradation phospho-IκBα 42 . Our data revealed that Mce1C and Mce1D can inhibit the activation of both MAPK and NF-κB pathways induced by N. farcinica (Fig. 9) in RAW264.7 cells. Previous studies showed that an intricate balance of ERK 1/2 and P38 MAPK pathways determines the levels of proinflammatory and anti-inflammatory cytokines and that activation of ERK1/2 mainly leads to expression of TNF-α by macrophages, whereas IL-10 secretion is generally dependent on P38 MAPK activation 43,44 . Interestingly, we observed that Mce1C induced phosphorylation of P38 MAPK relative to transfection of pMV261 vector in macrophages. We also detected the activation of P38 at 1 h after stimulation, and P38 phosphorylation was inhibited after 8 h of stimulation. As for ERK 1/2, The phosphorylation of ERK 1/2 in RAW264.7 cells expressing Mce1C protein was inhibited at the beginning of stimulation, but there was no significant difference in the phosphorylation level of ERK 1/2 relative to RAW264.7 cells transfected with empty vectors at 8 h after stimulation. Our results demonstrate that RAW264.7 cells transfected with Mce1C under the stimulation of N. farcinica induced a complex regulatory mechanism between ERK and P38. NF-κB and MAPK signaling pathways are downstream of TLR-2 and TLR-4, and TAK1, downstream of TLR, and affect MAPK and NF-κB signaling pathways by phosphorylating IKK and MAPK, thereby inducing the production of inflammatory factors 39 . Whether N. farcinica Mce1C or Mce1D suppresses the activation of NF-κB and MAPK signal pathways is related to TLR-2/TLR-4 is still unknown.
To our knowledge, this is the first report that N. farcinica Mce1C and Mce1D proteins suppress the expression of proinflammatory cytokines and inhibit the NF-κB and MAPK signaling pathways, thereby inhibiting the innate immune response. Whether the inhibition of the NF-κB and MAPK signaling pathways is directly related to the decrease of proinflammatory cytokine TNF and IL-6 expression needs to be further verified, and the mechanism by which the protein inhibits NF-κB and MAPK signaling pathways is under investigation.
In conclusion, the present work reveals that