Bacterial pathogens have evolved various mechanisms to modulate host immune responses for successful infection. In this study, RNA- sequencing technology was used to analyze the responses of human monocytes THP1 to Ye...Bacterial pathogens have evolved various mechanisms to modulate host immune responses for successful infection. In this study, RNA- sequencing technology was used to analyze the responses of human monocytes THP1 to Yersinia pestis infection. Over 6000 genes were differentially expressed over the 12 h infection. Kinetic responses of pathogen recognition receptor signaling pathways, apoptosis, antigen processing, and presentation pathway and coagulation system were analyzed in detail. Among them, RIG-I-like receptor （RLR） signaling pathway, which was established for antiviral defense, was significantly affected. Mice lacking MAVS, the adaptor of the RLR signaling pathway, were less sensitive to infection and exhibited lower IFN-13 production, higher Thl-type cytokines IFN-γ and IL-12 production, and lower Th2-type cytokines IL-4 and IL-13 production in the serum compared with wild-type mice. Moreover, infection of pathogenic bacteria other than E pestis also altered the expression of the RLR pathway, suggesting that the response of RLR pathway to bacterial infection is a universal mechanism.展开更多
Objective LcrV is an important component for the development of a subunit vaccine against plague.To reduce immunosuppressive activity of LcrV,a recombinant LcrV variant lacking amino acids 271 to 326 （rV270） was pre...Objective LcrV is an important component for the development of a subunit vaccine against plague.To reduce immunosuppressive activity of LcrV,a recombinant LcrV variant lacking amino acids 271 to 326 （rV270） was prepared by different methods in this study.Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a,or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a.After Co2＋ affinity chromatography,a purification strategy was developed by cleavage of His tag on column,following Sephacryl S-200HR column filtration chromatography.Results Removal of His tag by thrombin,enterokinase and factor Xa displayed a yield of 99.5%,32.4% and 15.3%,respectively.Following Sephacryl S-200HR column filtration chromatography,above 97% purity of rV270 protein was obtained.Purified rV270 that was adsorbed to 25% （v/v） Al（OH）3 adjuvant in phosphate-buffered saline （PBS） induced very high titers of antibody to rV270 in BALB/c mice and protected them （100% survival） against subcutaneous challenge with 106 CFU of Y.pestis virulent strain 141.Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa,but they exhibited extremely low cleavage activity to the corresponding recognition site.Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy.The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.展开更多
Objective To investigate genomic variations of two Chinese Yersinia pestis isolates that were isolated from different plague foci obtained from vaccine strain EV76 from the Yunnan province of China. Methods A microarr...Objective To investigate genomic variations of two Chinese Yersinia pestis isolates that were isolated from different plague foci obtained from vaccine strain EV76 from the Yunnan province of China. Methods A microarray containing 12 000 probes covering the entire genome of seven Yersinie pestis and two Yersinia pseudotuberculosis strains, was used. PCR assays were performed to confirm microarray results. Results The gene variations detected included the absence of five genes related to the synthesis of betaine in both EV76 and another sequenced attenuated strain, KIM D27. Several genes related to phage-related membrane proteins were found to be absent in the Antiqua biovar Yunnan strain, 485, which was isolated from a rodent plague foci. Conclusion These findings provide initial insight into the distinct strains isolated from natural foci, within their genomic context, including Yunnan Y. pestis strains. This information will be used therefore to establish subsequent comparisons of these sequences with published complete genomes of other strains.展开更多
Objective This study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis. Methods The transcriptional regulation of rovA by PhoP or via temperature upsh...Objective This study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis. Methods The transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 ~C, and with temperature shifting from 26 to 37 ~C, the wild-type （WT） strain or its phoP or crp null mutant （AphoP or Acrp, respectively） was subject to RNA isolation, and then the promoter activity of rovA or plo in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and AphoP to measure the promoter activity of rovA in these two strains with the ^-Galactosidase enzyme assay system. Results When Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that ofpla was stimulated by CRP. Conclusion The rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions （which are essential to triggering relevant gene regulatory cascades）, can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.展开更多
Objective To investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis(Y.pestis),as well as the roles of RyhBs in biofilm formation.Methods Regulatory relationships were assessed by a comb...Objective To investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis(Y.pestis),as well as the roles of RyhBs in biofilm formation.Methods Regulatory relationships were assessed by a combination of colony morphology assay,primer extension,electrophoretic mobility shift assay and DNase I footprinting.Results Fur bound to the promoter-proximal DNA regions of ryhB1 and ryhB2 to repress their transcription,while both RyhB1 and RyhB2 repressed the expression of Fur at the post-transcriptional level.In addition,both RyhB1 and RyhB2 positively regulated Y.pestis biofilm exopolysaccharide(EPS)production and the expression of hmsHFRS and hmsT.Conclusion Fur and the two RyhB homologs exert negative reciprocal regulation,and RyhB homologs have a positive regulatory effect on biofilm formation in Y.pestis.展开更多
The aim of this study is to carry out genotyping of 61 Y. pestts strmns tsolated trom lwarmota baibacina-Spermophilus undulates Plague Focus of Tianshan Mountains in China. Primer pairs targeting the 22 DFRs were desi...The aim of this study is to carry out genotyping of 61 Y. pestts strmns tsolated trom lwarmota baibacina-Spermophilus undulates Plague Focus of Tianshan Mountains in China. Primer pairs targeting the 22 DFRs were designed for detecting the genotypes of 61 strains. As a result, 61 strains of Y. pestis were divided into four genotypes 1, 2, 3, and 4. Genotypes 1, 2 and 3 were found from west part in Northern Tianshan Mountains Plague Focus, but type 1 was only from Nileke county. The type of strains from Aheqi was different from those of Atushi counties in Southern Tianshan Mountains and similar to strains in Northern Tianshan Mountains Plague Focus. The type 4 distributed over Atushi county, which was identical with that of strains from Marmota caudae Plague Focus of the Pamiri Plateau. It is concluded that geonotyping is identical with ecotyping made by Shuli Ji. Tianshan Mountains Plague Focus and Marmota caudae Plague Focus of the Pamiri Plateau have a cross spreading profile.展开更多
Plague is a zoonotic disease caused by the bacterium Yersinia pestis Lehmann and Neumann,1896.Although it is essentially a disease of rodents,plague can also be transmitted to people.Historically,plague has caused mas...Plague is a zoonotic disease caused by the bacterium Yersinia pestis Lehmann and Neumann,1896.Although it is essentially a disease of rodents,plague can also be transmitted to people.Historically,plague has caused mas-sive morbidity and mortality events in human populations,and has recently been classified as a reemerging dis-ease in many parts of the world.This public health threat has led many countries to set up wild and domestic an-imal surveillance programs in an attempt to monitor plague activity that could potentially spill over into human populations.Both China and the USA have plague surveillance programs in place,but the disease dynamics dif-fer in each country.We present data on plague seroprevalence in wildlife and review different approaches for plague surveillance in the 2 countries.The need to better comprehend plague dynamics,combined with the fact that there are still several thousand human plague cases per year,make well-designed wildlife surveillance pro-grams a critical part of both understanding plague risks to humans and preventing disease outbreaks in the future.展开更多
基金supported by the National Basic Research Program of China(Nos.2012CB518704 and 2013CB910804)the National Natural Science Foundation of China(No.31170122)the Basic Research Programs of Science and Technology Department Foundation of QingHai Province(No.2013-Z-748)
文摘Bacterial pathogens have evolved various mechanisms to modulate host immune responses for successful infection. In this study, RNA- sequencing technology was used to analyze the responses of human monocytes THP1 to Yersinia pestis infection. Over 6000 genes were differentially expressed over the 12 h infection. Kinetic responses of pathogen recognition receptor signaling pathways, apoptosis, antigen processing, and presentation pathway and coagulation system were analyzed in detail. Among them, RIG-I-like receptor （RLR） signaling pathway, which was established for antiviral defense, was significantly affected. Mice lacking MAVS, the adaptor of the RLR signaling pathway, were less sensitive to infection and exhibited lower IFN-13 production, higher Thl-type cytokines IFN-γ and IL-12 production, and lower Th2-type cytokines IL-4 and IL-13 production in the serum compared with wild-type mice. Moreover, infection of pathogenic bacteria other than E pestis also altered the expression of the RLR pathway, suggesting that the response of RLR pathway to bacterial infection is a universal mechanism.
基金supported by the National Key Program for Infectious Diseases of China (2009ZX10004-4001)
文摘Objective LcrV is an important component for the development of a subunit vaccine against plague.To reduce immunosuppressive activity of LcrV,a recombinant LcrV variant lacking amino acids 271 to 326 （rV270） was prepared by different methods in this study.Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a,or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a.After Co2＋ affinity chromatography,a purification strategy was developed by cleavage of His tag on column,following Sephacryl S-200HR column filtration chromatography.Results Removal of His tag by thrombin,enterokinase and factor Xa displayed a yield of 99.5%,32.4% and 15.3%,respectively.Following Sephacryl S-200HR column filtration chromatography,above 97% purity of rV270 protein was obtained.Purified rV270 that was adsorbed to 25% （v/v） Al（OH）3 adjuvant in phosphate-buffered saline （PBS） induced very high titers of antibody to rV270 in BALB/c mice and protected them （100% survival） against subcutaneous challenge with 106 CFU of Y.pestis virulent strain 141.Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa,but they exhibited extremely low cleavage activity to the corresponding recognition site.Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy.The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.
基金supported by a grant from the National High Technology Research and Development Program of China (863 Program) (No. 2006AA2Z4A7)a grant from the National Key Program for Infectious Diseases of China (No.2008ZX1004-002)a grant from China Mega-Project for Infectious Disease (No.2011ZX10004-001)
文摘Objective To investigate genomic variations of two Chinese Yersinia pestis isolates that were isolated from different plague foci obtained from vaccine strain EV76 from the Yunnan province of China. Methods A microarray containing 12 000 probes covering the entire genome of seven Yersinie pestis and two Yersinia pseudotuberculosis strains, was used. PCR assays were performed to confirm microarray results. Results The gene variations detected included the absence of five genes related to the synthesis of betaine in both EV76 and another sequenced attenuated strain, KIM D27. Several genes related to phage-related membrane proteins were found to be absent in the Antiqua biovar Yunnan strain, 485, which was isolated from a rodent plague foci. Conclusion These findings provide initial insight into the distinct strains isolated from natural foci, within their genomic context, including Yunnan Y. pestis strains. This information will be used therefore to establish subsequent comparisons of these sequences with published complete genomes of other strains.
基金supported by the National Natural Science Foundation of China (30930001 and 30900823)
文摘Objective This study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis. Methods The transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 ~C, and with temperature shifting from 26 to 37 ~C, the wild-type （WT） strain or its phoP or crp null mutant （AphoP or Acrp, respectively） was subject to RNA isolation, and then the promoter activity of rovA or plo in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and AphoP to measure the promoter activity of rovA in these two strains with the ^-Galactosidase enzyme assay system. Results When Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that ofpla was stimulated by CRP. Conclusion The rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions （which are essential to triggering relevant gene regulatory cascades）, can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.
基金supported by the Basic Application Research Project of Science and Technology Department of Qinghai province[2020-ZJ-788]the Key Laboratory of National Health Commission on Plague Control and Prevention[2019PT310004]+1 种基金the Key Laboratory for Plague Prevention and Control of Qinghai Province[p2020-ZJ-Y23]the Qinghai Province High-end Innovative Talents Thousand Talents Program(2019)。
文摘Objective To investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis(Y.pestis),as well as the roles of RyhBs in biofilm formation.Methods Regulatory relationships were assessed by a combination of colony morphology assay,primer extension,electrophoretic mobility shift assay and DNase I footprinting.Results Fur bound to the promoter-proximal DNA regions of ryhB1 and ryhB2 to repress their transcription,while both RyhB1 and RyhB2 repressed the expression of Fur at the post-transcriptional level.In addition,both RyhB1 and RyhB2 positively regulated Y.pestis biofilm exopolysaccharide(EPS)production and the expression of hmsHFRS and hmsT.Conclusion Fur and the two RyhB homologs exert negative reciprocal regulation,and RyhB homologs have a positive regulatory effect on biofilm formation in Y.pestis.
文摘The aim of this study is to carry out genotyping of 61 Y. pestts strmns tsolated trom lwarmota baibacina-Spermophilus undulates Plague Focus of Tianshan Mountains in China. Primer pairs targeting the 22 DFRs were designed for detecting the genotypes of 61 strains. As a result, 61 strains of Y. pestis were divided into four genotypes 1, 2, 3, and 4. Genotypes 1, 2 and 3 were found from west part in Northern Tianshan Mountains Plague Focus, but type 1 was only from Nileke county. The type of strains from Aheqi was different from those of Atushi counties in Southern Tianshan Mountains and similar to strains in Northern Tianshan Mountains Plague Focus. The type 4 distributed over Atushi county, which was identical with that of strains from Marmota caudae Plague Focus of the Pamiri Plateau. It is concluded that geonotyping is identical with ecotyping made by Shuli Ji. Tianshan Mountains Plague Focus and Marmota caudae Plague Focus of the Pamiri Plateau have a cross spreading profile.
文摘Plague is a zoonotic disease caused by the bacterium Yersinia pestis Lehmann and Neumann,1896.Although it is essentially a disease of rodents,plague can also be transmitted to people.Historically,plague has caused mas-sive morbidity and mortality events in human populations,and has recently been classified as a reemerging dis-ease in many parts of the world.This public health threat has led many countries to set up wild and domestic an-imal surveillance programs in an attempt to monitor plague activity that could potentially spill over into human populations.Both China and the USA have plague surveillance programs in place,but the disease dynamics dif-fer in each country.We present data on plague seroprevalence in wildlife and review different approaches for plague surveillance in the 2 countries.The need to better comprehend plague dynamics,combined with the fact that there are still several thousand human plague cases per year,make well-designed wildlife surveillance pro-grams a critical part of both understanding plague risks to humans and preventing disease outbreaks in the future.
