The Study On The Pathogenic Mechanism Of The Loss Of O Antigen And The Reservation Of Ail Gene During The Evolution Of Yersinia Pestis

Background and aim Lipopolysaccharides(LPS)is the major component of Gram-negative bacteria outer membrane,consists of three parts from the inside to outside: lipid A,core polysaccharide and O-antigen.Lipid A is the main component of endotoxin and can cause fever,microcirculatory disturbances and shock.The core polysaccharide also involved in the composition of endotoxin.O-antigen is the outermost part of lipopolysaccharide composed of dozens of the same oligosaccharide units,which cover the outer membrane,equivalent to a layer of coat worn by bacteria.It is a host antibody recognition target,which in addition to determine the serum lipopolysaccharide characteristics,and also to protect bacteria from phagocytosis by host cells and complement-mediated killings.It is known to all that Yersinia pestis is evolved from Yersinia enterocolitica and Yersinia pseudotuberculosis,especially share the high homologous of gene sequnce with Yersinia pseudotuberculosis.However,we know that Yersinia enterocolitica and Yersinia pseudotuberculosis are rich in O-antigen,while Yersinia pestis does not express O-antigen.For the reason that the O antigen protects the bacteria from the external environment,we think that the O antigen is an important virulence factor for the bacteria.However,why Yersinia pestis lost the virulent factor O-antigen during the evolution? Dose the loss of O-antigen contribute to the Yersinia pestis being a fatal strain of virulent evolution from a strain causing mild diarrhea? We know that the pathogenic mechanism of HIV infection is to attack CD4+ T lymphocytes,but CD4+ T lymphocytes are mainly found in lymph nodes.How does the HIV virus reach lymph nodes? Previous studies have shown that the envelope protein gp120 of HIV can interact with the receptor molecule h DC-SIGN on the surface of dendritic cells,so the HIV hijack antigen presenting cells.With the help of antigen presentation,the HIV so as to be captured and trafficked to lymph nodes to infect CD4+ T lymphocytes.Our previous studies showed that certain Gram-negative bacterium,such as E.coli,Neisseria gonorrhoeae,Haemophilus parasuis,Salmonella,Yersinia pestis,can interact with h DC-SIGN through core LPS,promoting bacterial adherence and phagocytosis.So we hypothesize that these gram-negative bacterium may use the same mechanisms as HIV infection,hijack antigen-presenting cells by the interaction of core LPS and h DC-SIGN,and transport to distant organs causes dissemination and infection.In addition,Professor Chen Tie first discovered that the core LPS of E.coli can interact with h DC-SIGN to promote bacterial adhesion and phagocytosis,and it is the specific oligosaccharide within the core,N-acetylglucose(N-Glc NAc),involved in the interaction with h DC-SIGN.Therefore,we used E.coli as a control strain in the following experimental design.Therefor this research will detect the invasion and dissemination role of the loss of O-antigen,and which kind of cell surface molecules can interact with the core LPS.Methods 1.Sequence the O-antigen gene of Yersinia pestis and Yersinia pseudotuberculosis Five strains of Yersinia pestis and 45 strains of Yersinia pseudotuberculosis were collected and six genes encoding O-antigen were sequenced to compare the diversity of these two kinds of strains.2.The cell invasion assay 2.1.The peritoneal macrophages of C57 mice were infected with Yersinia pestis KIM10(p PCP1+,pgm-,p Pst+),KIM10-O+,and the core LPS mutant strain of KIM10.E.coli with and without O antigen were used as control.The gentamicin protection assay was used to observe the difference of the invasion ability of these bacterium after two hours of infection.2.2.Repeat the cell invasion assay by using CHO cells expressing different cell surface molecules to interact with Yersinia pestis KIM10 and E.coli with O antigen exposed as a control,to find whether the cell surface molecular m DC-SIGN is the receptor for core LPS of Yersinia pestis.2.3.Repeat the cell invasion assay by using core LPS mutant of Yersinia pestis to observe whether the core LPS mutant strain can decrease the invasion ability of Yerseinia pestis.3.Cell phagocytosis inhibition test 3.1.The specific antibody to cell surface receptor molecule was used to interact with the bacteria prior cell invasion experiment to determine that the core LPS of Yersinia pestis can specifically bind to the corresponding cell surface receptor molecules.4.Animal infection experiment in vitro 4.1.C57BL/6 mice were infected by intraperitoneal injection of KIM10 / KIM 10-O+ bacterial suspension.Then mouse peritoneal macrophages were collected and treated with gentamicin protection assay as described previously 1.5 hours psot-infection to observe the invasion ability of KIM10 with and without O-antigen.5.Animal infection experiment in vivo 5.1.C57BL/6 mice were infected with Yersinia pestis 1418(p PCP1+,pgm-,p Pst+,Yersinia pestis containing virulent plasmid PYV,belong to biosafety level ?,non-pathogenic to human,but high dose of infection can cause mice death.)or 1418 O+ at appropriate doses via foot paws to mimic the bubonic plague infection.Then observe the bacterial load in different mice organs 48 hours post-infection.5.2.Transfer the fluorescent plasmid PXEN-18 by chemical to construct the Yersinia pestis strains 1418 PXEN-18 and 1418 O+ PXEN-18.Then use this pair of strains to infect C57BL/6 mice by foot paws to mimic bubonic plague and use the in vivo imaging system to observe the bacterial dissemination in the mice organs.5.3.Observe the survival portion of C57BL/6 mice infected Yersinia pestis 1418 / 1418 O+ by subcutaneous infection inoculated from foot paw path and intravenous infection from tail veil respectively.Results 1.The Yersinia.pseudotuberculosis may again lose the O antigen and evolve a new branch of Y.pestis.Five strains of Yersinia pestis and 45 strains of Yersinia.pseudotuberculosis were selected and six genes encoding O antigen were sequenced.The O antigen gene of Yersinia.pestis was found to be relatively stable and could be detected in all the five strains of Yersinia pestis.However,only a portion of the strain encoding the O antigen gene can be detected in Yersinia pseudotuberculosis,and the O antigen gene in some strains is lost.The result shows that Yersinia pseudotuberculosis may again lose the O antigen and evolves a new branch of Yersinia pestis,and under the conditions of global social turmoil,a fourth pandemic may occur.2.Yersinia pestis with core LPS covered with O-antigen can inhibit the invasion ability to macrophages.Compared to Yersinia pestis Kim10,the strain transformed with O-antigen gene Kim10 O+ and the outer core truncated strain Kim10 core-shows a decreased invasion ability to peritoneal macrophages of C57BL/6 mice.The result shows that the expression of O-antigen can inhibit the invasion ability of Yersinia pestis to host cells,and the core LPS of Yersinia pestis might be the ligand of some cell surface receptor molecules.3.Core LPS of Yersinia pestis can interact with m SIGN-R1 Yersinia pestis can interact with some C-type lectins such as m SIGN-R1,m DEC-205,m Langerin expressed on host cells.Here we use CHO cells express these molecules to phagocytosis the Yersina pestis without O-antigen.The CHO-m SIGN-R1 shows the strongest phagocytosis ability of Yersinia pestis.And this invasion ability can be inhibit by anti-m SIGN-R1 antibody and other molecules such as heparin polysaccharide and mannan.4.The exposure of core LPS of Yersinia pestis can promote the dissemination of the bacteria in mice body By counting the number of bacteria in the viscera of mice infected with bacteria,the number of bacteria in the liver and spleen of Yersinia pestis 1418 was much higher than Yersinia pestis 1418 O+.In vivo imaging results showed that Yersinia pestis 1418 could spread rapidly from the inoculation site to the liver and spleen,but the bacteria number of Yersina pestis 1418 O+ was significantly less than Yersinia pestis 1418 from the inoculated site to the distant organ during the same time.Inoculation of Yersinia pestis by foot pads,both 1418 and 1418 O + can lead to death of mice,but Yersinia pestis 1418 can cause the death of mice in a shorter time,and cause much more mouse death.The results above suggest that the loss of O-antigen helps the exposure of core LPS and promotes the invasion ability to host cells and the subsequently dissemination ability in the host of Yersinia pestis.Conclusion In this study,we demonstrated that core LPS of Yersinia pestis is the ligand of m SIGN-R1,and this core LPS-m SIGN-R1 interaction can promote the dissemination of Yersinia pestis in the mouse model.Background and aim The ail gene presented in the genome of the genus Yersinia,encoding a 17 k Da protein.Ail protein belongs to the outer membrane protein(OMPs)family,and it plays role in resisting to complement-mediated killing,promoting the invasion and adhesion ability of host cells and survival in macrophage,help Yop proteins inject into the host cells to inhibite the immune response.It is well known that the Ail gene of Yersinia pestis and Yersinia pseudotuberculosis are highly homologous,with the similarity up to 99%,but the role of Ail gene in this two strains is not the same.Researches have shown that the Ail gene in the Yersinia pestis has much stronger adhesion and invasion ability than Yersinia pseudotuberculosis.We know that the first key step for the pathogen to infect the host is to adhere and invade host epithelial cells.For Yersinia pseudotuberculosis this step is necessary,because Yersinia pseudotuberculosis needs adhere and invade host intestinal epithelial cells initiatively to cause subsequent intestinal infection.But this process is not necessary for Yersinia pestis.Because it is accepted that the infection routes for Yersinia pestis are only two.One is bubonic plague by fle bite,another is pneumonic plague by droplet.The original infection form is bubonic plague.However,in the bubonic plague,the human could be infected the Yersinia pestis by flea bites,allowing the bacteria directly penetrate into the host body,completely replace the adhesion and invasion function of ail gene.However,why Yersinia pestis still reserve the function of ail gene and optimized the adhesion and invasion ability of Ail from its ancestor? We hypothesis that the original infection form is oral plague,but not bubonic plague.The bubonic plague and pneumonic plague is developed from oral plague.For the reason that Yersinia pseudotuberculosis causes gastrointestinal infection requires the adherence and invasion ability of ail to invade epithelia cells.It is possible that the Yersinia pestis reserve the adhesion and invasion ability of ail gene from Yersinia pseudotuberculosis to infect human through the digestive tract.Studies have shown that the ail gene of Yersinia pseudotuberculosis is conducive to bacterial colonization in mice lung tissue.Therefore,we believe that the real purpose for Yersinia pestis to reserve the function of ail gene is not for bubonic plague,but for oral plague and pneumonic plague infection.It can promote the dissemination of Yersinia pestis in oral plague and pneumonic plague mouse model.Moreover,it is the adherence and invasion role of ail promotes the dissemination of Yersinia pestis in oral plague and pneumonic plague.Methods 1.Construction of Yersinia pestis expressing and not expressing Ail and Yersinia pestis contain the fluorescent plasmid PXEN-18 1.1.Use the suicide plasmid and the method of sucrose screening to delete ail gene of Yersinia pestis 1418 and 1418PYV(-)to obtain Yersinia pestis 1418Ail(-)and 1418PYV(-)Ail(-)strains.1.2.Polymerase chain reaction(PCR)assay was used to check whether ail gene was knocked out successfully,here Yersinia pestis 1418 was used as the positive control.And the function of ail to resist complement killing was verified by serum resistant assay.1.3.Construction of fluorescent plasmids into Yersinia pestis 1418 and 1418Ail(-)by electroporation or chemical transformation to obtain Yersinia pestis 1418PXEN-18 and 1418Ail(-)PXEN-18 expressing fluorescent plasmids for living body imaging experiment.2.Animal infection experiments in vivo 2.1.C57 mice were inoculated with the appropriate doses of 1418 and 1418Ail(-)to simulate pneumonic plague,bubonic plague,intranasal plague and systemic plague by different routes of infection,and the mortality of mice was observed.2.2.The C57 mice were inoculated with Yersinia pestis 1418 and 1418Ail(-)via intranasal,subcutaneous,intragastric and intravenous infection to imitate the pneumonic plague,bubonic plague,intragastric plague,and systemic plague to observe the dissemination of the bacterium from inoculation site to other organs at the early stage of infection.2.3.C57 mice were inoculated with Yersinia pestis 1418PXEN-18 and 1418Ail(-)PXEN-18 by using the four infection routs described above.And use the in vivo imaging system to further intuitively observe the dissemination situation of the bacterium at the early stage of infection.3.The cell invasion assay Chinese Hamster Ovarian epithelial cells(CHO)and C57 mouse peritoneal macrophages were infected with Yersinia pestis 1418PYV(-)and 1418PYV(-)Ail(-).Use the gentamincin protection experiment to observe the difference of invasion ability of these two strains two hours post infection.Results 1.The constructed Yersinia pestis lost the function of Ail protein and contain the fluorescent plasmid.Here we use the supernatant of boiled Yersinia pestis 1418 as positive control,the PCR result shows that the ail mutant constructed by suicide plasmid can not amplify the target band.And the PCR product was obtained by PCR with the primers according to the plasmid PXEN-18,suggesting that we successfully transformed the fluorescent plasmid PXEN-18 into the Y.pestis.The serum resistant experiment shows that the ail mutant strain of Yersinia pestis lost the ability to resist the serum complement killing of human.All the results above suggest that we successfully constructed the ail knockout strain of Yersinia pestis.2.The Ail protein can promote the dissemination of Yersinia pestis in pneumonic plague and intragastric plague.The C57 mice was infected by appropriate dose of Yersinia pestis 1418 and 1418Ail(-)through four different routes.In the pneumonic plague and intragastric plague,ail mutant strain can extend the time of death,furthermore,the same dose of ail mutant strain resulted in fewer mouse death than wild type strain.However,in the bubonic and systemic plague,there was no significant difference in mortality between the two strains.Only mice infected with ail mutant strain had a slightly slower death rate than that of wild type strain.We observed the distribution of bacteria in different organs at the early stage of infection by isolating,homogenizing the organs,and plating on agar plate to count the bacteria colonies.We found that in the pneumonic plague and intragastric plague models,the Yersinia pestis 1418 can disseminate from the inoculate site to distant organs(such as lymph nodes,liver,spleen,lungs).However,the dissemination ability of the ail gene knockout strain is lower than wild type strain.For bubonic plague and systemic plague,there is little difference of dissemination ability between these two strains,and the difference does not make statistic significance.By using the in vivo imaging system to observe the mice infected through four routes by constructed Yersinia pestis with fluorescent plasmid PXEN-18.The dissemination results showed the similar situation as the bacterial counting experiment described above.In the pneumonic and intragastric plague model,fluorescent signal can be detected in the viscera of Yersinia pestis 1418 infected mice during the early stage of infection.But there was no fluorescence signal observed in the organs of Yersinia pestis 1418Ail(-)infected mice.However,both groups of mice infected with ail mutant and wild type Yersinia pestis showed very strong fluorescent signal in the bubonic and systemic plague with little difference but without statistically significance.These results indicate that the Ail protein promotes the dissemination of Yersinia pestis in pneumonic and intragastric plague,but not bubonic and systemic plague.The invasion ability of Ail protein to penetrate the host epithelial cells may contribute to this phenomenon.3.Ail protein has the ability to invade epithelial cells When using the Yersinia pestis strain 1418PYV(-)and 1418PYV(-)Ail(-)to invade Chinese hamster ovary epithelial cells(CHO),the invasion ability of ail mutant strain was significantly lower than that of the wild type strain.This suggests that the ability of Ail protein to promote the spread of bacteria in pneumonic and intragastric plague is due to its ability to invade epithelial cells.Conclusions In this study,we demonstrated that Ail protein of Yersinia pestis can promote the dissemination in mouse pneumonic and intragastric plague model by adhere and invade host cells.