Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Background \ud Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. \ud \ud Methods \ud In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. \ud \ud Conclusion \ud This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies

Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome

Transfer Region of pO113 from Enterohemorrhagic Escherichia coli: Similarity with R64 and Identification of a Novel Plasmid-Encoded Autotransporter, EpeA

Enterohemorrhagic Escherichia coli (EHEC) is a prominent, food-borne cause of diarrhea, bloody diarrhea, and the hemolytic uremic syndrome in industrialized countries. Most strains of EHEC carry the locus for enterocyte effacement (LEE) pathogenicity island, but a proportion of isolates from patients with severe disease do not carry LEE and very little is known about virulence factors in these organisms. LEE-negative strains of EHEC typically express Shiga toxin 2 and carry a large plasmid that encodes the production of EHEC hemolysin. In this study, we determined the nucleotide sequence of the transfer region of pO113, the large hemolysin plasmid from LEE-negative EHEC O113:H21 (EH41). This 63.9-kb region showed a high degree of similarity with the transfer region of R64, and pO113 was capable of self-transmission at low frequencies. Unlike R64 and the related dot/icm system of Legionella pneumophila, however, pO113 was unable to mobilize RSF1010. In addition, the pO113 transfer region encoded a novel high-molecular-weight serine protease autotransporter of Enterobacteriaceae (SPATE) protein, termed EpeA. Like other SPATEs, EpeA exhibited protease activity and mucinase activity, but expression was not associated with a cytopathic effect on epithelial cells. Analysis of a second high-molecular-weight secreted protein revealed that pO113 also encodes EspP, a cytopathic SPATE identified previously in EHEC O157:H7. The nucleotide sequences encoding the predicted β-domains of espP and epeA were identical and also shared significant homology with a third SPATE protein, EspI. Both espP and epeA were detected in several LEE-negative clinical isolates of EHEC and thus may contribute to the pathogenesis of this subset of EHEC

Contribution of a Novel Gene, rpeA, Encoding a Putative Autotransporter Adhesin to Intestinal Colonization by Rabbit-Specific Enteropathogenic Escherichia coli▿

Rabbit-specific enteropathogenic Escherichia coli (REPEC) is an attaching and effacing pathogen of young rabbits. Using signature-tagged mutagenesis, we identified several known colonization factors of REPEC as well as a gene predicted to encode a novel autotransporter protein. This novel gene was termed rpeA for REPEC plasmid-encoded autotransporter

Dynein Light Chain Association Sequences Can Facilitate Nuclear Protein Import

Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures. We also find that P-protein DLC-AS, as well as analogous sequences from other proteins, acts as an independent module that can confer enhancement of nuclear accumulation to proteins carrying the P-protein NLS, as well as several heterologous NLSs. Photobleaching experiments in live cells demonstrate that the MT-dependent enhancement of NLS-mediated nuclear accumulation by the P-protein DLC-AS involves an increased rate of nuclear import. This is the first report of DLC-AS enhancement of NLS function, identifying a novel mechanism regulating nuclear transport with relevance to viral and cellular protein biology. Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application

A generalised module for the selective extracellular accumulation of recombinant proteins

BACKGROUND: It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. RESULTS: Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. CONCLUSIONS: The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications

A generalised module for the selective extracellular accumulation of recombinant proteins

Abstract Background It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. Results Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. Conclusions The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications.</p