| Salmonella enterica serovar Pullorum(S.Pullorum)is characterized by highly adaption to poultry.It is the causative agent of Pullorum disease(PD)with high morbidity and mortality,causing huge economic losses in poultry farming.Salmonella has its unique strategy to infect host,thus ensuring its survival within the host cells.Distinct from Shigella and Listeria,which escape from their nascent membrane-bound compartment and replicate in the cytoplasm,Salmonella forms a compartment called Salmonella-containing vacuole(SCV)in host cells and completes survival and proliferation in it.At present,the research on SCV mainly uses the model of S.Typhimurium infection,the research of S.Pullorum-containing vacuoles is unreported.This study mainly explored on the formation characteristics of S.Pullorum-containing vacuoles after infecting cell lines.We screened out the most suitable cell model and appropriate strain to study the development of SCV,which helped us to understand the interaction between S.Pullorum and host cell.At the same time,this study established a method for the identification of S.Pullorum T3SS effectors related to survival of Salmonella in host cells and provided a solution for in-depth study on the pathogenic mechanism of S.Pullorum.1.The establishment of S.Pullorum-infected cell modelFour kinds of avian cell lines HD-11,DF-1,DT-40 and LMH were infected by S06004 respectively,and the adhesion and invasion rate were then determined.The results showed that the adhesion and invasion rate of S06004 to avian macrophage cells HD-11 and chicken hepatocellular carcinoma cells LMH were higher than other cells.The adhesion rate of S06004 to DT-40 was high,but the invasion rate was low.Therefore,HD-11 and LMH were chose as the suitable cell models of S.Pullorum infection.50 of S.Pullorum strains isolated from 1960s-2010s were analyzed to compare their adhesion rate on HD-11 and LMH.The results showed that in HD-11 cells,the adhesion rate of most strains was similar to that of S06004,but 7101,B8605,S9804 and S9876 was higher than S06004,especially the S9876 showed nearly six times as much as S06004.In LMH cells,the adhesion rate of most strains was similar to S06004,except for S08026,S9876 and 1306 with higher infection ability than S06004,especially the S9876 showed nearly four times as much as S06004.Among 50 S.Pullorum strains,S9876 was the only one strain with higher adhesion rate than others in LMH and HD-11.Therefore,S9876 can be used as a candidate strain for exploring the formation of S.Pullorum-containing vacuoles.According to the cell infection results,10 out of 50 strains were further evaluated in the chicken embryo infection model to compare their pathogenic capacity.The 16-day-old HY-white chicken embryos were infected with a dose of 103 CFU/100?L by allantoic cavity inoculation.There were 3 strains screened preliminarily as the maximum number of deaths,which are C79-13,6803 and 6201.Taking account of the adhesion rate of three strains in HD-11 and LMH,C79-13 can be used as a candidate strain for exploring the formation of S.Pullorum-containing vacuoles.The invasion rate of C79-13 and S9876 on HD-11 and LMH were then measured to identify the most suitable strain for SCV study.The results showed that although the adhesion rate of S9876 was higher than C79-13,but the invasion ability of C79-13 was stronger than that of S9876 in two cell lines.Therefore,C79-13 was identified as the model strain for exploring the formation of S.Pullorum-containing vacuoles.2.The formation characteristics of S.Pullorum-containing vacuolesFirstly,a recombinant strain C79-13(pYA3334-GFP)expressing GFP protein was constructed successfully.We then chose the EEA1 as the nascent SCV marker,Rab7 and Lampl as the intermediate SCV markers,Cathepsin D as the mature SCV marker,and used the confocal microscopy to observe the formation of SCV at different infection stages in LMH and HD-11,respectively.The results showed that in LMH,about 80%of bacteria began to recruit EEA1 protein at 5 min post-infection to form the nascent SCV.At 15 min,Rab7 and Lampl were recruited by bacteria to form the intermediate SCV.The Rab7 positive SCV reached the highest rate at 30 min,while the Lampl positive SCV was at 15 min.From 30 min to 45 min,some bacteria recruited Cathepsin D which was the mature SCV marker,but at 60 min,the SCV stained with Cathepsin D protein was most clear and obvious.In macrophage HD-11,the nascent SCV marker EEA1 closely surrounded bacteria and the SCV increased from 5 min to 30 min.The intermediate SCV with Rab7 surrounded at 15min,and increased at 30 min and 45 min time points.The intermediate SCV marker Lamp 1 and the mature SCV marker Cathepsin D could not be obviously obtained by S.Pullorum,but some bacteria cells recruited Cathepsin D from 30 min,and reduced at 45 min and 60 min.In order to confirm the results of HD-11 observed by confocal microscopy,we observed the formation of SCV by transmission electron microscopy.At 30 min post-infection,there were uncompleted and unclosed SCV in cells.At 1 h,bacteria were separated and preserved in the SCV with complete membrane structure,and each SCV contains only one bacteria.At 5 h,SCV began to be damaged,and the replication and division of bacteria were observed in SCV.3.Function analysis of Salmonella Pullorum T3SS2 effector CigR to SCVCigR is a membrane-binding protein,one of the T3SS2 effectors encoded by SPI3.Previous studies have shown that the deletion of cigR gene can enhance the virulence of Salmonella,but some studies have shown that the deletion of cigR can lead to a significant reduction of the proliferation within mouse bone marrow macrophages.To analyze the function of CigR to S.Pullorum-containing vacuoles,a cigR mutant of S.Pullorum C79-13,named C79-13?cigR,was successfully constructed using ?-red recombinant system and analyzed the influence of CigR on formation of SCV.The results showed that loss of cigR gene enhanced the proliferation ability of S.Pullorum in the avian macrophage HD-11.And also,it can enhance the ability of S.Pullorum to recruit Rab7 in SCV. |
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