Objective:To evaluate the anti-shigellosis activity of the hydroethanol extract of Diospyros gilletii(D.gilletii)stem bark in Shigella flexneri(S.flexneri)-induced diarrheal mice.Methods:The hydroethanolic extract was...Objective:To evaluate the anti-shigellosis activity of the hydroethanol extract of Diospyros gilletii(D.gilletii)stem bark in Shigella flexneri(S.flexneri)-induced diarrheal mice.Methods:The hydroethanolic extract was obtained by maceration of D.gilletii stem bark in 70%hydroethanol(water꞉ethanol;30꞉70,v/v)solution.Then,mice pretreated with cyclophosphamide for immunosuppression were administered orally with an inoculum containing S.flexneri,and subsequently treated with 100,200,and 400 mg/kg of the hydroethanol extracts for 10 days.The bacterial colonies were enumerated and hematological and biochemical parameters were determined.Serum pro-inflammatory mediators including IL-1β,IL-18,and TNF-α,and nitric oxide levels were quantified by ELISA.Histological analyses of the kidney,liver,and colon were also conducted.Results:Treatment with 200 and 400 mg/kg of the hydroethanolic extracts markedly inhibited the growth of S.flexneri.Moreover,treatment with D.gilletii extract downregulated the levels of IL-1β,IL-18,and TNF-α,and restored hematological and biochemical parameters as well as histological architecture of the colon,liver,and kidneys.Additionally,the oral administration of 2000 mg/kg D.gilletii extract did not induce any sign of toxicity,with a median lethal dose greater than 2000 mg/kg.Conclusions:D.gilletii extract demonstrates the anti-shigellosis effects in S.flexneri-induced diarrheal mice,supporting the traditional use of this plant in treating diarrhea.展开更多
AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were sepa...AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were separated by two-dimensional gel electrophoresis (2-DE). Proteins were transferred to PVDF membrane and immunoblotted with sera from shigellosis patients. Reactive protein spots were matched to Coomassie stained gels run in parallel, cut out and trypsin digested. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was used to determine the peptide mass fingerprints, which were searched in the MASCOT database to identify the protein. RESULTS: A total of 8 immunoreactive proteins were successfully identified from the Coomassie stained gels in three repeats. Six of these proteins have not previously been reported as immunogenic in S. flexneri. These proteins could be potential candidates for vaccine or attenuation studies. CONCLUSION: Soluble and membrane proteins of S. flexneri 2457T have been screened by 2-DE and immunoblotting with sera from shigellosis patients. Eight proteins are identified as immunogenic.展开更多
AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogeni...AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogenic proteins of S. Aexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry （MALDI-TOF-MS） was performed to determine the molecular weight of peptides. Electrospray ionization （ESI-MS/MS） was performed to determine the sequences of the interesting peptides. RESULTS： A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein （YaeT） was detected, which might be a candidate target of vaccine. CONCLUSION： Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.展开更多
AIM To investigate the specific pathogenesis ofO-polysaccharide (O--PS) which is on the outermembrane of lipopolysaccharides (LPS) fromShigella fi^eri.METHODS The O--PS was isolated and purifiedfrom Shigella nexneri 5...AIM To investigate the specific pathogenesis ofO-polysaccharide (O--PS) which is on the outermembrane of lipopolysaccharides (LPS) fromShigella fi^eri.METHODS The O--PS was isolated and purifiedfrom Shigella nexneri 5 MgoT by enzymatichydrolysis and gel chromatography. Effects ofO--PS were observed by in vitro experiment,(HeLa cell Culture ), and in vivo experiment(rabbit lieal loop assay).RESULTS ID vitro and in vivo e-cP6riments withthe purified O--PS from Shigells flexnefi revealedthat the O--PS alone was toxic to Hela cells andcaused mucosal inflammation and hemorrhagicexudation in lieal loop of rabbit.DISCUSSION O--PS might b6 one of the factorscausing diarrhea and its mechanism wasdifferent from endotoxin reaction of LPS. Themolecular mechanism of O-PS need furtherstudies.展开更多
BACKGROUND Shigella flexneri(S.flexneri)is a major pathogen causing acute intestinal infection,but the systematic oxidative damage incurred during the course of infection has not been investigated.AIM To investigate t...BACKGROUND Shigella flexneri(S.flexneri)is a major pathogen causing acute intestinal infection,but the systematic oxidative damage incurred during the course of infection has not been investigated.AIM To investigate the incurred systemic RNA oxidative damage and the diagnostic value of RNA oxidative metabolites during S.flexneri-induced intestinal infection.METHODS In this study,a Sprague-Dawley rat model of acute intestinal infection was established by oral gavage with S.flexneri strains.The changes in white blood cells(WBCs)and cytokine levels in blood and the inflammatory response in the colon were investigated.We also detected the RNA and DNA oxidation in urine and tissues.RESULTS S.flexneri infection induced an increase in WBCs,C-reactive protein,interleukin(IL)-6,IL-10,IL-1β,IL-4,IL-17a,IL-10,and tumor necrosis factorα(TNF-α)in blood.Of note,a significant increase in urinary 8-oxo-7,8-dihydroguanosine(8-oxo-Gsn),an important marker of total RNA oxidation,was detected after intestinal infection(P=0.03).The urinary 8-oxo-Gsn level returned to the baseline level after recovery from infection.In addition,the results of a correlation analysis showed that urinary 8-oxo-Gsn was positively correlated with the WBC count and the cytokines IL-6,TNF-α,IL-10,IL-1β,and IL-17α.Further detection of the oxidation in different tissues showed that S.flexneri infection induced RNA oxidative damage in the colon,ileum,liver,spleen,and brain.CONCLUSION Acute infection induced by S.flexneri causes increased RNA oxidative damage in various tissues(liver,spleen,and brain)and an increase of 8-oxo-Gsn,a urinary metabolite.Urinary 8-oxo-Gsn may be useful as a biomarker for evaluating the severity and prognosis of infection.展开更多
Aim: Shigella flexneri (S. flexneri) is a gram-negative enterobacterium responsible for severe intestinal end systemic infection in humans. The bacteria can reach the liver due to degeneration of the colonic epitheliu...Aim: Shigella flexneri (S. flexneri) is a gram-negative enterobacterium responsible for severe intestinal end systemic infection in humans. The bacteria can reach the liver due to degeneration of the colonic epithelium. Hypoxia is present in many human diseases and can induce the expression of the transcription factor HIF-1alpha that may have a cell protective role. The influence of hypoxia and HIF-1alpha on bacterial infection, studied in this work, is unclear. Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor that acts as a master regulator of gene expression induced by hypoxia. Methods: We compared the ability of S. flexneri to invade rat hepatocytes in primary culture both in normoxic and hypoxic conditions. We evaluated TNF-alpha released by hepatocytes, apoptosis rate and HIF-1alpha expression by confocal microscopy as well as real time PCR technique. Results: We showed that S. flexneri invaded less hepatocytes previously submitted to 24 h hypoxia (6.5% O2) than those cultivated in normoxia (21% O2). S. flexneri also induced HIF-1α expression in hepatocytes, TNF-α secretion and apoptosis. Conclusion: a) Hypoxia alone was not a stimulus to TNF-α secretion, but induced cell apoptosis and HIF-1α expression;b) S. flexneri was able to invade rat hepatocytes and hypoxia apparently influenced significantly bacterial cell invasiveness;c) HIF-1α was expressed in hypoxic conditions, and it was also stimulated by S. flexneri.展开更多
Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger...Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.展开更多
This study investigated antibacterial and antibiofilm activity of the combined use of phenyllactic acid(PLA)and bacteriocin XJS01 against Shigella flexneri_14.The minimum inhibitory concentration(MIC)of PLA and XJS01 ...This study investigated antibacterial and antibiofilm activity of the combined use of phenyllactic acid(PLA)and bacteriocin XJS01 against Shigella flexneri_14.The minimum inhibitory concentration(MIC)of PLA and XJS01 against S.flexneri_14 was 2.45 mg/mL and 18.75μg/mL,respectively.Growth and kill kinetics assays showed that the combined use of 1/2MIC PLA plus 1/2MIC XJS01 had a better activity against planktonic S.flexneri_14 compared to treatment with PLA and XJS01 used singly(1/2MIC and 2MIC).Cellular biochemical and morphological analysis revealed the remarkable ability of the combination in disrupting cell appearance and promoting deformation of planktonic S.flexneri_14 compared to single use.Moreover,S.flexneri_14 biofilm formation was inhibited and degraded by the combination,which showed a more remarkable antibiofilm activity than PLA and XJS01 when used singly.This study demonstrates the synergistic antibacterial activity of PLA and XJS01 against S.flexneri_14 in either planktonic or biofilm states in foods.展开更多
In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442,Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study.With ...In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442,Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study.With this improved knockout system,we inactivated sitC gene,which is associated with iron transport in Shigella flexneri 2a strain 301,to yield the mutant,MTS.The functional detection of the mutant was performed at the level of culture medium,cell and animal experiment,respectively.The gene expression profiles were compared with DNA microarray between the mutant and the wild type under iron-restricted conditions.The results showed that MTS grew obviously less well than the wild-type strains in L broth containing 150μmol/L iron chelator DIP(2,2′-dipyridyl).Addition of iron or manganese to the cultures stimulated the growth of MTS to wild-type levels in rich culture medium.In either the experiment on the ability of intracellular multiplication and cell-to-cell spread in HeLa and U937 cell lines,or the experiment on keratoconjunctivitis in guinea pigs,MTS showed no obvious changes in virulence compared with the parental strain Sf301.When 65μmol/L DIP was added to the cultured HeLa cells,the ability of intracellular multiplication of MTS reduced about 51.6%as compared with that of Sf301.The analysis of expression profiles under iron-limited condition showed that MTS was more sensitive for the change of iron deficiency than Sf301.There are 106 more up-regulated genes in MTS than in wild-type strains,which are involved in membrane transportation,amino acid metabolism and uncategorized function genes,while down-regulated genes are mainly in-volved in energy and carbohydrate metabolism.Under low iron conditions,the expression levels of known iron-transport associated genes generally increased.Additionally,the number of these genes and their increase amplitude in MTS are more than those in Sf301.Together,these results confirmed that Sit iron-transport system is important for the growth of Shigella.展开更多
Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns ...Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. Interestingly, a putative outer membrane protein YciD （OmpW） was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced （such as AnsB, AspA, FrdAB, and KatG） or repressed （such as AceEF, OmpX, SodA, and SucAB） during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.展开更多
An in vivo expression technology(IVET)was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells,and virulence-related genes were further identified by mutational analysis.Thirteen intracellu...An in vivo expression technology(IVET)was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells,and virulence-related genes were further identified by mutational analysis.Thirteen intracellular induced genes were identified with a HeLa cell infection model.Of these,two were identified as alkylation-related genes;one was related to metabolism;one encoded a transcriptional regulator;three were identified as insertion elements;three ap-peared to be antisense to genes involved in the transmethylation,biosyntheseis,and phos-photransferase system;and three were predicted to encode polypeptides with unknown functions.Intracellular survival assavs showed that the mutants of alkA,citC and wcaJ genes had lower capability of intracellular replication or survival than the the wild-type strain.The results indicated that alkA,citC and wcaJ genes could take part in the intracellular survival or replication of S.flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements.However,the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay.Very possibly,yaiC gene was involved in the other mechanism of S.flexneri virulence.This study might lead to a better understanding of the intra-cellular survival or proliferation process of S.flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.展开更多
基金funded by the Yaounde-Bielefeld Bilateral Graduate School for Natural Products with Anti-parasite and Antibacterial Activity(YaBiNaPA)(grant number:57316173).
文摘Objective:To evaluate the anti-shigellosis activity of the hydroethanol extract of Diospyros gilletii(D.gilletii)stem bark in Shigella flexneri(S.flexneri)-induced diarrheal mice.Methods:The hydroethanolic extract was obtained by maceration of D.gilletii stem bark in 70%hydroethanol(water꞉ethanol;30꞉70,v/v)solution.Then,mice pretreated with cyclophosphamide for immunosuppression were administered orally with an inoculum containing S.flexneri,and subsequently treated with 100,200,and 400 mg/kg of the hydroethanol extracts for 10 days.The bacterial colonies were enumerated and hematological and biochemical parameters were determined.Serum pro-inflammatory mediators including IL-1β,IL-18,and TNF-α,and nitric oxide levels were quantified by ELISA.Histological analyses of the kidney,liver,and colon were also conducted.Results:Treatment with 200 and 400 mg/kg of the hydroethanolic extracts markedly inhibited the growth of S.flexneri.Moreover,treatment with D.gilletii extract downregulated the levels of IL-1β,IL-18,and TNF-α,and restored hematological and biochemical parameters as well as histological architecture of the colon,liver,and kidneys.Additionally,the oral administration of 2000 mg/kg D.gilletii extract did not induce any sign of toxicity,with a median lethal dose greater than 2000 mg/kg.Conclusions:D.gilletii extract demonstrates the anti-shigellosis effects in S.flexneri-induced diarrheal mice,supporting the traditional use of this plant in treating diarrhea.
文摘AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were separated by two-dimensional gel electrophoresis (2-DE). Proteins were transferred to PVDF membrane and immunoblotted with sera from shigellosis patients. Reactive protein spots were matched to Coomassie stained gels run in parallel, cut out and trypsin digested. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was used to determine the peptide mass fingerprints, which were searched in the MASCOT database to identify the protein. RESULTS: A total of 8 immunoreactive proteins were successfully identified from the Coomassie stained gels in three repeats. Six of these proteins have not previously been reported as immunogenic in S. flexneri. These proteins could be potential candidates for vaccine or attenuation studies. CONCLUSION: Soluble and membrane proteins of S. flexneri 2457T have been screened by 2-DE and immunoblotting with sera from shigellosis patients. Eight proteins are identified as immunogenic.
基金Supported by the Capital "248" Key Innovation Project, No. H010210360119, State Basic Research Development Program of China No. 973 Program, G1999054103 and 2005CB22904 and National Natural Science Foundation of China No. 30470101
文摘AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogenic proteins of S. Aexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry （MALDI-TOF-MS） was performed to determine the molecular weight of peptides. Electrospray ionization （ESI-MS/MS） was performed to determine the sequences of the interesting peptides. RESULTS： A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein （YaeT） was detected, which might be a candidate target of vaccine. CONCLUSION： Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.
基金Project supported by the National Natural Science Foundation of China,No.39370040.
文摘AIM To investigate the specific pathogenesis ofO-polysaccharide (O--PS) which is on the outermembrane of lipopolysaccharides (LPS) fromShigella fi^eri.METHODS The O--PS was isolated and purifiedfrom Shigella nexneri 5 MgoT by enzymatichydrolysis and gel chromatography. Effects ofO--PS were observed by in vitro experiment,(HeLa cell Culture ), and in vivo experiment(rabbit lieal loop assay).RESULTS ID vitro and in vivo e-cP6riments withthe purified O--PS from Shigells flexnefi revealedthat the O--PS alone was toxic to Hela cells andcaused mucosal inflammation and hemorrhagicexudation in lieal loop of rabbit.DISCUSSION O--PS might b6 one of the factorscausing diarrhea and its mechanism wasdifferent from endotoxin reaction of LPS. Themolecular mechanism of O-PS need furtherstudies.
基金Supported by the National Key R&D Program of China,No.2018YFC2000300and CAMS Innovation Fund for Medical Sciences,No.2018-I2M-1-002.
文摘BACKGROUND Shigella flexneri(S.flexneri)is a major pathogen causing acute intestinal infection,but the systematic oxidative damage incurred during the course of infection has not been investigated.AIM To investigate the incurred systemic RNA oxidative damage and the diagnostic value of RNA oxidative metabolites during S.flexneri-induced intestinal infection.METHODS In this study,a Sprague-Dawley rat model of acute intestinal infection was established by oral gavage with S.flexneri strains.The changes in white blood cells(WBCs)and cytokine levels in blood and the inflammatory response in the colon were investigated.We also detected the RNA and DNA oxidation in urine and tissues.RESULTS S.flexneri infection induced an increase in WBCs,C-reactive protein,interleukin(IL)-6,IL-10,IL-1β,IL-4,IL-17a,IL-10,and tumor necrosis factorα(TNF-α)in blood.Of note,a significant increase in urinary 8-oxo-7,8-dihydroguanosine(8-oxo-Gsn),an important marker of total RNA oxidation,was detected after intestinal infection(P=0.03).The urinary 8-oxo-Gsn level returned to the baseline level after recovery from infection.In addition,the results of a correlation analysis showed that urinary 8-oxo-Gsn was positively correlated with the WBC count and the cytokines IL-6,TNF-α,IL-10,IL-1β,and IL-17α.Further detection of the oxidation in different tissues showed that S.flexneri infection induced RNA oxidative damage in the colon,ileum,liver,spleen,and brain.CONCLUSION Acute infection induced by S.flexneri causes increased RNA oxidative damage in various tissues(liver,spleen,and brain)and an increase of 8-oxo-Gsn,a urinary metabolite.Urinary 8-oxo-Gsn may be useful as a biomarker for evaluating the severity and prognosis of infection.
文摘Aim: Shigella flexneri (S. flexneri) is a gram-negative enterobacterium responsible for severe intestinal end systemic infection in humans. The bacteria can reach the liver due to degeneration of the colonic epithelium. Hypoxia is present in many human diseases and can induce the expression of the transcription factor HIF-1alpha that may have a cell protective role. The influence of hypoxia and HIF-1alpha on bacterial infection, studied in this work, is unclear. Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor that acts as a master regulator of gene expression induced by hypoxia. Methods: We compared the ability of S. flexneri to invade rat hepatocytes in primary culture both in normoxic and hypoxic conditions. We evaluated TNF-alpha released by hepatocytes, apoptosis rate and HIF-1alpha expression by confocal microscopy as well as real time PCR technique. Results: We showed that S. flexneri invaded less hepatocytes previously submitted to 24 h hypoxia (6.5% O2) than those cultivated in normoxia (21% O2). S. flexneri also induced HIF-1α expression in hepatocytes, TNF-α secretion and apoptosis. Conclusion: a) Hypoxia alone was not a stimulus to TNF-α secretion, but induced cell apoptosis and HIF-1α expression;b) S. flexneri was able to invade rat hepatocytes and hypoxia apparently influenced significantly bacterial cell invasiveness;c) HIF-1α was expressed in hypoxic conditions, and it was also stimulated by S. flexneri.
基金supported by the State“973”Key Basic Research Program(Grant No.G1999054103)the State“863”High-Tech Project(Grant No.Z19-02-05-01)+1 种基金Beijing Innovation Engineering(Grant No.955020700)the North China Pharmaceutical Corporation(NCPC)
文摘Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.
基金supported by Yunnan Fundamental Research Projects(Grant No.202101BE070001-046)the Natural Science Foundation of China(31960286).
文摘This study investigated antibacterial and antibiofilm activity of the combined use of phenyllactic acid(PLA)and bacteriocin XJS01 against Shigella flexneri_14.The minimum inhibitory concentration(MIC)of PLA and XJS01 against S.flexneri_14 was 2.45 mg/mL and 18.75μg/mL,respectively.Growth and kill kinetics assays showed that the combined use of 1/2MIC PLA plus 1/2MIC XJS01 had a better activity against planktonic S.flexneri_14 compared to treatment with PLA and XJS01 used singly(1/2MIC and 2MIC).Cellular biochemical and morphological analysis revealed the remarkable ability of the combination in disrupting cell appearance and promoting deformation of planktonic S.flexneri_14 compared to single use.Moreover,S.flexneri_14 biofilm formation was inhibited and degraded by the combination,which showed a more remarkable antibiofilm activity than PLA and XJS01 when used singly.This study demonstrates the synergistic antibacterial activity of PLA and XJS01 against S.flexneri_14 in either planktonic or biofilm states in foods.
基金This work was supported by the High Technology Project(Grant No.2001AA223011)the State Key Basic Research Program(Grant No.G1999054105).
文摘In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442,Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study.With this improved knockout system,we inactivated sitC gene,which is associated with iron transport in Shigella flexneri 2a strain 301,to yield the mutant,MTS.The functional detection of the mutant was performed at the level of culture medium,cell and animal experiment,respectively.The gene expression profiles were compared with DNA microarray between the mutant and the wild type under iron-restricted conditions.The results showed that MTS grew obviously less well than the wild-type strains in L broth containing 150μmol/L iron chelator DIP(2,2′-dipyridyl).Addition of iron or manganese to the cultures stimulated the growth of MTS to wild-type levels in rich culture medium.In either the experiment on the ability of intracellular multiplication and cell-to-cell spread in HeLa and U937 cell lines,or the experiment on keratoconjunctivitis in guinea pigs,MTS showed no obvious changes in virulence compared with the parental strain Sf301.When 65μmol/L DIP was added to the cultured HeLa cells,the ability of intracellular multiplication of MTS reduced about 51.6%as compared with that of Sf301.The analysis of expression profiles under iron-limited condition showed that MTS was more sensitive for the change of iron deficiency than Sf301.There are 106 more up-regulated genes in MTS than in wild-type strains,which are involved in membrane transportation,amino acid metabolism and uncategorized function genes,while down-regulated genes are mainly in-volved in energy and carbohydrate metabolism.Under low iron conditions,the expression levels of known iron-transport associated genes generally increased.Additionally,the number of these genes and their increase amplitude in MTS are more than those in Sf301.Together,these results confirmed that Sit iron-transport system is important for the growth of Shigella.
基金This work was supported by the National Key Basic Research Program of China （973 Program, No. 2005CB522904） the National Natural Science Foundation of China （No. 30470101）.
文摘Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. Interestingly, a putative outer membrane protein YciD （OmpW） was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced （such as AnsB, AspA, FrdAB, and KatG） or repressed （such as AceEF, OmpX, SodA, and SucAB） during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.
基金supported by the National Key Basic Research Project(G1999054103)National High-technology Development Project(2001AA215211).
文摘An in vivo expression technology(IVET)was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells,and virulence-related genes were further identified by mutational analysis.Thirteen intracellular induced genes were identified with a HeLa cell infection model.Of these,two were identified as alkylation-related genes;one was related to metabolism;one encoded a transcriptional regulator;three were identified as insertion elements;three ap-peared to be antisense to genes involved in the transmethylation,biosyntheseis,and phos-photransferase system;and three were predicted to encode polypeptides with unknown functions.Intracellular survival assavs showed that the mutants of alkA,citC and wcaJ genes had lower capability of intracellular replication or survival than the the wild-type strain.The results indicated that alkA,citC and wcaJ genes could take part in the intracellular survival or replication of S.flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements.However,the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay.Very possibly,yaiC gene was involved in the other mechanism of S.flexneri virulence.This study might lead to a better understanding of the intra-cellular survival or proliferation process of S.flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.
