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Characterization of two homologous disulfide bond systems involved in virulence factor biogenesis in uropathogenic Escherichia coli CFT073.

ABSTRACT: Disulfide bond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic Escherichia coli (UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide and is in turn reoxidized by DsbB. DsbA has broad substrate specificity and reacts readily with reduced unfolded proteins entering the periplasm. The DsbLI system also comprises a functional redox pair; however, DsbL catalyzes the specific oxidative folding of the large periplasmic enzyme arylsulfate sulfotransferase (ASST). In this study, we characterized the DsbLI system of the prototypic UPEC strain CFT073 and examined the contributions of the DsbAB and DsbLI systems to the production of functional flagella as well as type 1 and P fimbriae. The DsbLI system was able to catalyze disulfide bond formation in several well-defined DsbA targets when provided in trans on a multicopy plasmid. In a mouse urinary tract infection model, the isogenic dsbAB deletion mutant of CFT073 was severely attenuated, while deletion of dsbLI or assT did not affect colonization.

SUBMITTER: Totsika M

PROVIDER: S-EPMC2698384 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Characterization of two homologous disulfide bond systems involved in virulence factor biogenesis in uropathogenic Escherichia coli CFT073.

Totsika Makrina M Heras Begoña B Wurpel Daniël J DJ Schembri Mark A MA

Journal of bacteriology 20090417 12

Disulfide bond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic Escherichia coli (UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide ...[more]

PMID: 19376849

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Project description:BACKGROUND:As one of human pathogens, the genome of Uropathogenic Escherichia coli strain CFT073 was sequenced and published in 2002, which was significant in pathogenetic bacterial genomics research. However, the current RefSeq annotation of this pathogen is now outdated to some degree, due to missing or misannotation of some essential genes associated with its virulence. We carried out a systematic reannotation by combining automated annotation tools with manual efforts to provide a comprehensive understanding of virulence for the CFT073 genome. RESULTS:The reannotation excluded 608 coding sequences from the RefSeq annotation. Meanwhile, a total of 299 coding sequences were newly added, about one third of them are found in genomic island (GI) regions while more than one fifth of them are located in virulence related regions pathogenicity islands (PAIs). Furthermore, there are totally 341 genes were relocated with their translational initiation sites (TISs), which resulted in a high quality of gene start annotation. In addition, 94 pseudogenes annotated in RefSeq were thoroughly inspected and updated. The number of miscellaneous genes (sRNAs) has been updated from 6 in RefSeq to 46 in the reannotation. Based on the adjustment in the reannotation, subsequent analysis were conducted by both general and case studies on new virulence factors or new virulence-associated genes that are crucial during the urinary tract infections (UTIs) process, including invasion, colonization, nutrition uptaking and population density control. Furthermore, miscellaneous RNAs collected in the reannotation are believed to contribute to the virulence of strain CFT073. The reannotation including the nucleotide data, the original RefSeq annotation, and all reannotated results is freely available via http://mech.ctb.pku.edu.cn/CFT073/. CONCLUSION:As a result, the reannotation presents a more comprehensive picture of mechanisms of uropathogenicity of UPEC strain CFT073. The new genes change the view of its uropathogenicity in many respects, particularly by new genes in GI regions and new virulence-associated factors. The reannotation thus functions as an important source by providing new information about genomic structure and organization, and gene function. Moreover, we expect that the detailed analysis will facilitate the studies for exploration of novel virulence mechanisms and help guide experimental design.

Project description:Bacterial microcompartments (MCPs) are widespread protein-based organelles composed of metabolic enzymes encapsulated within a protein shell. The function of MCPs is to optimize metabolic pathways by confining toxic and/or volatile pathway intermediates. A major class of MCPs known as glycyl radical MCPs has only been partially characterized. Here, we show that uropathogenic Escherichia coli CFT073 uses a glycyl radical MCP for 1,2-propanediol (1,2-PD) fermentation. Bioinformatic analyses identified a large gene cluster (named grp for glycyl radical propanediol) that encodes homologs of a glycyl radical diol dehydratase, other 1,2-PD catabolic enzymes, and MCP shell proteins. Growth studies showed that E. coli CFT073 grows on 1,2-PD under anaerobic conditions but not under aerobic conditions. All 19 grp genes were individually deleted, and 8/19 were required for 1,2-PD fermentation. Electron microscopy and genetic studies showed that a bacterial MCP is involved. Bioinformatics combined with genetic analyses support a proposed pathway of 1,2-PD degradation and suggest that enzymatic cofactors are recycled internally within the Grp MCP. A two-component system (grpP and grpQ) is shown to mediate induction of the grp locus by 1,2-PD. Tests of the E. coli Reference (ECOR) collection indicate that >10% of E. coli strains ferment 1,2-PD using a glycyl radical MCP. In contrast to other MCP systems, individual deletions of MCP shell genes (grpE, grpH, and grpI) eliminated 1,2-PD catabolism, suggesting significant functional differences with known MCPs. Overall, the studies presented here are the first comprehensive genetic analysis of a Grp-type MCP.IMPORTANCE Bacterial MCPs have a number of potential biotechnology applications and have been linked to bacterial pathogenesis, cancer, and heart disease. Glycyl radical MCPs are a large but understudied class of bacterial MCPs. Here, we show that uropathogenic E. coli CFT073 uses a glycyl radical MCP for 1,2-PD fermentation, and we conduct a comprehensive genetic analysis of the genes involved. Studies suggest significant functional differences between the glycyl radical MCP of E. coli CFT073 and better-studied MCPs. They also provide a foundation for building a deeper general understanding of glycyl radical MCPs in an organism where sophisticated genetic methods are available.

Dataset Information

Characterization of two homologous disulfide bond systems involved in virulence factor biogenesis in uropathogenic Escherichia coli CFT073.

Publications

Characterization of two homologous disulfide bond systems involved in virulence factor biogenesis in uropathogenic Escherichia coli CFT073.

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