m protein function

 

Microbes and Innate Immunity

 

ORIGINAL RESEARCH article

 

Introduction

Group A streptococcus (GAS), additionally known as Streptococcus pyogenes, is the most typical and versatile of human pathogens. Moreover, GAS is answerable for a large spectrum of human ailments, starting from superficial pores and skin infections to deadly ailments (Cunningham, 2000; Bryant et al., 2003; Liang et al., 2012). Particularly, GAS is understood to outlive for lengthy durations in hosts and for recurrent assaults, significantly within the aged and kids. Nevertheless, the technique of how GAS is ready to keep alive in hosts for lengthy durations is just not clear. Earlier research have reported that GAS exploits complement common elements to bind floor proteins, comparable to M and FbaA protein (Terao et al., 2001; Pandiripally et al., 2003; Ma et al., 2011), or to advertise the cleavage of cell surface-bound C3b to iC3b to inhibit opsonization by C3, which in flip promotes bacterial entry into epithelial cells and protects in opposition to antibiotics (Cue et al., 1998; Collin and Olsén, 2002; Collin et al., 2002). As the primary line of protection in innate immunity, macrophages can set off an inflammatory response following the recruitment of neutrophils and extra macrophages, to eradicate invasive pathogens. Nevertheless, the mechanisms underlying the best way wherein GAS can escape innate immunity cell assault and survive within the physique are poorly outlined. We speculated that GAS ought to have a reverse impact on macrophages and be capable to inhibit their operate. Our earlier experiments have screened a spread of unfavorable regulatory proteins comparable to SOCS1, SOCS2, COCS3, and A20 in macrophages following GAS an infection and located that each SOCS1 (Wu et al., 2015) and A20 have been activated. Nevertheless, the mechanism answerable for the inhibition of A20 is totally different from that of SOCS1. Within the current research, we centered on the interplay between GAS and A20 in macrophages.

A20 is a crucial unfavorable regulator of immune cells, and its expression will be induced by all kinds of stimuli, together with tumor necrosis issue (TNF), interleukin (IL)-1, phorbol esters, hydrogen peroxide, lipopolysaccharide (LPS), viruses, and the cell floor receptor CD40. A20 can alsorestrain nuclear factor-κB alerts mediated by Toll-like receptors, tumor necrosis issue receptor 1 (TNFR1), and NOD2 (nucleotide oligomerization domain-2)-like receptor to create suggestions that inhibits the extreme launch of TNF-α, IL-1, IL-6, IL-8, intercellular adhesion molecules, and different proinflammatory elements. On this method, A20 can management cell apoptosis and necrosis (Opipari et al., 1992; Wertz et al., 2004; Hitotsumatsu et al., 2008).

On this research, we discovered GAS induced decrease ranges of p65, a key transcription issue of inflammatory cytokines, and inflammatory elements in macrophages, when in comparison with these inducedby E. coli. As well as, A20 expression induced by GAS appeared earlier and was larger than that induced by E. coli. GAS is understood to provide quite a lot of exocrine proteins and specific a collection of cell floor proteins. Due to this fact, we tried to establish the possible molecular mechanisms of interplay between GAS and A20, and particularly, which main elements of GAS have been concerned within the manufacturing of A20 by macrophages.

 

Supplies and Strategies

Mice

Feminine C57BL/6 mice, aged 7–8 weeks, have been bought from Beijing Laboratory Animal Middle. MyD88−/− mice have been additionally bought from the Mannequin Animal Analysis Middle of Nanjing College. All mice have been housed and manipulated in accordance with the Care and Use of Laboratory Animals (China), and have been maintained below particular pathogen-free situations. The protocol for animal experiments was authorized by the animal experimental ethics committee of Hebei Medical College (Reference quantity: 2015046).

Micro organism

Streptococcal pressure GAS SF370 M1 and different mutant GAS have been saved in our laboratory at −80°Cand routinely grown at 37°C in Todd-Hewitt Broth supplemented with 0.5% yeast extract (THY) (Difco, MI, USA). Briefly, cryopreserved GAS have been reanimated, inoculated into agar plates containing sheep blood, and incubated at 37°C for 18 h. A single colony was then transferred to three ml of THY broth at 37°C and shaken in a single day at 250 rpm. Lastly, the micro organism have been collected in 1.5-ml microcentrifuge tubes by centrifugation at 4,000 rpm for five min, washed 3 times, resuspended in 1 ml phosphate-buffered saline (PBS), and counted. E. coli was routinely cultivated at 37°C in LB tradition media.

Cells

The murine macrophage cell line RAW264.7 (RAW cells) was bought from the Cell Centre of Chinese language Academy of Medical Sciences and was maintained in DMEM supplemented with 100 U/ml penicillin and streptomycin, and 10% FBS (Gibco BRL, USA) (full medium). Mouse bone marrow-derived macrophages (BMDMs) have been generated from bone marrow cells obtained from the femurs of 15-week-old wild kind (WT) or MyD88−/− C57 BL/6 mice. Bone marrow cells (3 × 106) have been cultured with DMEM full medium containing 10 ng/ml M-CSF. After culturing for 7 days, the totally differentiated BMDMs have been used for experiments.

GAS An infection of Macrophages

RAW cells or BMDMs have been seeded at 1 × 106/nicely in 6-well plates containing tradition medium with out antibiotics. The following day, GAS and E. coli have been harvested throughout the mid-logarithmic progress section and added to cells at a multiplicity of an infection (MOI) equal to 10. After 2 h of incubation at 37°C, non-adherent extracellular micro organism have been eradicated by eradicating the tradition medium and washing utilizing phosphate buffer saline (PBS). Adherent extracellular micro organism have been subsequently killed by incubation with recent medium containing 10 μg/mL penicillin G/streptomycin at 37°C for two h. At particular time factors after an infection, supernatants have been collected for ELISA, and the cells have been ready for quantitative real-time polymerase chain response (qPCR) or Western blot evaluation.

qPCR

Cells collected at numerous time factors have been used to isolate complete RNA utilizing the RNeasy equipment (Takara, Bio. Inc., Japan) in accordance with the producer’s protocol. qPCR evaluation was then carried out utilizing oligo (dT) and random primers by a modified protocol. Briefly, cDNA samples transformed from 1 μg complete RNA was diluted at a number of concentrations. Diluted cDNA was blended with a pair of primers (10 μM) focusing on mouse IL-1β, IL-6, TNF-α, and β-actin cDNA sequences at an annealing temperature of 60°C and with 35 amplification cycles following the producer’s directions. The next primers have been used for PCR amplification: IL-6-F: AAG GAG TGG CTA AGG ACC AA; IL-6-R: GTT TGC CGA GTA GAT CTC AAA; IL-1β-F: TTC CTT GTG CAA GTG TCT GAA G; IL-1β-R: CAC TGT CAA AAG GTG GCA TTT; TNF-α-F: TGA CGT GGA ACT GGC AGA AGA; TNF-α-R: TGG GCC ATA GAA CTG ATG AGA G; β-actin-F: TAC CCA GGC ATT GCT GAC AGG; β-actin-R: ACT TGC GGT GCA CGA TGG A. The two-ΔΔCt methodology of relative quantification was used to calculate modifications within the expression of goal genes (Kubista et al., 2006). Information are offered because the imply ± customary deviation of triplicate samples and are consultant of three impartial experiments.

ELISA

Supernatants from RAW cells have been harvested at totally different factors after GAS stimulation and saved at −70°C, after which enzyme-linked immunosorbent assay (ELISA, eBioscience Inc. USA) was carried out to measure the expression of IL-6, TNF-α, or IL-1β, in accordance with the producer’s directions. Briefly, 50 μl of every pattern, and 50 μl of pattern diluents, have been added into pre-washed wells and a typical dilution was added to all customary wells. Subsequently, 50 μl biotin-conjugate was added to all wells adopted by incubation at room temperature for two h. After washing, streptavidin-HRP after which TMB substrate resolution have been successively added to every nicely. The absorbance of every nicely was then learn at a wave size of 450 nm. Information are offered because the imply ± customary deviation of triplicate samples and are consultant of three impartial experiments.

Histology

C57BL/6 mice have been anesthetized with 1% pentobarbital sodium, and challenged by intranasal (i.n.) instillation of 108 CFU 50% tissue tradition infective doses (TCID50)/50 μl of GAS or E. coli, with PBS appearing as a unfavorable management. After 48 h, the handled mice have been sacrificed and the best lung was remoted and instantly mounted in 4% paraformaldehyde. The left lung was perfused with 1 ml PBS to gather bronchoalveolar lavage fluid (BALF), which was centrifuged. A lot of the supernatant was eliminated and cell smears of centrifugal sedimentation have been stained with Wright Giemsa. The variety of leukocytes current was then counted below oil microscopy. Tissue samples have been subsequently processed, embedded in paraffin, skinny sectioned, and positioned on slides coated with L-lysine. Sections have been stained with hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) to show the inflammatory response.

Western Blotting

Complete cell protein was extracted from cells contaminated by GAS or E. coli utilizing the RIPA protein extraction equipment (Beyotime, CHN) and was then used for Western blotting to calculate the modifications in A20, TRAF6, and p-P65. Briefly, cells contaminated by GAS or E. coli have been washed twice, blended with RIPA lysis buffer, after which centrifuged at 8,000 g for five min at 4°C. Then, the supernatant was collected and protein focus was decided in keeping with the Bradford methodology (Bradford, 1976). Complete cell lysates or M protein have been then separated utilizing sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking, blots have been incubated with rabbit monoclonal antibodies in opposition to A20/TNFAIP3 (Cell signaling Inc.), TRAF6 (Cell signaling Inc. USA), and p-NF-κB p65 (Cell signaling Inc. USA); β-actin (Cell signaling Inc.) was used as an inner management. Lastly, blots have been hybridized with horseradish-peroxidase-(HRP-)-conjugated goat anti-rabbit immunoglobulin G (IgG), incubated with enhanced chemiluminescence (ECL) resolution (PerkinElmer Life Sciences, USA), and at last uncovered to X-ray movie.

SiRNA

For SiRNA remedy, 2 × 105 RAW264.7 cells have been seeded per nicely on a six-well tissue tradition plate for 18 h at 37°C in a CO2 incubator. SiA20 transfection medium was then added to the pre-washed cells in accordance with a siRNA transfection protocol (Santa Cruz Biotechnology, Inc. USA) after which incubated for six h at 37°C in a CO2 incubator. After the transfection combination was eliminated, the cells have been stimulated with heat-killed GAS for 7 h, and dwell GAS for 1 h, respectively. Lastly, the supernatant of the transfected cells was collected for ELISA. Cells have been washed as soon as with PBS, after which mounted with 4% paraformaldehyde for immunoflurescence research, or harvested for Western blot evaluation or qRT-PCR evaluation.

Immunoflurescence

Cells have been grown on glass cowl slips and incubated in a single day at 37°C and 5% CO2 in an incubator. After being washed 4 instances, cells have been mounted in 4% paraformaldehyde and blocked in 1% BSA (Saint Louis, USA) for 1 h at 37°C. Subsequently, cells have been incubated with monoclonal anti-p-P65 (1: 100) and anti-A20 (1:100) antibodies in a single day at 4°C. After 4 rinses, FITC-conjugated and secondary antibodies have been used to visualise the goal proteins by fluorescence microscopy (Tokyo, Japan). Nuclei have been counter-stained with DAPI.

Statistical Evaluation

SPSS statistical software program (model 21.0) was used for knowledge evaluation. The usual deviation (SD) of the imply is proven until in any other case indicated. Statistical significance is indicated as both *p < 0.05 or **p < 0.01.  

Outcomes – “m protein function”

Gasoline Induced a Weaker Inflammatory Response Than E. coli

RAW cells have been stimulated by GAS or E. coli, respectively, and the proinflammatory cytokines IL-1β, TNF-α, and IL-6, have been detected by qRT-PCR and ELISA at totally different time factors. The relative mRNA expression ranges of IL-1β, TNF-α, and IL-6 in GAS-infected RAW cells have been all weaker than these in E. coli-infected RAW cells at 1, 3, and 5 h post-stimulation (P < 0.05) (Figures 1A–C). Accordingly, the protein ranges of those proinflammatory cytokines have been additionally decrease than these produced by E. coli-infected RAW cells till 9 h post-stimulation (P < 0.05) (Figures 1A′–C′), suggesting that GAS induced a decrease stage of proinflammatory cytokine response in RAW cells than E. coli at an early stage after an infection. It was not clear whether or not these decrease ranges of proinflammatory cytokines would induce a weaker state of irritation in response to GAS in contrast with E. coli. We due to this fact examined this in a mouse mannequin contaminated with 1 × 107 CFU of GAS or E. coli, or with PBS as a management for 48 h, after which carried out histopathological evaluation utilizing H&E staining and PAS staining. As anticipated, H&E staining confirmed that the E. coli-challenged lungs of mice contained ample inflammatory cell infiltration within the alveolar partitions, whereas PAS staining revealed intra-alveolar edema, and hemorrhage, accompanied by bronchiolitis with shed epithelium and mucus within the bronchus (Figures 2A,B). In distinction, GAS-challenged lungs confirmed a dramatic discount of inflammatory cells together with decreased ranges of mucus and alveolitis (Figures 2A,B). Lungs from the group of mice handled with PBS confirmed infiltration of only some inflammatory cells and no mucus (Figures 2A,B). There have been far fewer inflammatory cells within the BALF of the GAS group in comparison with the E. coli group (P < 0.0001), though, there was a big distinction between the GAS group and the PBS management (P = 0.0004; Determine 2C). These outcomes indicated that when challenged with GAS, mice elicited a weaker inflammatory response in contrast with mice challenged with E. coli.

A20 Expression Was Stronger and Lasted Longer in GAS-Stimulated RAW Cells and BMDM Than That of E. coli RAW cells have been harvested following an infection with GAS or E. coli (MOI of 10:1) at totally different time factors, and the expression of A20 was analyzed by Western blotting. The outcomes confirmed that A20 expression regularly elevated in RAW cells from 2 h and peaked at 6–8 h following stimulation by GAS (Determine 3A). Accordingly, the expression of TRAF6, the down-stream goal protein of A20, was decreased from 1 to six h and elevated once more from 8 h. The expression of A20 in RAW cells stimulated by E. coli was delayed and was decrease than that of cells stimulated by GAS. Accordingly, TRAF6 expression fell from 2 to 4 h after which elevated regularly from 6 h. We additionally investigated these mechanisms in BMDM cells (Determine 3B) and noticed the same sample of outcomes. Consequently, the expression of A20 expression in GAS-infected macrophages was larger and lasted longer than that of BMDM cells contaminated with E. coli; moreover, the degrees of TRAF6 have been decrease than these of BMDW cells contaminated with E. coli. Subsequent, we tried to make use of an A20 SiRNA assay to substantiate the above outcomes. Following silencing of the A20 gene by SiA20 (Figures 4A,C), the extent of TRAR6 clearly elevated (Determine 4C). Correspondingly, p-P65 activation elevated considerably (Figures 4B,C) in GAS-stimulated RAW cells for six h. Concurrently, the expression ranges of IL-1β, TNF-α, and IL-6 within the supernatant of GAS-stimulated RAW cells have been all considerably enhanced in contrast with different teams (Determine 5). These outcomes prompt that A20, a unfavorable regulatory protein, is more likely to play an important function in inhibiting the expression of inflammatory elements in macrophages following GAS an infection. GAS-Induced A20 Expression in Macrophages Primarily Trusted the MyD88-Signaling Pathway however Partially Trusted TNFR1 With a view to decide whether or not MyD88 was concerned within the expression of A20, BMDM cells, harvested from MyD88−/−knock-out mice, have been contaminated with both GAS or E. coli for six h after which complete cell protein was analyzed by Western blotting utilizing WT BMDM cells as a management. As anticipated, the expression of A20 was a lot decrease in MyD88−/− BMDM cells than in WT teams (P < 0.05); moreover, we did not detect the downstream protein p-P65, in MyD88−/− BMDM cells stimulated by GAS (Determine 6A), suggesting that the expression of A20 is especially depending on the MyD88 signaling pathway in BMDM cells following GAS or E. coli an infection. Subsequent, RAW cells have been blocked with anti-TNFR1 monoclonal antibody previous to GAS an infection. Information confirmed that A20 expression was barely decreased when the dose of anti-TNFR1 antibody was 10 μg/ml or much less (5 μg/ml), and was even larger following blockage with a fold dose (20 μg/ml) than the unfavorable management (P < 0.001), though considerably decreased expression was noticed (Determine 6B), indicating that GAS-induced A20 expression is partially depending on TNFR1. The M Protein of GAS Performs an Important Position within the Induction of the A20 Gene As a result of, the M protein of GAS performs an necessary function in pathogenic mechanisms, we used qPCR and Western blotting to guage its function on the induction of the A20 gene in BMDM cells. Curiously, we discovered that the expression of A20 induced by M−/− GAS was a lot decrease than with WT GAS (P < 0.05; Determine 7A). Streptococcal erythrogenic toxin B (SpeB), a cysteine protease, is able to degrading M protein. Consequently, we investigated BMDM cells that had been contaminated with SpeB−/− GAS; Curiously, A20 expression was vastly improved in contrast with WT GAS (Determine 7B), suggesting that the M protein may be associated to GAS-induced A20 expression. To validate this supposition, we used M-GST protein to stimulate BMDM cells for six h to additional take a look at its operate by way of A20 gene induction, and located that M-GST induced larger A20 expression than the GST tag (P < 0.00196)or PBS management (P < 0.002; Determine 7C). Subsequently, we eliminated the GST tag from the M protein, after which used it to stimulate BMDM cells, and measure A20 expression over totally different time factors. Outcomes confirmed that prime ranges of A20 expression appeared from 2 to 12 h (Determine 7D), demonstrating that it's the M protein of GAS that performs an important function within the expression of A20 by macrophages.  

Dialogue

GAS is a crucial scientific pathogen that may trigger a large spectrum of ailments in people, together with pharyngitis, necrotizing fasciitis, erysipelas, poisonous shock syndrome, sepsis, and even mortality. Moreover, there isn’t a efficient prophylactic vaccine in opposition to GAS. Therefore, it is vitally necessary to discover mechanisms underlying pathogenesis and resistance to immunity with the intention to establish novel therapeutic targets. The pathogenic impact of GAS is expounded to its robust toxicity and resistance to immunity (Horstmann et al., 1988), which is partially attributable to the GAS-mediated up-regulation of unfavorable regulators in host cells. Host innate immune mechanisms play an important function in controlling GAS progress and limiting the additional unfold of a pathogen past the positioning of an infection (Goldmann et al., 2005). Resident macrophages have been proven to be crucial in controlling an infection in a mouse mannequin (Goldmann et al., 2004). Usually, macrophages are activated by phagocytosis and specific proinflammatory mediators comparable to TNF-α, IL-1β, and IL-6 to recruit phagocytic cells, comparable to neutrophils, extra macrophages, and dentritic cells, which, in flip, induce irritation at native websites (Park et al., 2008; Netea et al., 2009; Rojas et al., 2010). Upon activation of macrophages by a pathogen, a number of signaling cascades are triggered, resulting in activation of nuclear factor-κB (NF-κB) which induces the expression of genes related to proinflammatory cytokines and chemokines (Topley et al., 1996). Contemplating that NF-κB activation is central to many mobile processes and is closely-related to the expression of inflammatory cytokines, we analyzed NF-κB activation and the expression of proinflammatory mediators in macrophages contaminated with GAS. Surprisingly, outcomes confirmed that in contrast with E. coli, GAS induced a decrease expression stage of inflammatory cytokines inside 7 h of stimulation, and slight irritation within the lung (Determine 2A). Accordingly, our earlier outcomes confirmed that the extent of NF-κB activation induced by GAS was a lot decrease than that by Staphylococcus aureus, however was just like that induced by E. coli (Wu et al., 2016). It was not clear as to why the degrees of inflammatory mediators induced by GAS have been a lot decrease than these induced by E. coli. Nevertheless, we speculated that unfavorable suggestions proteins doubtlessly performed an important function within the regulation of inflammatory mediators. To deal with this query, we investigated the expression of such unfavorable elements, together with SOCS-1 (Wu et al., 2015), SOCS-3, and A20. Particularly, A20 is understood to play a key function within the termination of NF-κB signaling, and inhibits activation of NF-κB (Boone et al., 2004; Wertz et al., 2004). TNF receptor related issue (TRAF) 6, which mediates interplay with the NF-κB important modulator (NEMO), is a goal protein for A20 and will be degraded by the deubiquitination of A20, ensuing within the inhibition of NF-κB alerts (Hitotsumatsu et al., 2008). The expression of A20 will be induced by numerous stimulators of irritation, together with bacterial endotoxins, exotoxins, and proinflammatory cytokines (TNF-α and IL-1). In flip, A20 can inhibit the extreme launch of assorted inflammatory elements and adhesion molecules (Wertz et al., 2004; Liu et al., 2005). Our current outcomes confirmed that the extent of A20 expression induced by GAS was a lot larger than that induced by E. coli in RAW264.7 cells or BMDM, accordingly, the expression stage of the goal protein TRAF6 was decrease than that induced by E. coli. Thus, the activation of NF-κB was clearly inhibited from 2 to six h after GAS stimulation, which led to a discount within the expression of proinflammatory cytokines. To additional show these factors, we subsequent carried out A20 SiRNA evaluation, which confirmed that the extent of p-P65 was considerably elevated (Figures 4B,C), importantly, each mRNA and protein ranges of proinflammatory cytokines have been additionally considerably elevated after silencing of the A20 gene, as proven in Determine 5.

MyD88 is a crucial adaptor protein in inflammatory pathways and signaling pathways mediated by Toll-like receptors (TLRs). Excluding TLR3, all TLRs are MyD88 dependent (Kawai and Akira, 2010). We contaminated MyD88−/− BMDMs with GAS to additional examine the correlation between MyD88 and A20, and located that there was no A20 expression in MyD88 knock-out cells, whereas A20 was detected readily in WT BMDM cells. This implies that A20 expression is MyD88 dependent in each GAS- or E. coli-infected BMDM cells. Nevertheless, there was no vital distinction between the 2 teams in WT BMDM cells, indicating that MyD88 is just not the principle cause for differential A20 expression between the 2 teams.

In a earlier research, Dixit et al. reported that binding of TNF-α to its receptor TNFR1 is the principle signaling pathway mediating A20 expression after cytokine synthesis (Dixit et al., 1990; Opipari et al., 1992). Within the current research, we detected excessive expression of TNF-α in GAS-induced macrophages. Nevertheless, the blockade of RAW cells with anti-TNFR1 antibody confirmed that the expression of A20 was solely partially blocked in GAS-infected RAW cells, indicating that the expression of A20 was partially depending on the TNFR1 pathway. Consequently, we analyzed some necessary floor proteins of GAS with the intention to consider whether or not they had an impact on A20 expression. M protein and streptococcal erythrogenic toxin B (SpeB) of GAS have been included on this evaluation, largely as a result of M protein is a very powerful membrane protein and the important thing virulence issue of GAS (Lancefield, 1962). This protein can exhibit a robust proinflammatory impact and contributes to the best way wherein GAS can evade phagocytosis through macrophages (Ma et al., 2009; Metzgar and Zampolli, 2011). SpeB is a cysteine protease recognized to cleave M protein from GAS (Raeder et al., 1998; Chaussee et al., 2000; Wei et al., 2005), whereas M protein impacts the maturation of SepB. Curiously, we discovered that A20 expression induced by M−/−GAS was considerably decreased in contrast with that induced by WT GAS. As an alternative, SpeB−GAS induced A20 expression was a lot larger than that of WT GAS in BMDM cells (Figures 7A,B), suggesting that when the SpeB gene of GAS was knocked out, the soundness of the M protein was enhanced, thus resulting in a better stage of A20 expression than that induced by WT GAS. This principle was confirmed by utilizing purified M protein to stimulate macrophages (Figures 7C,D).

In abstract, GAS induces a excessive stage of A20 expression by means of its floor M protein which inhibits the inflammatory sign pathways, thus resulting in a lower within the manufacturing of proinflammatory cytokines (Determine 8). Thus, fewer inflammatory cells have been recruited to the native an infection web site, which, in flip, may be conducive to GAS progress and additional unfold of the pathogen past the positioning of an infection. In a earlier paper, Liu et al. reported that the E3 ubiquitin ligase, TRIM29, inhibited interferon-regulatory elements and signaling through the transcription issue NF-κB of alveolar macrophages following an infection with virus comparable to influenza virus (Xing et al., 2016). Collectively, our knowledge and different research implied that the operate of macrophage could possibly be regulated by a number of regulatory elements when contaminated by pathogens. This will have important implications for our understanding of pathogenesis, in addition to interactions between the host and the pathogen.

 

Creator Contributions

LW and CM conceived and designed the experiments. CM, XG, and SW carried out the experiments. LZ, JW, ZZ, ZY, and XS analyzed the info. WL, XW, and HF contributed reagents, supplies, evaluation instruments. CM and XG wrote the manuscript. All authors learn and authorized the ultimate model of the manuscript.

“m protein function”