Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin‐derived protein
2018; Springer Nature; Volume: 37; Issue: 17 Linguagem: Inglês
10.15252/embj.201798045
ISSN1460-2075
AutoresHang Yang, Jingjing Xu, Wuyou Li, Shujuan Wang, Junhua Li, Junping Yu, Yuhong Li, Hongping Wei,
Tópico(s)Microbial infections and disease research
ResumoArticle23 July 2018free access Transparent process Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin-derived protein Hang Yang orcid.org/0000-0001-6750-1465 Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Jingjing Xu The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China Search for more papers by this author Wuyou Li The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China Search for more papers by this author Shujuan Wang Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Junhua Li Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Junping Yu Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Yuhong Li Corresponding Author [email protected] orcid.org/0000-0002-6435-2027 The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China Search for more papers by this author Hongping Wei Corresponding Author [email protected] orcid.org/0000-0002-9948-8880 Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Hang Yang orcid.org/0000-0001-6750-1465 Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Jingjing Xu The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China Search for more papers by this author Wuyou Li The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China Search for more papers by this author Shujuan Wang Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Junhua Li Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Junping Yu Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Yuhong Li Corresponding Author [email protected] orcid.org/0000-0002-6435-2027 The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China Search for more papers by this author Hongping Wei Corresponding Author [email protected] orcid.org/0000-0002-9948-8880 Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Author Information Hang Yang1, Jingjing Xu2, Wuyou Li2, Shujuan Wang1, Junhua Li1, Junping Yu1, Yuhong Li *,2 and Hongping Wei *,1 1Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China 2The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China *Corresponding author. Tel: +86 27 87647443; Fax: +86 27 87647443; E-mail: [email protected] *Corresponding author. Tel: +86 27 51861076; Fax: +86 27 87199492; E-mail: [email protected] EMBO J (2018)37:e98045https://doi.org/10.15252/embj.201798045 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract New anti-infective approaches are much needed to control multi-drug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S. aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S. aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S. aureus were reduced 2.4- to 23.4-fold as response to V12CBD. More significantly, V12CBD could activate macrophages through NF-κB pathway and enhance phagocytosis against S. aureus. As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS. aureus infections. Synopsis The recombinant phage lysin-derived protein, V12CBD, attenuates Staphylococcus aureus virulence and enhances host innate immunity via different mechanisms, thus serving as a potential therapeutic and prophylactic candidate for anti-virulence therapy. V12CBD reduces adhesion and invasion of S. aureus to epithelial cells, and sensitizes S. aureus to macrophage killing. V12CBD downregulates transcriptions of multiple virulence factors of S. aureus. V12CBD activates macrophage through NF-κB pathway. Introduction The emergence and increasing prevalence of bacterial strains that are resistant or tolerant to available antibiotics pose serious threats to the public health worldwide, demanding the discovery of new therapeutic approaches (Spellberg et al, 2008). Classical antibiotics are usually screened from natural products and compound libraries that have bacteriostatic or bacteriocidal activity against the whole organisms (Martinez, 2008). However, bacterial pathogens can easily evolve to generate variants that withstand the immediate life-or-death pressure placed by such selected drugs, leading to the useless of these drugs, including the last-line antibiotics (Walsh, 2003; Projan & Bradford, 2007). Consequently, there is an urgent need for anti-infective agents with new modes of action different from classical antibiotics (Vicente et al, 2006). Targeting bacterial virulence is an alternative approach that means to attenuate the pathogenesis of a target bacterium to aid clearance by the host immune defense (Cegelski et al, 2008; Rasko & Sperandio, 2010). Bacterial pathogens usually express a large repertoire of different virulence factors that help them to survive under various conditions. In addition, bacterial pathogens evolve to use different factors to interplay with the host at different times over the course of a complex infectious cycle (Miller et al, 1989). Usually, the successful infection is a result of complicated interactions between the time-resolved factors' expression profile of the pathogen and its microenvironment encountered during infection, but not a single one (Monack et al, 2004; Thanert et al, 2017). Therefore, the anti-virulence strategy can target multiple therapeutic windows to intervene the outcome of an infection, such as targeting bacterial factors mediating adhesion to the host (Krachler & Orth, 2013), secretion systems (Baron, 2010), regulatory systems (Rasko et al, 2008), quorum-sensing signaling (Starkey et al, 2014), elements that involved in evading host defense (Wang et al, 2015), and toxin trafficking and function (Saenz et al, 2007). Other strategies based on the opsonophagocytic activity of antibodies derived from passive or active immunity have also been pursued as promising options for bacterial diseases (DiGiandomenico & Sellman, 2015). Although successful for the treatment of several infections, such as pneumococcal pneumonia, meningococcal meningitis, and tuberculosis (Casadevall et al, 2004), the antibody-based therapies are not available yet for many notorious multi-drug-resistant (MDR) pathogens, for instance methicillin-resistant Staphylococcus aureus (MRSA). Encouragingly, several vaccine candidates, including a monoclonal antibody binding to S. aureus toxin, are currently in clinical trials (Hua et al, 2014; DiGiandomenico & Sellman, 2015; Giersing et al, 2016). A recent report also showed that chimeric antibodies by fusing IgG Fc with binding domains from cell wall hydrolases were found having opsonophagocytic activities against S. aureus in mice models (Raz et al, 2017), which may represent a new approach for developing therapeutic antibodies. However, opsonophagocytic antibodies that perform well in animal models may have rare or no protective efficacy in humans as evidenced by the failure of all previous S. aureus vaccines that have been proven to be protective in experimental models (Proctor, 2012; Jansen et al, 2013; Fowler & Proctor, 2014). Lysins, expressed by bacteriophages to digest the host bacterial cell wall for the release of newly assembled progeny virions, have attracted much attention as alternative anti-infectives against infectious diseases (Nelson et al, 2012; Pastagia et al, 2013). Lysins can lyse the peptidoglycan layer of target bacteria, resulting in their death within minutes. Lysins are modular in structure with a peptidoglycan hydrolytic catalytic domain and a cell wall binding domain (CBD) recognizing the conserved element in wall carbohydrates of a target bacterium (Fischetti, 2005). Researchers have consensus on high affinity and low resistance of lysin CBDs. However, nothing is known about the effects of CBDs on virulence and infection of target pathogens so far. In the present work, we found that a protein derived from CBD of lysin PlyV12 (Liu et al, 2015), not the whole lysin, has surprising anti-virulence capacity against methicillin-resistant S. aureus (MRSA). MRSA now represents an enormous public health burden that is not adequately addressed by current antibiotics (Boucher & Corey, 2008; DeLeo & Chambers, 2009; Tong et al, 2015). Here, we found for the first time that V12CBD could repress expression of S. aureus virulence genes, sensitize S. aureus to the killing of macrophage, promote phagocytosis of macrophage, and eventually protect mice from lethal MRSA infections in therapeutic and prophylactic models. Unlike current antibiotics, V12CBD can act not only on reducing virulence of bacteria, but also on activating host immunity. These unique functions would render V12CBD a novel molecule to control MDR S. aureus infections. Results Binding with V12CBD suppresses the invasion of Staphylococcus aureus to epithelial cell in vitro V12CBD can bind to the cell wall of S. aureus to form complex (Dong et al, 2015), without interfering with the growth or the morphology of S. aureus (Appendix Fig S1). Because the primary site of S. aureus infection is often a breach in epithelial cells that may lead to colonization and internalization, causing acute and chronic infections, we tested whether V12CBD could inhibit adhesion and internalization of S. aureus into epithelial A549 cells. As shown in Fig 1A, the V12CBD-treated S. aureus cells displayed a reduced adhesion to A549 cells in comparison with the PBS-treated controls (1.09 × 106 CFU/well at 100 μg/ml V12CBD vs. 1.66 × 106 CFU/well in control, P < 0.05). After using gentamycin to kill the bacteria adhered to the cell surface, the number of intracellular staphylococci was also found lower in V12CBD-treated wells (2.67 × 105 CFU/well at 100 μg/ml V12CBD vs. 3.98 × 105 CFU/well in control, P < 0.05, Fig 1B). To assess the specificity of V12CBD, we examined the effects of V12CBD on the adhesion of Listeria monocytogenes (Lmo), Streptococcus agalactiae (Sag), and Salmonella enteritidis (Sen) to A549 cells. No significant differences were observed in V12CBD-treated groups and the PBS-treated groups (Fig 1C). Moreover, we tested the effects of the CBD (CBD511) of Listeria phage lysin Ply511 (Loessner et al, 1995), which has similar charge and hydrophobic properties to V12CBD (MWV12CBD = 18.4 kDa, pIV12CBD = 9.96, ZV12CBD = +14.1; MWCBD511 = 15.5 kDa, pICBD511 = 9.96, ZCBD511 = +13.9), on the adhesion and internalization of S. aureus N315 to A549 cells. Results showed that treatment with CBD511 reduces the adhesion and internalization of Lmo to A549 cells but not S. aureus (Appendix Fig S2). These results suggest that binding with V12CBD specifically suppresses adhesion and invasion of S. aureus to the epithelial cells. Figure 1. Binding with V12CBD sensitizes immune clearance of Staphylococcus aureus A, B. Effects of V12CBD on the adhesion (A) and internalization (B) of S. aureus by A549 cells. The total number of viable bacteria within each well (Nt) is determined by plating series dilutions on TSA plates. The internalized bacteria (Ni) is determined by treating each well with 200 μg/ml gentamycin for 2 h, followed by plating assay. The bacteria adhered on the cell surface (Na) is expressed as the difference between the total bacteria and the internalized ones (Na = Nt−Ni). n = 5. *P < 0.05. C. Effects of V12CBD on adhesion of Listeria monocytogenes, Staphylococcus agalactiae, and Staphylococcus enteritidis to A549 cells. n = 3. n.s., not significant. D. Example FACS plot for phagocytosis of Staphylococcus aureus by macrophages. WGA-conjugated S. aureus N315 is pretreated with various concentrations of V12CBD (red line) or PBS (cyan line) and then cocultured with macrophage RAW264.7 cells for 1 h at 37°C. After treatment with trypsin and washed with PBS at 300 g for six times, cells are analyzed using a BD LSRFortessa analyzer flow cytometer. Images shown are representative of two independent repeats (n = 3 per group). E. Summary data of phagocytosis of S. aureus by macrophages. The extent of phagocytosis of each treatment is calculated using the CFlow software. n = 6. n.s., not significant. F, G. Effect of V12CBD on the susceptibility of S. aureus to killing by RAW264.7 and peritoneal macrophages. Staphylococcus aureus N315 is pre-bound with V12CBD and exposed either to macrophage RAW264.7 (F) or to peritoneal macrophages from BALB/c mouse for 4 h (G). n = 9 (F) and n = 6 (G). **P < 0.01, ***P < 0.001. H. Effect of V12CBD on the virulence of S. aureus in vivo. Staphylococcus aureus T23 is pre-bound with varied V12CBD (0 μg/ml, black line; 100 μg/ml, green line; 200 μg/ml, red line; 400 μg/ml, blue line) and injected intraperitoneally to mice. n = 5. *P < 0.05, ***P < 0.001. Data information: Data are expressed as mean ± SEM. Pairwise comparison is performed using two-tailed Student's t-test. Download figure Download PowerPoint Binding with V12CBD sensitizes Staphylococcus aureus to killing by macrophages and attenuates the virulence of S. aureus in a mouse model Because wall carbohydrates shield S. aureus from host immune defense and oxidant killing (O'Riordan & Lee, 2004), we next examined whether binding with V12CBD could promote phagocytosis of S. aureus by macrophages. After incubation with wheat germ agglutinin (WGA) conjugated, V12CBD (0, 25, 50, and 100 μg/ml) pretreated S. aureus N315 at 37°C for 1 h, macrophages RAW264.7 cells were treated with trypsin to detach the bacteria adhered on cell surface, washed, and analyzed by flow cytometry to determine the extent of phagocytosis (Fig 1D). The flow cytometry showed that V12CBD does not promote phagocytosis of WGA-conjugated S. aureus by macrophage RAW264.7 (Fig 1E). However, survival rate of V12CBD-treated S. aureus was ~ 4.3 times lower than that of the PBS-treated bacteria (24.6% at 100 μg/ml V12CBD vs. 105.5% in control, Fig 1F) after incubation with RAW264.7 cells. When the concentration of V12CBD increased to 400 μg/ml, the survival rate of V12CBD-treated S. aureus was ~ 10.3 times lower than that of the PBS-treated one (10.2% vs. 105.5%, Appendix Fig S3). Moreover, the fate of S. aureus after incubation with the peritoneal macrophages from BALB/c mouse showed similar survival trends (Fig 1G). Since binding with V12CBD sensitized S. aureus to killing by macrophages in an ex vivo assay, a virulent MRSA isolate S. aureus T23 pretreated with various concentrations of V12CBD (0, 100, 200, and 400 μg/ml) was injected intraperitoneally to mice at a final dose of 2.5 × 108 CFU/mouse. As shown in Fig 1H, the mice infected with PBS-treated S. aureus (bacteria alone, n = 5) died within 1 day after the challenge, whereas the mice infected with V12CBD-treated S. aureus showed dose-dependent survival rates. Eighty percentages of the mice (4/5) were alive at day 10 after infection with 400 μg/ml V12CBD-treated S. aureus. Binding with V12CBD suppresses multiple important virulence factors expressed by Staphylococcus aureus To further understand the possible reasons behind the virulence attenuation, a RNA-Seq approach was used to investigate the impact of binding with V12CBD on the transcriptional response of S. aureus T23. In summary, a total of 225 differentially expressed genes (DEGs) were identified (at least twofold change, with a P-value < 0.05) after V12CBD treatment (Appendix Table S1). Of those, transcripts of 111 genes were found upregulated and 114 downregulated in response to V12CBD. Predominantly, genes involved in translation (76.9%), nucleotide metabolism (80%), and cell division (100%), as well as replication and repair (82%), were repressed in S. aureus treated with V12CBD (Fig 2A). The GO enrichment analysis showed that the DEGs were scattered in 34 GO terms, specifically, a total of three items related to cellular components, 25 items for biological processes, and six items for molecular functions (Appendix Fig S4). Functional classification of the DEGs using KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis revealed that a large group of S. aureus genes expressed differentially in response to V12CBD belonged to the membrane transport pathway, the environmental information processing category (Fig 2B). Particularly, genes belonging to ABC transports such as pstA, cycB, opuA, abcA/bmrA, opuBD, msbA, and nikC, genes involved in phosphotransferase system such as lacE, as well as genes involved in bacterial secretion system including secD and secY, were upregulated in S. aureus (Appendix Table S1). Because the ABC transports play significant roles in nutrient uptake and in secretion of toxins and antimicrobial agents in bacteria (Davidson & Chen, 2004), the increased expression of the genes encoding these pathways in the presence of V12CBD indicates a certain degree of nutrient limitation of S. aureus. This nutrient limitation may lead to the upregulation of genes that enable S. aureus to uptake amino acids (opuBD), phosphate ion (pstA), lipids (abcA/bmrA), and sugars (cycB) from the extracellular microenvironment. Also, several genes involved in ABC transports such as modB, znuA, oppA/mppA, phnD, and metN were found downregulated (Appendix Table S1), most of which were substrate-binding proteins in iron complex transport system. Figure 2. Gene expression analysis of Staphylococcus aureus in response to V12CBD eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) functional categories of the DEGs expressed in S. aureus in the presence of V12CBD. Enrichment of DEGs in KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. *P < 0.05. Data information: Significance of enrichment in KEGG pathways was performed using Fisher's exact test. Download figure Download PowerPoint Several genes encoding important virulence factors, such as surface protein-encoding gene sdrC, which is a well-characterized surface-associated protein that mediates the adhesion of S. aureus to biomaterial and host cell surface (Barbu et al, 2014), protein arginine kinase encoding gene mcsB, which is required for stress tolerance and virulence of S. aureus (Wozniak et al, 2012), persistent infection-associated genes (putP and oppF2), and the genes involved in capsular polysaccharide biosynthesis (tarI and SAOUHSC_00125), were downregulated in the presence of V12CBD (Table 1 and Appendix Table S1). Since most S. aureus strains are L-proline auxotrophs, PutP catalyzes the sodium-dependent uptake of extracellular L-proline that contributes to in vivo survival and persistent infection of S. aureus (Schwan et al, 1998; Bayer et al, 1999). The gene product of oppF2 is involved in oligopeptide uptake that has been proven important for S. aureus growth and survival in multiple infection environments (Coulter et al, 1998). And capsular polysaccharides are important for S. aureus to evade the host innate immune defenses (Thakker et al, 1998; Cunnion et al, 2001). Table 1. Virulence-associated DEGs expressed by Staphylococcus aureus in the presence of V12CBD Locus Gene Description Fold change RNA-Seq qRT–PCR SAOUHSC_00544 sdrC Serine-aspartate repeat-containing protein C −2.42 −2.71 (−23.4) SAOUHSC_00125 Cap5L protein/glycosyltransferase −3.25 −3.57 (−12.42) SAOUHSC_00225 tarI Ribitol-5-phosphate cytidylyltransferase 1 −4 −9.43 (−7.55) SAOUHSC_00504 mcsB Protein arginine kinase −3 −5.61 (−5.36) SAOUHSC_02119 putP Sodium/proline symporter −3.93 −4.22 (−3.01) SAOUHSC_01377 oppF2 Putative oligopeptide transport ATP-binding protein −2.62 – SAOUHSC_00020 walR Transcriptional regulatory protein −2.45 −3.35 (−93.05) SAOUHSC_01586 srrA Transcriptional regulatory protein −3.83 – DEGs, differentially expressed genes. The fold changes in qRT–PCR represent the expression levels of S. aureus in the presence of 25 and 100 μg/ml V12CBD, respectively. Two of 16 two-component systems (TCSs; Beier & Gross, 2006; Stock et al, 2000) of S. aureus (WalKR and SrrAB) were also found downregulated in the presence of V12CBD (Table 1 and Appendix Table S1). The WalKR TCS, originally identified in Bacillus subtilis (Fabret & Hoch, 1998), is highly conserved and specific to low G+C Gram-positive bacteria, including S. aureus (Martin et al, 1999). The WalKR system is considered as the only essential TCS for the viability of S. aureus and plays a central role in controlling cell wall metabolism, oxidative stress resistance, and virulence regulation (Dubrac et al, 2007; Ji et al, 2016). The walKR operon of S. aureus comprises five-jointed gene locus, that is, walR, walK, yycH, yycI, and yycJ, under a Sigma A-dependent promoter (Dubrac et al, 2008). Although the genes encoding yycI and yycJ were not DEGs, the expression levels of walR and yycH were greatly repressed in V12CBD-treated S. aureus (Table 1 and Appendix Table S1). The reduced expression of walR indicates reduced virulence, since walR positively regulates the expression of major virulence genes involved in host matrix interactions, cytolysis, and host inflammatory response (Delaune et al, 2012). SrrAB (staphylococcal respiratory response AB) is another well-known TCS of S. aureus that helps survival of S. aureus (Kinkel et al, 2013) and protects S. aureus from neutrophil killing in low-oxygen conditions (Ulrich et al, 2007). The repressed expression of srrAB system may lead to reduced ica gene transcription and polysaccharide intercellular adhesion expression in S. aureus (Ulrich et al, 2007), which also imply reduced virulence. Furthermore, to confirm the effect of V12CBD on the expression of virulence factors of S. aureus, the RNA-Seq results were validated by quantitative real-time PCR (qRT–PCR). In general, there was a positive correlation between the data obtained from qRT–PCR and that from the Illumina RNA-Seq (Table 1). Interestingly, the expression level of walR in S. aureus was strikingly declined (fold change from −3.35 to −93.05) when V12CBD concentration increased from 25 to 100 μg/ml (Table 1), indicating that the WalKR TCS may be the main system that senses the change of V12CBD in the environment. V12CBD enhances phagocytosis and killing of Staphylococcus aureus by macrophages In order to minimize the possible effects of residual V12CBD on the RAW264.7 cells in the above experiments, we examined the phagocytic capacity of V12CBD-treated macrophages against WGA-conjugated S. aureus N315 by flow cytometry (Fig 3A). Unexpectedly, treatment with V12CBD indicated substantial phagocytosis of S. aureus by macrophage RAW264.7 in a dose-dependent manner (Fig 3B). After treatment with 100 μg/ml V12CBD, phagocytosis of staphylococci increased about 2.9-fold (47.6% vs. 16.5%). Accordingly, the survival rates of S. aureus in the macrophages declined along with the increase in the treatment concentration of V12CBD (Fig 3C). Additional experiments showed that V12CBD (labeled with Alexa Fluor 488) could not permeate A549 cells (Appendix Fig S5), but can be taken up by the macrophages in a time-dependent manner (Fig 3D and Appendix Fig S5). Figure 3. V12CBD activates macrophages and promotes their phagocytosis and killing against Staphylococcus aureus Example FACS plot for phagocytosis of S. aureus by V12CBD-pretreated macrophages. RAW264.7 cells are pretreated with V12CBD (cyan line) or PBS (red line) for 24 h before the experiment. Then, WGA-conjugated S. aureus N315 is added and cocultured with RAW264.7 cells for 1 h. After treatment with trypsin and washing with PBS at 300 g for six times, RAW264.7 cells are analyzed using a BD LSRFortessa flow cytometer. Images shown are representative of two independent repeats (n = 3 per group). Summary data of phagocytosis of S. aureus by macrophages. The extent of phagocytosis after each treatment is analyzed using the CFlow software. n = 6. **P < 0.01, ***P < 0.001. Survival of S. aureus in RAW264.7 cells pretreated with V12CBD for 24 h. n = 6. *P < 0.05, ***P < 0.001. Images of RAW264.7 cells after cocultured with Alexa Fluor 488-labeled V12CBD for 1 and 24 h. Scale bar: 5 μm. Images shown are representative of two independent repeats (n = 3 per group). Expression levels of Il1b, Il6, Tnfa, and iNos in macrophage RAW264.7 after exposure to 0, 25, and 50 μg/ml V12CBD for 8 and 24 h. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001. Expression levels of TNF-α and IL-6 in the supernatants of RAW264.7 after treated with 100 μg/ml V12CBD for 24 h. Cells treated with a mixture of 15 ng/ml IFN-γ and 15 ng/ml LPS are used as positive controls. n = 3. **P < 0.01, ***P < 0.001. Expression levels of Il1b, Il6, Tnfa, and iNos in BHK-21 cells after transfection with pcDNA3.1 or pcDNA3.1-V12CBD for 48 h. n = 6. *P < 0.05. Data information: Data are shown as mean ± SEM. Pairwise comparison is performed using two-tailed Student's t-test, unless stated otherwise. All genes' expression levels are compared with that of Gadph gene in the same condition. The PBS-treated cells were used as blank controls. Download figure Download PowerPoint Because the innate immune system provides the first line of defense to the host by initiating a sequence of events that result in the production and secretion of a wide range of inflammatory cytokines, the activation of macrophages, and the initiation of adaptive immunity (Fournier & Philpott, 2005), in order to further understand the mechanisms behind the finding that V12CBD can enhance phagocytosis and killing of S. aureus by macrophages (Fig 3A–C), we examined the effects of V12CBD on the expression of several cytokines of macrophage by qRT–PCR. Because V12CBD was expressed by Escherichia coli, to exclude the possible effects of contaminated LPS, we detected the expressions of these genes in the presence of LPS inhibitor polymyxin B. It was found that polymyxin B could completely block the stimulation effect of LPS (Appendix Fig S6A), but not the effects of V12CBD on the gene expression profiles of macrophage (Appendix Fig S6B). Further results showed that the expression of genes encoding a core set of proinflammatory cytokines, i.e., interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), as well as inducible nitric oxide synthase (iNOS), was upregulated in V12CBD dose-dependent manners after treating macrophage RAW264.7 with V12CBD for 24 h (Fig 3E), while the expression of the gene encoding Toll-like receptor 4 (TLR4, the receptor of LPS) did not have significant changes (Appendix Fig S7). Correspondingly, increased accumulations of TNF-α (from 0.51 to 1.25 μg/ml) and IL-6 (from 0.0084 to 0.10 μg/ml) were
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