Timothy J. Wells

A commensal gone bad: Complete genome sequence of the prototypical enterotoxigenic escherichia coli strain H10407

In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.

EhaA is a novel autotransporter protein of enterohemorrhagic Escherichia coli O157:H7 that contributes to adhesion and biofilm formation

Autotransporter (AT) proteins have been identified in many Gram-negative pathogens and are unique in that their primary sequence is sufficient to direct their transport across the bacterial membrane system. Where characterized they are uniformly associated with virulence. Using conserved AT motifs as a search tool, four putative AT proteins were identified in the Enterohemorrhagic Escherichia coli O157:H7 EDL933 genome. The genes encoding these proteins (z0402/ehaA, z0469/ehaB, z3487/ehaC and z3948/ehaD) were PCR amplified, cloned and expressed in an E. coli K-12 MG1655flu background. Preliminary characterization revealed that ehaA, ehaB and ehaD encode proteins associated with increased biofilm formation. One of these genes (ehaA) resides on a genomic island in E. coli O157:H7 strains EDL933 and Sakai. Over-expression of EhaA in E. coli K-12 demonstrated it is located at the cell surface and resulted in the formation of large cell aggregates, promoted significant biofilm formation and mediated adhesion to primary epithelial cells of the bovine terminal rectum. The expression of ehaA was demonstrated in E. coli EDL933 by RT-PCR. An EhaA-specific antibody revealed the EhaA protein was expressed in 24/50 generic Shiga toxin-producing E. coli (STEC) strains of various serotypes including O157:H7. However, the deletion of ehaA from E. coli EDL933 and a STEC strain from serotype O111:H(-) did not affect biofilm growth. Our results suggest that EhaA may contribute to adhesion, colonization and biofilm formation by E. coli O157:H7 and possibly other STEC serotypes.

UpaH Is a Newly Identified Autotransporter Protein That Contributes to Biofilm Formation and Bladder Colonization by Uropathogenic Escherichia coli CFT073

ABSTRACT Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.

Size and conformation limits to secretion of disulfide-bonded loops in autotransporter proteins

Background: There is a general paucity of cysteine residues within the passenger domains of autotransporter proteins. Results: Distantly spaced cysteines forming disulfide-bonded loops or those enclosing structural elements are secretion-incompetent. Conclusion: Only closely spaced cysteine pairs are compatible with the autotransporter pathway. Significance: Secretion of folded peptides by the autotransporter pathway is limited; hence autotransporters lack large disulfide-bonded loops to remain secretion-competent. Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.

SadA, a Trimeric Autotransporter from Salmonella enterica Serovar Typhimurium, Can Promote Biofilm Formation and Provides Limited Protection against Infection

ABSTRACT Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against Salmonella.

Autotransporters of Escherichia coli: a sequence-based characterization

Autotransporter (AT) proteins are found in all Escherichia coli pathotypes and are often associated with virulence. In this study we took advantage of the large number of available E. coli genome sequences to perform an in-depth bioinformatic analysis of AT-encoding genes. Twenty-eight E. coli genome sequences were probed using an iterative approach, which revealed a total of 215 AT-encoding sequences that represented three major groups of distinct domain architecture: (i) serine protease AT proteins, (ii) trimeric AT adhesins and (iii) AIDA-I-type AT proteins. A number of subgroups were identified within each broad category, and most subgroups contained at least one characterized AT protein; however, seven subgroups contained no previously described proteins. The AIDA-I-type AT proteins represented the largest and most diverse group, with up to 16 subgroups identified from sequence-based comparisons. Nine of the AIDA-I-type AT protein subgroups contained at least one protein that possessed functional properties associated with aggregation and/or biofilm formation, suggesting a high degree of redundancy for this phenotype. The Ag43, YfaL/EhaC, EhaB/UpaC and UpaG subgroups were found in nearly all E. coli strains. Among the remaining subgroups, there was a tendency for AT proteins to be associated with individual E. coli pathotypes, suggesting that they contribute to tissue tropism or symptoms specific to different disease outcomes.

The Escherichia coli O157:H7 EhaB autotransporter protein binds to laminin and collagen I and induces a serum IgA response in O157:H7 challenged cattle

Enterohaemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing E.  coli that cause gastrointestinal disease with the potential for life-threatening sequelae. Cattle serve as the natural reservoir for EHEC and outbreaks occur sporadically as a result of contaminated beef and other farming products. While certain EHEC virulence mechanisms have been extensively studied, the factors that mediate host colonization are poorly defined. Previously, we identified four proteins (EhaA,B,C,D) from the prototypic EHEC strain EDL933 that belong to the autotransporter (AT) family. Here we characterize the EhaB AT protein. EhaB was shown to be located at the cell surface and overexpression in E.  coli K-12 resulted in significant biofilm formation under continuous flow conditions. Overexpression of EhaB in E.  coli K12 and EDL933 backgrounds also promoted adhesion to the extracellular matrix proteins collagen I and laminin. An EhaB-specific antibody revealed that EhaB is expressed in E.  coli EDL933 following in vitro growth. EhaB also cross-reacted with serum IgA from cattle challenged with E.  coli O157:H7, indicating that EhaB is expressed in vivo and elicits a host IgA immune response.

Laboratory adapted Escherichia coli K-12 becomes a pathogen of Caenorhabditis elegans upon restoration of O antigen biosynthesis

Escherichia coli has been the leading model organism for many decades. It is a fundamental player in modern biology, facilitating the molecular biology revolution of the last century. The acceptance of E. coli as model organism is predicated primarily on the study of one E. coli lineage; E. coli K‐12. However, the antecedents of todays laboratory strains have undergone extensive mutagenesis to create genetically tractable offspring but which resulted in loss of several genetic traits such as O antigen expression. Here we have repaired the wbbL locus, restoring the ability of E. coli K‐12 strain MG1655 to express the O antigen. We demonstrate that O antigen production results in drastic alterations of many phenotypes and the density of the O antigen is critical for the observed phenotypes. Importantly, O antigen production enables laboratory strains of E. coli to enter the gut of the Caenorhabditis elegans worm and to kill C. elegans at rates similar to pathogenic bacterial species. We demonstrate C. elegans killing is a feature of other commensal E. coli. We show killing is associated with bacterial resistance to mechanical shear and persistence in the C. elegans gut. These results suggest C. elegans is not an effective model of human‐pathogenic E. coli infectious disease.

A generalised module for the selective extracellular accumulation of recombinant proteins

BackgroundIt 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.ResultsHere, 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.ConclusionsThe 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.

Increased severity of respiratory infections associated with elevated anti-LPS IgG2 which inhibits serum bactericidal killing

An antibody directed against the O-antigen of Pseudomonas aeruginosa LPS can block complement-mediated bacterial killing and contributes to the severity of respiratory infection.