Murine SIGN-R1 Is A Receptor For Yersinia Pestis That Promotes Phagocytosis By Macrophages

Objective: Yersinia pestis is the Gram-negative bacterium that causes human plague, but its natural hosts are rodent species. It is hypothesized that Y. pestis hijacks antigen presenting cells (APCs) in order to be delivered to lymph nodes to initiate disease, but how APCs initially catch the bacterium remains unknown. There are evidences that several Gram-negative bacterial strains use their core LPS to interact with C-type lectin receptor. Therefore, Y. pestis might also use its core LPS to interact with one kind of C-type lectin receptor to invade APCs, such as mouse macrophages. Our study is to test this mechanism and find correlative receptor and ligand.Methods: 1 Establishment of C-type lectin transfectants. CHO-mDC-SIGN, CHO-mSIGN-R1, CHO-mSIGN-R3, CHO-mDEC-205 (CD205) and CHO- mLangerin (CD207), were generated by transfecting CHO cells with 5 mouse corresponding C-type lectin cDNAs which stored in expression vector pcDNA3.1, following selection by long trem G418 for stable surface expression. The expression of receptors is affirmed by flow cytometry.2 Construction of two Y. pestis strains: smooth and deep rough type strains. Wild type Y. pestis is rough strain and its core LPS is exposed outside. Deletion the phosphoheptose isomerase which catalyzes the first step in the core LPS biosynthesis pathway by suicide plasmid allelic exchange will truncate the outer core of LPS and get deep rough mutant. Expression of O-antigen in the outside of core LPS will get smooth strain.3 Adherence and phagocytosis assays: Cells were suspended in RPMI with 2% FCS and plated in 24 or 96-well plates. 1:50 volume of bacteria were added into cell suspensions at a concentration of 1 x 107 CFU /ml. The cells were allowed to incubate for 2.5 h at 37℃in the presence of 5% CO2. The cells were washed then lysed by 0.5% saponin. The amount of bacteria including adherence and invasion will be calculated by spreading plates after dilution of lysed solution.To determine the internalization of bacteria, gentamiciin, which kills extracellular bacteria but cannot penetrate into host cells, was added into each well to a final concentration of 100 ug/ml, and the cultures were incubated for 60 min. Cells were washed to remove the antibiotics. Then, the cells were lysed by 0.5% saponin and spreaded on plates. The level of internalization of bacteria in these host cells was calculated by determining the CFU recovered from lysed cells. All experiments were performed in triplicatet.4 Determination of phagocytosis by flow cytometry: Bacteria were dyed by fluorescent reagent CFDA-SE and washed 2x to remove the excess dye. Labelled bacteria were added to cell cultures for 2 h. Cell cultures were washed to remove unbound bacteria then were fixed with 2% paraformaldehyde. Before flow cytometry, a 1:10 dilution of Trypan blue (0.4%) was added to the fixed cell cultures and the mixture was incubated at ambient temperature for 10 min to quench the fluorescence from extracellular labelled bacteria. Trypan blue blocks fluorescence but cannot penetrate host cells. The rate of bacterial internalization was determined by comparing the intensity. The higher of the fluorescence-intensity shows, the more of bacteria are phagocytosed by cells.5 The inhibition experiment of exogenous reagents: anti-SIGN-R1 antibody, mannan, peptides and oligosaccharides were added to block phagocytosis assays to verify the specificity of the interaction of bacteria and cells6 Phagocytosis assays in vivo: Bacterial suspensions were directly injected into intraperitoneal cavity of live mice. After 1.5 h of infection, killed mice and purified peritoneal macrophages. Following the procedures of phagocytosis assays in vitro. Results: Y. pestis wild type strains promoted a higher level of invasion of mouse macrophages and CHO-SIGN-R1 than deep rough and smooth strains. In five C-type lectin transfectants, only CHO-SIGN-R1 got the ability of phagocytosis to Y. pestis and this reaction can be inhibited by anti-SIGN-R1 antibody.Conclusions:This study has demonstrated that murine R1 is a cellular receptor for Y. pestis. The mechanism is wild type Y. pestis expose core LPS naturally which can be recognized by the murine SIGN-R1 on mouse macrophage. The knowledge acquired from this study may allow us to develop novel strategies to combat this bacterial pathogen by blocking the interaction between Y. pestis and host receptors.