Loss Of Virulence Plasmid Promotes The Interactions Between Shigella Sonnei With CD209 And CD207 Receptors

Background and aimSince the 21st century,bacillary dysentery mainly caused by Shigella sonnei worldwide.The ingestion of as few as 100 bacteria is sufficient to cause bacillary dysentery,due to inflammatory reaction induced by Shigella invasion on the colonic and rectal mucosa.According to the epidemiological study,there are 160 million cases and one million people die of dysentery every year in the world,mainly in children under five years old.It has been shown that S.sonnei encoding Shigella toxin and producing extended-spectrum beta-lactamase(ESBL)multiple drug resistance could be also transmitted by sexual transmission,and S.sonnei is closely related to the occurrence of non-calculous cholecystitis.Therefore,it is urgent to study the characteristics and pathogenic mechanism of Shigella.S.sonnei contains a 210-kb virulence plasmid that encodes an O-antigen gene cluster of lipopolysaccharide(LPS).However,this virulent plasmid is frequently lost during replication.What is the advantage in infection for S.sonnei to lose its virulent plasmid?Most Gram-negative enteropathogenic bacteria express a full LPS contains lipidA,core and O antigen.After losing the O-antigen and becoming a rough strain,the Gram-negative bacteria express a LPS core(LOS)on its surface.Previous studies suggested that by using the LPS core,Gram-negative bacteria are able to interact with several C-type lectin receptors that are expressed on antigen presenting cells(APCs).Such as Neisseria gonorrhoeae and Yersinia pestis that do not express O antigen can interact with CD209through LPS core.Moreover,Yersinia pseudotuberculosis,Salmonella typhimurium and other pathogens with O antigen expression artificially can invade antigen presenting cells(APCs)by the interaction of CD209 and LPS core.As for S.sonnei,could it also be possible to interact with immune cells in the same way as Yersinia pestis by naturally throwing away the O antigen genes and exposing the LPS core during its replication?This study puts forward the lost 210kb of virulence plasmid in S.sonnei(rough)to its outer membrane protein core LPS as ligands from the perspective of the interaction between the host-pathogenic interaction,with the host cell surface of C-type lectin receptors in combination with each other,promoted S.sonnei adhesion and invasion of APCs.Although the APCs-S.sonnei interactions most likely result in the clearance of rough S.sonnei during infection,it is our speculation that it’s likely that very small number of S.sonnei may utilize the same mechanism to hijack APCs and escape the immune system surveillance.Methods1.The Congo red plate was used to isolate S.sonnei smooth type and rough typeThe smooth type and rough type S.sonnei were obtained by using the characteristic that S.sonnei could naturally lose the toxic plasmid during replication,and the Congo red could be combined with the virulent proteins encoded by T3SS to verify that the virulent strains showed red colonies,while the avirulent ones showed colorless colonies.2.Genetic testing of S.sonnei smooth type and rough typePrimers were selected to encode characteristic genes,210kb virulence plasmid genes and O antigen specific sequence genes of S.sonnei,and polymerase chain reaction(PCR)was used to identify the smooth and rough type.3.Analysis of LPS phenotype of S.sonneiLPS was extracted and purified,and the expression of O antigen was detected by SDS-PAGE silver staining.4.Cell invasion assays4.1 Escherichia coli K12 rough strain CS180 and the O antigen expressing strain CS1861were used as control.S.sonnei smooth(wild type)and the parallel rough strain(lost 210kb virulence plasmid)were used to interact with the peritoneal macrophages of C57BL/6 mice.The gentamicin protecting assay was used to observe the invasion ability of S.sonnei rough strain 1.5 hours post infection.4.2 Escherichia coli K12 rough strain CS180 and the O antigen expressing strain CS1861 were selected as control,S.sonnei smooth(wild type)and it’s parallel rough strain(lost 210kb virulence plasmid)were used to interact with dendritic cells(DCs)isolated from human gut.The gentamicin protecting assay was used to observe the infection ability of S.sonnei to invade DCs 1.5 hours post infection.4.3 Escherichia coli K12 rough strain CS180 and Yersinia pseudotuberculosis(Y1)were used as control,we used S.sonnei smooth(wild type)/rough(lost 210kb virulence plasmid)/rough O~+(rough with pSS37)to interact with CHO cell lines expressing CD209/CD207 receptors.The gentamicin protecting assays was used to observe whether S.sonnei rough strain can invade antigen presenting cells by interacting with CD209/CD207receptors.5.Inhibition assaysBacterial lipopolysaccharide(LPS)analogues and specific antibodies were used to interact with cells first,and then cell invasion assays was carried out to determine whether S.sonnei rough strain can bind to CD209/CD207 receptors specifically,and whether the LPS core plays a role in the interaction.6.Animal assaysIntraperitoneal injection route:bacteria were injected directly into C57BL/6 mice with the appropriate dose of S.sonnei smooth/rough/rough O~+.To confirm the bacterial colonization in infected tissues,spleens and MLNs were collected from intraperitoneally challenged mice 24 hours post infection.Results1.Screening and identification of smooth and rough strains of S.sonnei.The wild type S.sonnei strain was cultured with Congo red plates,and several colonies were randomly selected and identified with specific primers to obtain the rough type of S.sonnei.2.S.sonnei did not express O antigen after the loss of virulence plasmid.The results of silver staining showed that the Ss-rough did not express O antigen.3.Rough S.sonnei invades human DCs and mouse macrophages.S.sonnei WT and its rough strain were phagocytosed by DCs to some extent,and S.sonnei rough strains were uptaken more than WT.4.CD209 and CD207 are receptors for phagocytosis of rough S.sonnei.S.sonnei rough invaded CHO cell lines expressing mSIGNR1,hDC-SIGN and hLangerin effectively,and this kind of interaction was blocked by O antigen,demonstrating that S.sonnei rough can interact with mSIGNR1,hDC-SIGN,and hLangerin.5.Inhibition experiment of receptors-mediated phagocytosis of S.sonnei rough byadding anti-mSIGNR1/anti-hLangerin antibodyThe phagocytosis ability of S.sonnei rough by transfected CHO cell lines was significantly reduced after antibodies were applied.This suggested that the mSIGNR1 and hLangerin played a role in the interaction between DCs and S.sonnei rough bacteria.However,mannan did reduce the interaction of S.sonnei rough and CD209,but not hCD207 receptor,suggesting that besides the LPS core,other sugar residues exposed on the surfaces of S.sonnei rough may also mediate the interactions.6.Expression of O-antigen reduces the ability of S.sonnei rough to be disseminatedto mesenteric lymph nodes and spleens.To compare the bacterial colonization in infected tissues,spleens and MLNs were collected after 24 hours from intraperitoneally challenged mice,it was observed that S.sonnei rough could still invade the peripheral tissues when losing the virulence plasmid.However,O antigen could inhibit the invasion of S.sonnei rough into mesenteric lymph nodes and spleens.ConclusionIn this study,we certified that S.sonnei rough strains could invade APCs through the interactions with C-type lectin human Langrin(CD207),human DC-SIGN(CD209a)and mouse SIGNR1(CD209b).