Secreted proteins of Yersinia pestis 201
Ontology highlight
ABSTRACT:
ORGANISM(S): Yersinia Pestis Co92
SUBMITTER: 
Ruifu Yang
PROVIDER: PXD020062 | iProX | Wed Dec 16 00:00:00 GMT 2020
REPOSITORIES: iProX
Publications
Secretome and Comparative Proteomics of Yersinia pestis Identify Two Novel E3 Ubiquitin Ligases That Contribute to Plague Virulence.
Cao Shiyang S Chen Yuling Y Yan Yanfeng Y Zhu Songbiao S Tan Yafang Y Wang Tong T Song Yajun Y Deng Haiteng H Yang Ruifu R Du Zongmin Z
Molecular & cellular proteomics : MCP 20210222
Plague is a zoonotic disease that primarily infects rodents via fleabite. Transmission from flea to host niches requires rapid adaption of Yersinia pestis to the outer environments to establish infection. Here, quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking the two typical niches, i.e., the mammalian host (Mh) and the flea vector (Fv), were performed to understand the adaption strategies of this deadly pathogen. A secretome of Y. pestis containing 308 ...[more]
Similar Datasets
Project description:Y. pestis strain 201 and mutant ΔyscI cultures grown under Mh (37°C, TMH with no calcium) condition were used as the experimental and control groups, respectively. After 8 h of incubation, the cultures supernatants were collected by centfugation and concentrated using centrifugal filters. The concentrations of proteins were determined using a Pierce BCA Protein Assay Kit. Tandem mass tag labeling was used to obtain accurate quantitative information to minimize variability between samples. For quantitative analysis, at least two unique peptides were used for the identification and quantification of proteins.
2020-12-16 | PXD020060 | iProX
Project description:The secreted proteome and bacteria proteome of yersinia pestis at 37℃ mimic human. Y. pestis strain 201 cultures grown under Mh (37°C, TMH with no calcium) condition were used as the experimental and control groups, respectively. After 8 h of incubation, the cultures supernatants were collected by centfugation and concentrated using centrifugal filters. The concentrations of proteins were determined using a Pierce BCA Protein Assay Kit. Tandem mass tag labeling was used to obtain accurate quantitative information to minimize variability between samples. For quantitative analysis, at least two unique peptides were used for the identification and quantification of proteins.
2020-12-16 | PXD023183 | iProX
Project description:Y. pestis strain 201 cultures grown under Mh (37°C, TMH with no calcium) and Fv (26°C, TMH with 2.5M calcium) conditions were used as the experimental and control groups, respectively. After 8 h of incubation, the bacterial pellets were collected by centfugation, re-suspended in lysis buffer (8 M urea in PBS, pH 7.4) and lysed by sonication in an ice bath. The concentrations of proteins were determined using a Pierce BCA Protein Assay Kit. Tandem mass tag labeling was used to obtain accurate quantitative information to minimize variability between samples. For quantitative analysis, at least two unique peptides were used for the identification and quantification of proteins.
2020-12-16 | PXD020063 | iProX
Project description:The secreted proteome and bacteria proteome of yersinia pestis at 26 ℃ mimic flea. Y. pestis strain 201 cultures grown under Fv (26°C, TMH with 2.5M calcium) condition. The bacteria pellets and supernant were used as the experimental and control groups, respectively. After 8 h of incubation, the cultures supernatants were collected by centfugation and concentrated using centrifugal filters. The concentrations of proteins were determined using a Pierce BCA Protein Assay Kit. Tandem mass tag labeling was used to obtain accurate quantitative information to minimize variability between samples. For quantitative analysis, at least two unique peptides were used for the identification and quantification of proteins.
2020-12-16 | PXD023157 | iProX
Project description:Y. pestis strain 201 and mutant ΔyscI cultures grown under Mh (37°C, TMH with no calcium) condition were used as the experimental and control groups, respectively. After 8 h of incubation, the bacterial pellets were collected by centfugation, re-suspended in lysis buffer (8 M urea in PBS, pH 7.4) and lysed by sonication in an ice bath. The concentrations of proteins were determined using a Pierce BCA Protein Assay Kit. Tandem mass tag labeling was used to obtain accurate quantitative information to minimize variability between samples. For quantitative analysis, at least two unique peptides were used for the identification and quantification of proteins.
2020-12-16 | PXD023156 | iProX
Project description:The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis under zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2. The wild-type (WT) Y. pestis strain 201 belongs to a newly established Y. pestis biovar, Microtus, which was thought to be avirulent to humans, but highly virulent to mice. An in-frame deletion of the zur gene was constructed by using one step inactivation method based on the lambda phage recombination in which PCR primers provide the homology to the target gene, as described previously by Datsenko and Wanner. The entire coding region of zur was replaced by a kanamycin resistance (KnR) cassette, which was verified by PCR and DNA sequencing. The resulting mutant strain was referred to as Îzur. Both the WT strain and the Zur mutant were pre-cultivated at 26 ºC to the middle exponential growth phase (OD620 about 1.0) in TMH medium. The cell cultures were then diluted 1:20 in fresh TMH medium and grown at 26°C until an OD620 of about 1.0. Finally, 5mM ZnCl2 was added into each cell culture to ensure zinc rich conditions. Growth was continued for 30 min at 26°C before harvested for total RNA isolation. Gene expression profiles were compared between WT and Îzur. RNA samples were isolated from four individual bacterial cultures, as biological replicates, for each strain. The dual-fluorescently (Cy3 or Cy5 dye) labeled cDNA probes, for which the incorporated dye was reversed, were synthesized from the RNA samples, and then hybridized to four separated microarray slides, respectively.
2010-05-18 | E-GEOD-15183 | biostudies-arrayexpress
Project description:Background The osmotic regulator OmpR in Escherichia coli regulates differentially the expression of major porin proteins OmpF and OmpC. In Yersinia enterocolitica and Y. pseudotuberculosis, OmpR is required for both virulence and survival within macrophages. However, the phenotypic and regulatory roles of OmpR in Y. pestis are not yet fully understood. Results Y. pestis OmpR is involved in building resistance against phagocytosis and controls the adaptation to various stressful conditions met in macrophages. The ompR mutation likely did not affect the virulence of Y. pestis strain 201 that was a human-avirulent enzootic strain. The microarray-based comparative transcriptome analysis disclosed a set of 224 genes whose expressions were affected by the ompR mutation, indicating the global regulatory role of OmpR in Y. pestis. Real-time RT-PCR or lacZ fusion reporter assay further validated 16 OmpR-dependent genes, for which OmpR consensus-like sequences were found within their upstream DNA regions. ompC, F, X, and R were up-regulated dramatically with the increase of medium osmolarity, which was mediated by OmpR occupying the target promoter regions in a tandem manner. Conclusion OmpR contributes to the resistance against phagocytosis or survival within macrophages, which is conserved in the pathogenic yersiniae. Y. pestis OmpR regulates ompC, F, X, and R directly through OmpR-promoter DNA association. There is an inducible expressions of the pore-forming proteins OmpF, C, and X at high osmolarity in Y. pestis, in contrast to the reciprocal regulation of them in E. coli. The main difference is that ompF expression is not repressed at high osmolarity in Y. pestis, which is likely due to the absence of a promoter-distal OmpR-binding site for ompF. Bacterial strains The wild-type (WT) Y. pestis biovar microtus strain 201 is avirulent to humans but highly lethal to mice [1]. The 43 to 666 base pairs of ompR (720bp in total length) were replaced by the kanamycin resistance cassette using the one-step inactivation method based on the lambda Red phage recombination system, with the helper plasmid pKD46, to generate the ompR mutants of Y. pestis (designated as ΔompR) [2]. Chromosomal integration of the mutagenic cassette was confirmed by PCR and sequencing using oligonucleotides external to the integrated cassette (data not shown). The elimination of pKD46 in ΔompR was verified by PCR. Bacterial growth and RNA isolation Overnight cultures (an OD620 of about 1.0) of WT or ΔompR in the chemically defined TMH medium [3] were diluted 1:20 into the fresh TMH. Bacterial cells were grown at 26°C to the middle exponential growth phase (an OD620 of about 1.0). To trigger the high osmolarity conditions in OmpR-related experiments, a final concentration of 0.5 M sorbitol was added, after which the cell cultures were allowed to grow for another 20 min. Total RNA of bacterial cells was extracted using the TRIzol Reagent (Invitrogen) without the DNA removal step (for RT-PCR and primer extension) or by using MasterPureTM RNA Purification kit (Epicenter) with the removal of contaminated DNA (for microarray). Immediately before harvesting, bacterial cultures were mixed with RNAprotect Bacteria Reagent (Qiagen) to minimize RNA degradation. RNA quality was monitored by agarose gel electrophoresis, and RNA quantity was determined using a spectrophotometer. Microarray expression analysis Gene expression profiles were compared between WT and ΔompR using a Y. pestis whole-genome cDNA microarray as described in a previous work [4]. RNA samples were isolated from four individual bacterial cultures as biological replicates for each strain. The dual-fluorescently (Cy3 or Cy5 dye) labeled cDNA probes, for which the incorporated dye was reversed, were synthesized from the RNA samples. These were then hybridized to 4 separated microarray slides. A ratio of mRNA levels was calculated for each gene. Significant changes of gene expression were identified using the SAM software [5]. After the SAM analysis, only genes with at least two-fold changes in expression were collected for further analysis. References 1. Zhou D, Tong Z, Song Y, Han Y, Pei D, et al. (2004) Genetics of metabolic variations between Yersinia pestis biovars and the proposal of a new biovar, microtus. J Bacteriol 186: 5147-5152. 2. Zhan L, Han Y, Yang L, Geng J, Li Y, et al. (2008) The cyclic AMP receptor protein, CRP, is required for both virulence and expression of the minimal CRP regulon in Yersinia pestis biovar microtus. Infect Immun 76: 5028-5037. 3. Straley SC, Bowmer WS (1986) Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins. Infect Immun 51: 445-454. 4. Zhou D, Qin L, Han Y, Qiu J, Chen Z, et al. (2006) Global analysis of iron assimilation and fur regulation in Yersinia pestis. FEMS Microbiol Lett 258: 9-17. 5. Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116-5121.
2011-10-23 | E-GEOD-26601 | biostudies-arrayexpress
Project description:Microarrays were used to identify Y. pestis genes that are differentially regulated under conditions of phoP overexpression. RNA samples were isolated from cultures of two isogenic Y. pestis strains, one overexpressing phoP (KIM10+phoP(delta)/PhoP) and the other not (KIM10+phoP(delta)/MMB67EH), and their gene expression profiles were compared.
2006-07-01 | E-MEXP-712 | biostudies-arrayexpress
Project description:Microarrays were used to identify Y. pestis genes that are transcriptional targets of phoP. RNA samples were isolated from cultures of two isogenic Y. pestis strains, one carrying wild-type phoP (KIM6+) and the other carrying a deletion within phoP (KIM6+phoP(delta)), and their gene expression profiles were compared.
2006-07-01 | E-MEXP-713 | biostudies-arrayexpress
Project description:Yersinia pestis, the etiological agent of plague, is able to sense cell density by quorum sensing. The function of quorum sensing in Y. pestis is not clear. Here, the process of autoinducer-2 (AI-2) quorum sensing was investigated by comparing transcript profiles when AI-2 quorum-sensing signal is added in to control. The strain Îpgm (pigmentation-negative) mutant was used as wild type.The control consisted of cells grown and treated under the same conditions without added signals. Six independent RNA samples from Y. pestis CO92 Îpgm cultures were paired with six independent RNA samples from AI-2 signal added in cultures for hybridization to six two-color microarrays. A dye-swap design was used to remove the Cy5 and Cy3 dye bias.
2016-07-03 | E-GEOD-30237 | biostudies-arrayexpress