Study On The Pathogenesis Of Salmonella Typhimurium

?Background and Objective? Salmonella typhimurium,a Gram-negative bacterium,is an important zoonotic pathogen which can cause intestinal infectious diseases.Salmonella typhimurium is mainly transmitted through fecal-oral route by contaminated food or water.Once entering the intestine,it proliferates in the intestine and adheres to the intestinal mucosal epithelial then invades the laminae propria.It can cause infectious diseases ranging from gastroenteritis to septicemia and multiple organ failure caused by systemic dissemination and infection.However,the molecular mechanisms underlying this bacterial dissemination have yet to be elucidated.Studies indicated that HIV promotes viral dissemination by combining with the C-type lectin receptor DC-SIGN(CD209)expressed on human antigen presenting cells(APCs)to hijack APCs.Our previous study indicated that a mutant strain of Salmonella typhimurium,which exposures core oligosaccharide by knocking out the O-antigen of lipopolysaccharide(LPS)expressing gene,can also bind to DC-SIGN(CD209).In this study,we showed that S.typhimurium interacted with CD209 s,leading to the invasion of APCs and potentially the dissemination to regional lymph nodes,spleen and liver in mice.Shielding of the exposed LPS core through the expression of O-antigen reduces dissemination and infection.Thus,we propose that S.typhimurium may also utilize APCs via interactions with CD209 s as a way to disseminate to the lymph nodes,spleen and liver to initiate host infection,similar to HIV.In this study,we show that the S.typhimurium released from macrophages,which suppressed the expression of O-antigen,could interact with CD209 s and therefore lead to invasion of APCs and potential dissemination to lymph nodes,liver and spleen.Thus,we propose that S.typhimurium may also utilize APCs via interaction with CD209 s as a way for dissemination and infeciton,similar to HIV.?Methods? 1.In vitro assay for plasminogen activation of Pgt E in S.typhimurium after phagocytosis by macrophage cell line RAW264.7 S.typhimurium was phagocytosed and released by mouse macrophage-like cell line RAW264.7(ST-treated).The plasminogen activation of Pgt E in ST-treated or S.typhimurium untreated by RAW264.7(ST)were examined.2.In vitro assay for the invasion of human and mice APCs by S.typhimurium released from macrophages-and untreated S.typhimurium a)Dendritic cells(DCs)in human lamina propria is puritfied.Then ST-treated and ST interacted with human intestinal DCs.E.coli expressing and not expressing O-antigen were used as the controls.Invasive ability by gentamicin protection assay was examined.b)The plasmid p Ac GFP1 which expresses green fluorescent protein was transferred into S.typhimurium.The invasion of mouse peritoneal macrophages by ST-treated and ST was examined.E.coli Expressing and not expressing O-antigen were used as the controls.3.In vitro assay for the invasion of CHO,CHO-m SIGN-R1 and CHO-h DC-SIGN by treated and untreated S.typhimurium The stable transfected cell lines,expressing h DC-SIGN(CHO-h DC-SIGN)and m SIGN-R1(CHO-m SIGN-R1),were tested for their ability to phagocytose S.typhimurium and ST-treated,respectively.Yersinia pseudotuberculosis(Y1)incubated at 26 °C,E.coli expressing and not expressing O-antigen were used as controls.Invasive ability by gentamicin protection assay was determined.4.Cell invasion inhibition assay ST-treated bacteria were incubated with CHO-m SIGN-R1 and CHO-h DC-SIGN cells with or without mannan.The rate of phagocytosis of ST-treated bacteria was evaluated by the recovery of bacteria from gentamicin protection.Yersinia pseudotuberculosis(Y1)incubated at 26 ° C,E.coli expressing and not expressing O-antigen used as controls.5.In vivo infection assay for dissemination Plasmid p BR322 and plasmid p AY100.1 which expresses O-antigen was transferred into S.typhimurium.After intragastric inoculation of ST-pBR322 or ST-pAY100.1,C57BL/6J mice were euthanized on Day 4 post-infection.Then,the spleen,liver and MLNs of mice were separated,homogenized and spread onto LB agar plates.The recovered colony forming units(CFU)value was regarded as the rate of bacterial dissemination.6.In vivo infection assay for survival rates a)Survival rates of mice after intragastric infection with ST-p BR322 or ST-p AY100.1.b)Up to 10 mg of the C-type lectin receptor antagonist mannan was orally administered to the mice.Survival rates of mice after intragastric infection of ST-p BR322 with or without mannan were observed.c)Survival rates of mice after peritoneal infection with ST-p BR322 or ST-p AY100.1 cultured at 37 oC were boserved.?Results? 1.The LPS core of S.typhimurium LT2 may be exposed after phagocytosis by the macrophage cell line RAW264.7 S.typhimurium possesses outer membrane protein Pgt E,which however is not functional under the suppression of O-antigen.After treatment of macrophage cell line RAW264.7,Pgt E can activate the plasminogen.The enzymatic activity of Pgt E can be inhibited in S.typhimurium with p AY100.1,which expresses O-antigen stably.These suggest that the LPS core of S.typhimurium is possibly exposed after treatment of macrophage cell line RAW264.7 2.LPS core-dependent invasion of S.typhimurium into human and mouse APCs Given the possible exposure of the LPS core after phagocytosis by macrophages,we examined bacterial invasion of APCs,human DCs from the lamina propria and mouse peritoneal macrophages,with and without phagocytosis of ST.ST-treated invaded the primary peritoneal macrophages and human intestinal lamina propria DCs more effectively than S.typhimurium without phagocytosis,indicating that S.typhimurium released from macrophage can invaded human and mouse APCs.3.Human DC-SIGN and mouse SIGN-R1 are receptors for S.typhimurium phagocytosed by macrophages ST-treated invaded stably transfected cell lines,expressing h DC-SIGN(CHO-h DCSIGN)and m SIGN-R1(CHO-m SIGN-R1),more effectively than CHO.S.typhimurium without treatment show poor efficiency of invasion,suggesting that human DC-SIGN and mouse SIGN-R1 are receptors for macrophage-treated S.typhimurium.4.The h DC-SIGN and m SIGN-R1 mediated phagocytosis of S.typhimurium released from macrophages was inhibited by mannan oligosaccharides After the addition of mannan oligosaccharides,the invasiveness of S.typhimurium released from macrophages to h DC-SIGN-expressing cells(CHO-h DC-SIGN)but not m SIGN-R1-expressing cells(CHO-m SIGN-R1)was significantly reduced.This suggests that the mannan can inhibite interaction between h DC-SIGN and macrophage-treated S.typhimurium,but possesses a very limited ability to inhibit m SIGN-R1-mediated interaction,and indicates that h DC-SIGN(h CD209a)and m SIGN-R1(m CD209b)vary in their interactions with S.Typhimurium.5.Expression of O-antigen inhibits the dissemination of S.typhimurium in mice Mice were infected via intragastric administration with S.typhimurium contains plasmid p AY100.1 which stably expresses O-antigen(ST-p AY100.1)and S.typhimurium contains plasmid p BR322(ST-p BR322)and sacrificed.Liver,MLNs and spleen were then recovered and homogenized.The dissemination rates of the bacteria into the different organs were calculated by counting CFU.Higher numbers of ST-p BR322 than ST-p AY100.1 were isolated from the spleens,MLNs and livers,indicating that expression of O-antigen inhibits the dissemination of S.typhimurium in mice.6.The expression of O-antigen and addition of mannan reduces the infectivity of S.typhimurium The mortality rate of mice infected with S.typhimurium expressing O-antigen or in the presence of mannan were significantly reduced.suggesting that the expression of O-antigen and addition of mannan reduces the infectivity of S.typhimurium.?Conclusions? This study demonstrates that S.typhimurium released from macrophage invades the host antigen presenting cells by binding CD209 s to promote its dissemination and infection.?Background and Objective? Yersinia pestis(Y.pestis)is the pathogen of plague.According to the route of infection,it can cause bubonic plague,pneumonic plague and septicemic plague.This clonal species is emerged from mild gastrointestinal pathogen Y.pseudotuberculosis,which only causes mild,self-limiting gastrointestinal disease yersinosis in humans and is transmitted by ingestion of contaminated food.An apparent distinction during the evolution of Y.pestis from Y.pseudotuberculosis is the gain of plasmid p PCP1 and p MT.The only one major virulence determinants encoded on the p PCP1 is plasminogen activator(Pla),a member of the omptin family.Y.pestis was capable of causing pneumonic plague after acquisition of pla.Protein Pla has been shown to cleave a number of host substrates in vitro including plasminogen,?2-antiplasmin,plasminogen activator inhibitor 1 and complement protein C3.Our previous study found that Pla can utilize m DEC205(CD205)as a receptor to promote its dissemination and infection.Although the activity of Pla is unnecessary for death via septicemia,it activates host fibrinolysis during bubonic plague to enable dissemination from the skin to distal sites and required for the massive outgrowth of bacteria during pneumonic plague.Thus,we can say that Pla is required for the full virulence of Y.pestis during both bubonic and pneumonic plague.However,the exactly origination of pla during the evolution of Y.pestis is still uncertain.Both Pla and Pgt E of enteropathogen Salmonella typhimurium(S.typhimurium)belong to the outer membrane protein family.Although they have different substrates,their function are similarities and both of them can be inhibited by O-antigen.What's more,the degrees of homology based on DNA sequences within the coding regions were high to 69% between them.The degrees of homology between the proteins based on the alignment of the deduced amino acid sequences were75%.According to these,we hypothesize that there may be relationship between the acquisition of pla by enteropathogenic Y.pseudotuberculosis and pgtE of Salmonella.This study intends to detect the plasminogen activator activity of Pgt E expressing Y.pestis,and wehter it can aslo bind m DEC205 to hijack APCs for infection in vitro and in vivo,to explore whether the pgtE of Salmonella typhimurium is similar to the pla of Yersinia pestis for promoting the infection of Yersinia pestis by binding CD205,and offer the new evidence for the relationship between them.?Methods? 1.In vitro assay for plasminogen activation of Pgt E in Y.pestis Transfecting plasmids expressing Pgt E and Pla respectively into Y.pestis and E.coli,then detect the plasminogen activation of Pgt E and Pla in Y.pestis by plasminogen activator colorimetric assay?The activation was determined by the absorbance of substrate.2.In vitro assay for the invasion of mouse alveolus macrophages by Pgt E expressing Y.pestis and E.coli Detect the invasion of mouse alveolus macrophages by E.coli-pla,1419-pla(Y.pestis express Pla),1419-pgtE(Y.pestis express Pgt E).1419 and 1418 were used as controls.Invasive ability by gentamicin protection assay was determined.3.In vitro assay for the invasion of CHO and CHO-m DEC205 by Pgt E expressing Y.pestis and E.coli The stably transfected cell lines expressing m DEC205(CHO-m DEC205)were tested for their ability to phagocytose Pgt E expressing Y.pestis and E.coli.Yersinia pseudotuberculosis(Y1)incubated at 26 °C,DH5?and DH5?-pla were used as controls.Invasive ability by gentamicin protection assay was determined.4.In vivo infection assay a)Plasmid p SE380,pla and pgtE were transferred into wide-type Y.pestis 91001 and 91001 p PCP1-respectively.Infecting mice by intranasal route to mimic the pneumonic plague.Lungs were taken after 48 hours of infection and HE staining was used to detect the inflammation.b)Plasmid p SE380,pla and pgtE were transferred into wide-type Y.pestis 91001 and 91001 p PCP1-respectively.Infecting mice by intranasal route to mimic the pneumonic plague.The mortality of the mice was detected.?Results? 1.Pgt E in Y.pestis expressed plasminogen activation.Transfecting plasmids expressing Pgt E and Pla respectively into Y.pestis and E.coli and detect the plasminogen activation.The Yersinia pestis 1418,which encode Pla,the plasminogen activator,was used as the positive control.The enzyme activity of 1419-pla(Y.pestis knock out the p PCP1)and E.coli-pla is similar to 1418,and the absorbance of substrate treated by 1419-pgtE was higher than 1419,suggesting Pgt E in Y.pestis expressed plasminogen activation.2.Pgt E-expressing Y.pestis invade mouse alveolar macrophages After transforming plasmid pla and pgtE into Y.pestis and E.coli,we examined the interaction between them and mouse alveolar macrophages.E.coli-pgtE invaded the mouse alveolar macrophages more effectively than E.coli,at the same time 1419-pla and 1419-pgtE invaded the mouse alveolar macrophages more effectively than 1419,indicating that Pgt E-expressing Y.pestis invade mouse alveolar macrophages.3.m DEC205 is the receptor for Pgt E-expressing Y.pestis E.coli-pgtE,1419-pla and1419-pgtE invaded stably transfected cell line expressing mouse DEC205(CHO-m DEC205)more effectively than CHO.1419 show poor efficiency of invasion,suggesting that mouse DEC205 is the receptor for Pgt E-expressing Y.pestis.4.Expressin of Pgt E in Y.pestis stimulate the inflammation in lungs of mice Lungs of mice infected by intranasal infection with 91001 p PCP1--pla and 91001p PCP1--pgtE show more inflammatory cell infiltration through HE stains,suggesting that like Pla,Pgt E in Y.pestis stiumulate the inflammation in lungs of mice.?Conclusions? This study demonstrates that similar to Pla,Pgt E can utilize mouse DEC205 as the receptor to hijack hostantigen presenting cells for infection of Y.pestis and offer the new evidence for the relationship between pla of Y.pestis and pgtE of S.typhimurium.