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Hongqing Zhao,
Chen Chen,
Yanwen Xiong,
Xuefang Xu,
Ruiting Lan,
Haiyin Wang,
Xinyue Yao,
Xiangning Bai,
Xuetong Liu,
Qiong Meng,
Xiaoai Zhang,
Hui Sun,
Ailan Zhao,
Xuemei Bai,
Yuli Cheng,
Qiang Chen,
Changyun Ye,
Jianguo Xu
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ABSTRACT: Escherichia coli O157:H7 is an important food-borne pathogen that can cause hemorrhagic colitis and hemolytic-uremic syndrome in humans. pO157_Sal, a novel conjugative plasmid is present in a Chinese O157:H7 outbreak strain Xuzhou21. Here we investigated the phenotypic and transcriptional differences between the wild type strain Xuzhou21 and the pO157_Sal cured mutant strain Xuzhou21m. RNA-Seq analysis found that all 52 ORFs encoded on pO157_Sal were transcribed. One hundred and sixty eight chromosomal and pO157 genes were differentially expressed (≥2 fold difference) between Xuzhou21 and Xuzhou21m. Sixty-seven and 101 genes were up-regulated and down-regulated respectively by pO157_Sal including genes related to stress response, adaption and virulence. The plasmid-cured mutant Xuzhou21m grew slower than wild type Xuzhou21 and pO157_Sal plasmid complemented strain Xuzhou21c in M9 medium under the condition of high NaCl or presence of sodium deoxycholate (NaDC), corroborating with the RNA-Seq data. Seven differentially expressed genes are associated with NaDC resistance, including the adenine-specific DNA-methyltransferase gene (dam), multidrug efflux system subunit gene mdtA, hyperosmotically inducible periplasmic protein gene osmY and oxidation-reduction related genes while two differentially expressed genes (osmY and pspD) are likely to be related to resistance to osmotic pressure. A number of differentially expressed genes were virulence associated including four genes encoding T3SS effectors from the chromosome and ehxD from pO157. Through complementation of Xuzhou21m with a plasmid construct carrying the pO157_Sal hha homolog we further showed that the pO157_Sal hha represses the expression of T3SS effectors. These findings demonstrated that the plasmid pO157_Sal affects the transcription of the chromosomal and pO157 plasmid genes and contributes to the enhanced ability to resist stress. We conclude that pO157_Sal plays an important role in regulating global gene expression and affects the virulence and adaptation of E. coli O157:H7.
PLoS ONE 01/2013; 8(5):e65466. · 4.09 Impact Factor
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ABSTRACT: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major foodborne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome. The role of EHEC O157:H7-enterohemolysin (Ehx) in the pathogenesis of infections remains poorly defined. In this study, we used gene deletion and complement methods to confirm its putative functions. Results demonstrated that, in THP-1 cells, EHEC O157:H7-Ehx is associated with greater production of extracellular interleukin (IL)-1β than other cytokines. The data also showed that EHEC O157:H7-Ehx contributed to cytotoxicity in THP-1 cells, causing the release of lactate dehydrogenase (LDH). Although we observed a positive correlation between IL-1β production and cytotoxicity in THP-1 cells infected with different EHEC O157:H7 strains, our immunoblot results showed that the majority of IL-1β in the supernatant was mature IL-1β and not the pro-IL-1β that can be released after cell death. However, EHEC O157:H7-Ehx had no detectable effect on biologically inactive pro-IL-1β at the mRNA or protein synthesis levels. Neither did it affect the expression of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, or NOD-like receptor family pyrin domain containing 3 (NLRP3). RNA interference experiments showed that EHEC O157:H7-induced IL-1β production required the involvement of ASC, caspase-1, and NLRP3 expression in THP-1 cells. Our results demonstrate that Ehx plays a crucial role in EHEC O157:H7-induced IL-1β production and its cytotoxicity to THP-1 cells. NLRP3 inflammasome activation is also involved in EHEC O157:H7-stimulated IL-1β release.
PLoS ONE 01/2012; 7(11):e50288. · 4.09 Impact Factor
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Qiangzheng Sun,
Ruiting Lan,
Yiting Wang,
Ailan Zhao,
Shaomin Zhang,
Jianping Wang,
Yan Wang,
Shengli Xia,
Dong Jin,
Zhigang Cui, Hongqing Zhao,
Zhenjun Li,
Changyun Ye,
Shuxia Zhang,
Huaiqi Jing,
Jianguo Xu
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ABSTRACT: Shigella flexneri is the major Shigella species that causes diarrheal disease in developing countries. It is further subdivided into 15 serotypes based on O-antigen structure. Serotyping of S. flexneri is important for epidemiological purposes. In this study, we developed a multiplex PCR assay targeting the O-antigen synthesis gene wzx and the O-antigen modification genes gtrI, gtrIC, gtrII, oac, gtrIV, gtrV, and gtrX for molecular serotyping of S. flexneri. The multiplex PCR assay contained eight sets of specific PCRs in a single tube and can identify 14 of the 15 serotypes (the exception being serotype Xv) of S. flexneri recognized thus far. A nearly perfect concordance (97.8%) between multiplex PCR assay and slide agglutination was observed when 358 S. flexneri strains of various serotypes were analyzed, except that 8 strains were carrying additional cryptic and/or defective serotype-specific genes. The multiplex PCR assay provides a rapid and specific method for the serotype identification of S. flexneri.
Journal of clinical microbiology 08/2011; 49(11):3766-70. · 4.16 Impact Factor
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Ping Wang,
Yanwen Xiong,
Ruiting Lan,
Changyun Ye,
Hua Wang,
Jun Ren,
Huaiqi Jing,
Yiting Wang,
Zhemin Zhou,
Zhigang Cui, [......],
Dong Jin,
Xuemei Bai,
Ailan Zhao,
Yan Wang,
Shaomin Zhang,
Hui Sun,
Juan Li,
Tao Wang,
Lei Wang,
Jianguo Xu
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ABSTRACT: In addition to the large virulence plasmid pO157, a novel 38-kb conjugative plasmid, pO157_Sal, was identified and sequenced from an Escherichia coli O157:H7 outbreak-associated Chinese isolate that shares high similarity with a plasmid in Salmonella enterica serovar Agona. The plasmid was found in 15 of 326 isolates, 12 of which were of the same pulsed-field gel electrophoresis type.
Journal of clinical microbiology 02/2011; 49(4):1594-7. · 4.16 Impact Factor
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ABSTRACT: Based on a proteomic reference map of the important probiotic organism Bifidobacteria longum NCC2705 constructed by our previous research, we compared the proteomic profiles of Bifidobacteria longum strain NCC2705 grown on lactose or glucose to identify the catabolic route allowing lactose fermentation.
We considered the proteins differentially expressed if their relative volume deviated more than 3-fold with ImageMaster 2D Elite version 5.0 software. Interesting spots were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, and phosphorylation analysis of proteins with mobility changes by Pro-Q Diamond Stain.
The identified spots represent 31 protein entries, 14 up-regulated proteins, 17 down-regulated proteins. These identified proteins, which were hydrophilic proteins and their genes with CAI value above 0.5 represented the most abundant proteins, included key stress proteins, metabolism-related proteins, and proteins related to translation. Two proteins including Tal (BL0715, transaldolase, L3) and Pyk (BL0988, pyruvate kinase, G9) exhibited clear post-translational modification.
Proteomic comparison of glucose- and lactose-grown cells revealed that lactose and glucose were catabolized via the same degradation pathway, and the rate of glucose assimilation was higher than that of lactose. Spot and protein analysis revealed that post-translational modifications might be common in these proteins. Pro-Q Diamond staining analysis revealed that lactose trigger Tal phosphorylation at 43 T /47 S, and inhibited Pyk phosphorylation at 65 S. These proteins were identified for the first time as bifidobacterial phosphoproteins.
ACTA MICROBIOLOGICA SINICA 12/2008; 48(11):1451-8.
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ABSTRACT: To demonstrate the fructose metabolism pathway in Bifidobacterium Longum NCC2705 and to construct its fermentation model, we explored the comparative proteome cultivating the strain on glucose or fructose, based on a proteomic reference map of B. longum NCC2705 constructed earlier. Then, we used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and electro-spray ionization tandem mass spectrometry (ESI-MS/MS) for differently expressed proteins identification. Furthermore, with semi-quantitative RT-PCR we determined the distinctively expressed proteins at the level of transcription. Proteomic comparison of glucose- and fructose-grown cells demonstrated much similarity. On the page of fructose there were all the enzymes and proteins that exist during the process of glucose degradation. We observed a greater variation of more than three-fold for the identified 9 spots representing 5 protein entries by MALDI-TOF MS. The sugar-binding protein specific to fructose (BL0033) and an ABC transporter ATP binding protein (BL0034) showed higher expression level from cells grown on fructose. It was also determined by semi-quantitative RT-PCR subsequently. BL0033 time course and concentration experiments showed that the induction time correlated to higher fructose concentration, and increased expression of BL0033. Fructose was catabolized via the same degradation pathway as glucose at the level of proteomics. BL0033 was induced by fructose. All results suggest that the uptake of fructose into the cell may be conducted by a specific ABC transport system, in which BL0033 and BL0034 as components might have played an important role.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 09/2008; 24(8):1401-6.
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Jing Yuan,
Bin Wang,
Zhongke Sun,
Xin Bo,
Xitong Yuan,
Xiang He, Hongqing Zhao,
Xinying Du,
Fang Wang,
Zheng Jiang,
Ling Zhang,
Leili Jia,
Yufei Wang,
Kaihua Wei,
Jie Wang,
Xuemin Zhang,
Yansong Sun,
Liuyu Huang,
Ming Zeng
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ABSTRACT: To investigate the molecular mechanisms underlying the adaptation of Bifidobacterium longum to the intestinal tract, we utilized a new model for rabbit intestinal culture of B. longum and reported the changes in proteomic profiles after incubation in the in vivo environment. By 2D-PAGE coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses, proteomic profiles of B. longum strain NCC2705 grown in the in vivo and in vitro environments were compared. Confirmed by semiquantitative RT-PCR, which exhibited at least a 3-fold change or greater, 19 up-regulated proteins, 14 down-regulated proteins, and 4 proteins with mobility changes were identified during intestinal growth. These identified proteins include key stress proteins, metabolism-related proteins, and proteins related to translation. Our results indicate that some useful proteins are expressed at higher levels in cells during intestinal growth. These proteins reflected the adaptation of B. longum NCC2705 to the intestine, such as EF-Tu which contributes to the retention or attachment as a Bifidobacterium adhesin-like factor, bile salt hydrolase (BSH) which might play an important role in the molecular mechanisms for the initial interaction of probiotic with the intestinal environment, and stress proteins which defend B. longum against the action of bile salts and other harmful ingredients of the gastrointestinal tract (GIT). The most striking fact of our observation was that four proteins GlnA1, PurC, LuxS, and Pgk exhibit clear post-translational modification. Western blot (WB) analysis and Pro-Q Diamond staining revealed that substances of the GIT trigger Pgk and LuxS phosphorylation at Ser/Thr residues for bacteria grown in vivo. These proteins were identified for the first time as bifidobacterial phosphoproteins. Our data suggest that the phosphorylated autoinducer-2 production protein LuxS of B. longum NCC2705 (LuxS-P) is the active form of LuxS and that LuxS-P may play a key role in the regulation of quorum sensing.
Journal of Proteome Research 02/2008; 7(1):375-85. · 5.11 Impact Factor
