Impact Of Salmonella Enteritidis Effector Protein AvrA On The Host Inflammatory Response

Salmonella spp.are Gram-negative,facultative anaerobes and intracellular pathogens in both humans and animals that not only lead to diseases and death in livestock and poultry,causing a serious economic loss,and the contaminated products of livestock and poultry are also posing a major public health problem worldwide.In the past 20 years,Salmonella enterica serovar Enteritidis(Salmonella Enteritidis)has been one of the most common serotypes in salmonellosis in humans despite the implementation of control and prevention measures.Salmonella species possess a range of effector proteins that are translocated into host cells via a type ? secretion system(T3SS).These bacterial effector proteins display a large repertoire of biochemical activities and modulate the function of host regulatory molecules,including tight junction(TJ)proteins and their upstream regulators.Humans can be infected with Salmonella via consumption of contaminated pork,beef,poultry,and eggs or contact with fecal matter in places with poor sanitation.Salmonellosis in humans is characterized by abdominal pain,diarrhea,nausea,vomiting,fever,and headache.Salmonella Enteritidis is also increasingly reported in cases of invasive and extra-intestinal infections,such as septicemia,arthritis,endocarditis,meningitis and urinary tract infections.After internalized with the host,Salmonellae possess a range of effector proteins through T3SS that coordination or antagonism to inducing inflammatory or anti-inflammatory function.It is very important to investigation of the inflammatory interaction between Salmonellae and host.AvrA is a Salmonella effector protein secreted by the Salmonella pathogenicity island 1(SPI-1)T3SS.The AvrA is an anti-inflammatory protein possesses acetyltransferase activity toward specific host mitogen-activated protein kinase kinases(MAPKKs),inhibits the host c-Jun N-terminal kinase(JNK)/AP-1 and NF-kB signaling pathways.AvrA also possesses a deubiquitinase activity to stabilize ?-catenin and I?B?,thus inhibits NF-?B pathways and activate the ?-catenin pathway.Previous studies on AvrA were based on Salmonella Typhimurium and performed using in vitro and mouse models.However,the role of the bacterial effector AvrA in Salmonella Enteritidis remains unknown.In this study,we generated the avrA gene deletion mutant C50336?avrA in Salmonella Enteritidis strain C50336 by ?-Red mediated recombination,and it's recovery strain complemented by plasmid pBR322-avrA.Using in vitro and mouse models,we evaluated the general pattern of host inflammatory response after C50336 and C50336?avrA mutant infection that may be provides a novel concept in microbial host interaction and host inflammatory response.1.Construction and characterization of an avrA deletion mutant of Salmonella EnteritidisIn this section,we constructed an avrA deletion mutant in Salmonella Enteritidis C50336 strain,using ?-Red-mediated recombination,and it's recovery strain complemented by plasmid pBR322-avrA.The biochemical characteristics,adhesion,invasion and intracellular colonization were evaluated in avrA mutant and complemented strain.The result showed that,Salmonella Enteritidis deletion with avrA:(1)the growth and biochemical characteristics were unchanged;(2)no avrA mRNA and AvrA protein were detected in C50336?avrA mutant,while avrA mRNA and AvrA protein were overexpressed in complemented strain;(3)no affection on mRNA transcription of other effector proteins;(4)the LD50 of C50336AavrA mutant was 6.9 × 103,wild-type C50336 and complemented strain was 5.0 × 105 and 2.0 ×105 respectively.The virulence of C50336AavrA mutant was 70 fold higher than that of wild type;(5)leads to increased invasion in human epithelial Caco-2 BBE cells;(6)leads to decreased intracellular colonization in human epithelial Caco-2 BBE cells.In addition,we constructed an avrA prokaryotic expression vector,purified the recombinant AvrA protein and then prepared specific anti-AvrA polyclonal antibodies.Moreover,we established a eukaryotic expression vector pCMV-HA-AvrA and mutant pCMV-HA-AvrA(C186A)that with high transfection efficiency in Caco-2 BBE cell.The research provided the biologic materials for studying the role of AvrA in the process of Salmonella Enteritidis host inflammatory reaction.2.Inflammatory responses induced by Salmonella Enteritidis avrA deletion mutant in vitro and in vivoUsing the Salmonella infected human epithelial Caco-2 BBE cells model and streptomycin pretreated mice model,we evaluated the host inflammatory responses after Salmonella Enteritidis infection both in vitro and in vivo.In vitro assay,relative mRNA level of inflammatory factors in Caco-2 BBE cells infected with Salmonella,and the concentration of those inflammatory factors in culture supernatants were determined.In mouse model,we detected the relative mRNA levels of inflammatory factors in mice organ and concentration of inflammatory factors in mice sera at 8 hours and 4 days post Salmonella infection.The results showed that,Caco-2 BBE cells infected with Salmonella Enteritidis wild type strain,the mRNA level of IL-1?,COX-2,IL-8,iNOS,TNF-?,IFN-y,IL-6 and IL-10 were increased at early stage of infection,and among them IL-8 was increased greatly.While the mRNA level of IL-17A,IL-4,IL-12?,IL-18,LC3A and LC3B were slightly increased at the middle and late period of infection.At 6 hours post Salmonella infection,the concentration of IL-6,TNF-a,IL-8,GM-CSF,IP-10,IL-1R? and IL-1? in culture supernatant were greatly increased in avrA mutant infected cells compared with groups of wild type and complemented strain.IL-12,IFN-y and MCP-1 were increased after Salmonella infection,but no difference among the three strains infected cells.It's indicated that Salmonella Enteritidis infection induces dramatic inflammatory responses,and avrA deletion mutants leads to increased stronger inflammatory responses.In mouse infection model,the body weight loss and mortality in C50336?avrA group were higher than wild type and complemented strain infected mice.That confirms the above data,Salmonella Enteritidis C50336?avrA mutant leads increased virulence in mice.The results of bacterial load in mice organ showed that Salmonella colonization reached the highest level at 3 days post infection.At 8 hours post infection,no bacteria were detected in mice liver and spleen,but large amounts of Salmonella were detected in mice digestive tract.At 4 days post infection,Salmonella colonization in spleen and colon were greatly higher in C50336?avrA mutant infected group than that in wild type and complemented strain infected mice.It's indicated that avrA deletion leads to increased capacity of Salmonella dissemination in mice.Inflammatory factor expression in mice organ data showed that,in ileum,mRNA level of IFN-?,IL-1?,IL-10 and IL-6 were higher in C50336AavrA mutant infected group than that in wild type and complemented strain infected mice at 8 hours post infection.mRNA level of IL-18 was higher at 4 days post infection than that at 8 hours post infection.In colon,mRNA level of TNF-a and IL-17A were increased at 8 hours post infection,but no difference among the three strains infected mice.However,at 4 days post infection,mRNA level of IFN-?,IL-1?,IL-6,IL-10,IL-18 and TGF-?1 were increased and higher in C50336AavrA mutant infected group compared to that in wild type and complemented strain infected mice.Those results were similar with above in vitro data,Salmonella Enteritidis avrA deletion leads to increased inflammatory responses in mice organ.In addition,Salmonella induced inflammatory factor expression was more intensity in mice colon than ileum,indicated that colon was more sensitivity in response to Salmonella infection.In mice liver,the mRNA levels of inflammatory factors were increased at 4 days post infection.mRNA levels of IFN-y,IL-1?,IL-6,iNOS,COX-2 and TNF-? were higher in C50336AavrA mutant infected group than that in wild type and complemented strain infected mice.In spleen,mRNA levels of TNF-a,IL-17A,IFN-y,IL-1? IL-6,IL-18,iNOS and COX-2 were higher in C50336AavrA mutant infected group than that in wild type and complemented strain infected mice at 4 days post infection.In mice sera,the concentration of GM-CSF,IFN-?,IL-1?,IL-1?,IL-2,IL-6,IL-10,IL-13,IL-17,TNF-?,IP-10,KC,MCP-1 and VEGF was higher in C50336AavrA mutant infected group than that in wild type and complemented strain infected mice at 4 days post infection.Concentration of MIG was increased at 4 days post infection,but no difference among the three strains group.Salmonella induces inflammatory responses in mice intestines,and Salmonella disseminates to mice organ through intestines barrier,also induces systemic inflammatory factor expression in mice.avrA deletion leads to increased Salmonella dissemination and colonization in mice organ,also increased inflammatory factor expression.This study provided the foundation for studying the role of AvrA in the process of Salmonella Enteritidis host inflammatory reactions.3.Salmonella Enteritidis protein AvrA stabilizes intestinal tight junction via block JNK pathway and attenuates host inflammatory responseIn previous study,we constructed an avrA mutant and complemented strain in Salmonella Enteritidis C50366,and evaluated the host inflammatory responses after Salmonella infection both in vitro and in vivo.On this basis,we analyzed the changes of intracellular signaling pathway after Salmonella infection.The results showed that,in human epithelial Caco-2 BBE cells,the protein expressions of p-JNK,Cleaved-Caspase-3,P53,Beclin-1,Bax,LC3B,P62,ATG16L1,p-P38,p-P65,P65 and p-mTOR were increased after Salmonella infection.The protein expression level of p-JNK,Cleaved-Caspase-3,P53,Beclin-1,Bax and LC3B were higher in C50336?avrA mutant infected group than that in wild type and complemented strain infected cells.The other protein including VPS34,P38,p-P38,VDR,ATG-7,JNK,Caspase-3,P70,p-IKBa and IKBa were no significant changes after Salmonella Enteritidis infection.Apoptosis cells number(early stage,Annenxin V positive and PI negative cells)were greatly higher in avrA mutant infected group than that in wild type or complemented strain infected cells.p-JNK expression level in Caco-2 BBE transfected with pCMV-HA-AvrA was decreased,this confirmed the previous Western bloting data that AvrA inhibits phosphorylation of JNK.After Salmonella infection,there was greater JNK phosphorylation in avrA mutant infected cells than in wild type or complemented strain infected cells.In parallel with increased p-JNK,the tight junction protein ZO-1 was decreased in avrA mutant infected cells.While Occludin,Caludin-1,Claudin-7 and adhesion junction protein E-Cadherin did not changed.Those results were confirmed in human epithelial HCT116 and SKC015 cells.We further determined the distribution of ZO-1 protein in Caco-2 BBE cells by immunofluorescence.The results showed that,ZO-1 localization was disrupted in avrA mutan infected cells but was unchanged in complemented strain or wild type infected cells.There was no change in the adhesion junction protein E-cadherin with or without Salmonella Enteritidis colonization.We next measured the trans-epithelial electrical resistance(TEER)in human epithelial Caco-2 BBE cells.There was a dramatic decrease in TEER at 1-8 hours after Salmonlla infection,and this decrease was greater in avrA mutant infected cells than in complemented strain or wild type infected cells.There was a significant difference between avrA mutant infected cells and wild type or complemented infected cells 2-8 hours after Salmonella infection.Those results confirmed previous data,Salmonella Enteritidis deletion with avrA leads to increased bacterial invasion result in disruption of epithelia tight junction.Morphological changes were observed at 8 hours post Salmonella Enteritidis infection,including goblet cell loss,polymorphonuclear cell infiltration into the lamina propria,and epithelial damage.Mice infected with avrA mutant showed a marked increase in the pro-inflammatory response compared with mice infected with the wild type or complemented strain.In colonic samples from avrA mutant infected mice,ZO-1 protein expression was significantly decreased in parallel with increased JNK phosphorylation compared to samples from wild type or complemented strain infected mice.No changes were observed in the expression levels of claudin-7.Occludin or E-cadherin.qRT-PCR showed that ZO-1 mRNA levels in the mice colon were not altered by avrA mutant,wild type or complemented strain infection.This result is consistence with our previous in vitro study.Immunofluorescence analysis of mouse colonic tissues further confirmed that ZO-1 localization was disrupted in avrA mutant infected mice compared to complemented strain or wild type infected mice.Similar to the Western blotting results,there was no change in claudin-7 or E-Cadherin by immunofluorescence.Previous Western blotting results showed that,expression of Cleaved-Caspase-3 was increased in avrA mutant infected mice intestines.Immunofluorescence analysis further confirmed that apoptosis cells were greatly increased at 4 DPI in avrA mutant infected mice compared to complemented strain or wild type infected mice.JNK inhibitor pretreated Caco-2BBE cells showed that,after treatment with the SP600125,phosphorylated JNK was nearly undetectable in Salmonella infected cells.However,the JNK inhibitor did not disrupt ZO-1 expression in avrA mutant infected cells.qRT-PCR supported the observation that the JNK inhibitor had no effect on ZO-1 mRNA expression.Inhibiting the JNK pathway decreased the expression of the JNK-dependent pro-inflammatory cytokine IL-8,even after Salmonella infection,and no difference was observed among the three strains infected cells.Together,AvrA-induced suppression of the JNK pathway and TJ protein disassembly increases ZO-1 stability following Salmonella Enteritidis infection.We also examined downstream molecules in the JNK pathway,including FoxOs and c-Jun.FoxO and total c-jun levels were not changed by AvrA,whereas p-c-Jun was regulated by AvrA.Similar to ZO-1,the activation of p-c-Jun by avrA mutant was abolished by treatment with the JNK inhibitor.Dual luciferase assay showed that,both in Caco-2 BBE and HCT116 cells,AP-1 transcriptional activity was significantly increased in avrA mutant infected cells,compared with that of wild type or complemented strain infected cells.The activation of AP-1 by avrA mutant was abolished by treatment with the JNK inhibitor.The research investigated the mechanism of host inflammatory responses induced by Salmonella Enteritidis Confirmed that Salmonella Enteritidis effector protein AvrA stabilizes epithelial tight junction and prevents bacterial invasion.We also identified some differences on AvrA in host inflammatory response between Salmonella Enteritidis and Salmonella Typhimurium infection.It defined the mechanism of AvrA in the process of host inflammatory reaction after Salmonella infection.