Rodney A. Welch

Genome sequence of enterohaemorrhagic Escherichia coli O157:H7

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions—all of which could be targets for surveillance.

Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli

We present the complete genome sequence of uropathogenic Escherichia coli, strain CFT073. A three-way genome comparison of the CFT073, enterohemorrhagic E. coli EDL933, and laboratory strain MG1655 reveals that, amazingly, only 39.2% of their combined (nonredundant) set of proteins actually are common to all three strains. The pathogen genomes are as different from each other as each pathogen is from the benign strain. The difference in disease potential between O157:H7 and CFT073 is reflected in the absence of genes for type III secretion system or phage- and plasmid-encoded toxins found in some classes of diarrheagenic E. coli. The CFT073 genome is particularly rich in genes that encode potential fimbrial adhesins, autotransporters, iron-sequestration systems, and phase-switch recombinases. Striking differences exist between the large pathogenicity islands of CFT073 and two other well-studied uropathogenic E. coli strains, J96 and 536. Comparisons indicate that extraintestinal pathogenic E. coli arose independently from multiple clonal lineages. The different E. coli pathotypes have maintained a remarkable synteny of common, vertically evolved genes, whereas many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain.

Pore-forming cytolysins of gram-negative bacteria.

A great deal is known about the structure, function and metabolic effects of enzymatic bacterial toxins such as the diphtheria, pertussis and cholera toxins. By comparison, our understanding of the pore‐forming, cytolytic toxins, particularly those produced by Gram‐negative bacterial pathogens, is far less complete. The genetics and biochemistry of a large, newly discovered family of calcium‐dependent, pore‐forming cytotoxins (RTX toxins) produced by different genera of the Enterobacteriaceae and Pasteurellaceae are discussed in this review. This toxin family is especially noteworthy because the individual toxins often exhibit different cell‐ and host‐specificity. A brief review is also included of two ancestrally unrelated groups of calcium‐independent, pore‐forming toxins, the haemolysins produced by Proteus mirabilis and Serratia marcescens and the aerolysins secreted by species of Aeromonas. Their structure and function are contrasted with those of the RTX family members. Emerging questions about the role of cytolysins in pathogenesis are presented. Perhaps the most important issue raised is whether or not less attention should be paid to the the lytic capacity of these cytotoxins, with more energy being devoted to the understanding of their non‐lytic Inhibitory activities against host cells.

Transcriptome of Uropathogenic Escherichia coli during Urinary Tract Infection

ABSTRACT A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any E. coli pathotypes transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.

RTX Toxin Structure and Function: A Story of Numerous Anomalies and Few Analogies in Toxin Biology

It can be agreed that RTX toxins contribute to the pathogenesis of different diseases by causing dysfunction of the general cellular reactions of the immune response. The suggestion that RTX toxins induce cytokine production in nonimmune cells that would ultimately cause tissue damage is an expansion of their role in disease pathogenesis (Uhlen et al. 2000). Investigators in the RTX toxin field may not agree with me, but precise and satisfactory answers to the following questions are not yet available. How do RTX toxins mechanistically damage a cell? Do RTX toxins have receptors in the classic sense, in which there is a reversible ligand and receptor complex? What is responsible for the common Ca2+ ion influx in affected cells? The recent observation that an RTX toxin stimulates host-cell-mediated Ca2+ ion oscillation in part challenges the long held concept that these toxins damage cells by the direct formation of pores. Are the Ca2+ ion fluxes truly the noxious cellular insult? What is the final molecular structure of RTX toxins at the time they cause cellular death? How does the common requirement for acyl modification among RTX toxins fit into the toxin structure and mechanism of cellular killing, particularly when mixtures of unusual fatty acids are used by some toxins? There are a number of outstanding laboratories throughout the world that are seeking answers to these questions. We can reasonably expect that during the next decade research on the structure and function of RTX toxins will lead to new chemotherapeutic targets and reagents for basic cell biology and biotechnology.

StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor.

Escherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1‐INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn), cleaves C1‐INH to produce (unique) ≈ 60–65 kDa fragments. StcE does not digest other serine protease inhibitors, extracellular matrix proteins or universal protease targets. We also observed that StcE causes the aggregation of cultured human T cells but not macrophage‐like cells or B cells. Substitution of aspartic acid for glutamic acid at StcE position 435 within the consensus metalloprotease active site ablates its abilities to digest C1‐INH and to aggregate T cells. StcE is secreted by the etp type II secretion pathway encoded on pO157, and extracellular StcE levels are positively regulated by the LEE‐encoded regulator, Ler. StcE antigen and activity were detected in the faeces of a child with an E. coli O157:H7 infection, demonstrating the expression of StcE during human disease. Cleavage of C1‐INH by StcE could plausibly cause localized pro‐inflammatory and coagulation responses resulting in tissue damage, intestinal oedema and thrombotic abnormalities.

Coordinate Expression of Fimbriae in Uropathogenic Escherichia coli

ABSTRACT Uropathogenic Escherichia coli is the most common etiological agent of urinary tract infections. Bacteria can often express multiple adhesins during infection in order to favor attachment to specific niches within the urinary tract. We have recently demonstrated that type 1 fimbria, a phase-variable virulence factor involved in adherence, was the most highly expressed adhesin during urinary tract infection. Here, we examine whether the expression of type 1 fimbriae can affect the expression of other adhesins. Type 1 fimbrial phase-locked mutants of E. coli strain CFT073, which harbors genes for numerous adhesins, were employed in this study. CFT073-specific DNA microarray analysis of these strains demonstrates that the expression of type 1 fimbriae coordinately affects the expression of P fimbriae in an inverse manner. This represents evidence for direct communication between genes relating to pathogenesis, perhaps to aid the sequential occupation of different urinary tract tissues. While the role of type 1 fimbriae during infection has been clear, the role of P fimbriae must be further defined to assert the relevance of coordinated regulation in vivo. Therefore, we examined the ability of P fimbrial isogenic mutants, constructed in a type 1 fimbrial-negative background, to compete in the murine urinary tract over a period of 168 h. No differences in the colonization of these mutants were observed. However, comparison of these results with previous studies suggests that inversely coordinated expression of adhesin gene clusters does occur in vivo. Interestingly, the mutant that was incapable of expressing either type 1 or P fimbriae compensated by synthesizing F1C fimbriae.

Assays of hemolytic toxins

The ability to produce a cytolytic toxin contributes to the success of many organisms in a particular niche by such diverse means as lysis of a phagolysosomal membrane of the macrophage by hemolysin from the intracellular parasite Trypanosoma cruzi, disruption of leukocyte activity by the Escherichia coli hemolysin, and destruction of invading bacteria by hemolysin from the annelid Glycera dibranchiata. The relative contribution of erythrocyte lysis to survival of the cytolysin producer is still under investigation. Nevertheless, the hemolytic phenotype is both a powerful tool for identifying novel cytolysins and a convenient marker for studying cytolytic activity in established toxins.

The StcE protease contributes to intimate adherence of enterohemorrhagic Escherichia coli O157:H7 to host cells.

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a diarrheal pathogen that causes attaching and effacing (A/E) lesions on intestinal epithelial cells. Strains of the O157 serogroup carry the large virulence plasmid pO157, which encodes the etp type II secretion system that secretes the genetically linked zinc metalloprotease StcE. The Ler regulator controls expression of many genes involved in A/E lesion formation, as well as StcE, suggesting StcE may be important at a similar time during colonization. Our laboratory has previously demonstrated that StcE cleaves C1-esterase inhibitor, a regulator of multiple inflammation pathways. Here we report two new substrates for StcE, mucin 7 and glycoprotein 340, and that purified StcE reduces the viscosity of human saliva. We tested the hypothesis that StcE contributes to intimate adherence of EHEC to host cells by cleavage of glycoproteins from the cell surface. The fluorescent actin stain (FAS) test was used to observe the intimate adherence represented by fluorescently stained bacteria colocalized with regions of bundled actin formed on HEp-2 cells. An E. coli O157:H7 strain with a stcE gene deletion was not affected in its ability to generally adhere to HEp-2 cells, but it did score threefold lower on the FAS test than wild-type or complemented strains. Addition of exogenous recombinant StcE increased intimate adherence of the mutant to wild-type levels. Thus, StcE may help block host clearance of E. coli O157:H7 by destruction of some classes of glycoproteins, and it contributes to intimate adherence of E. coli O157:H7 to the HEp-2 cell surface.

Pore-formation by Escherichia coli hemolysin (HlyA) and other members of the RTX toxins family.

Escherichia coli hemolysin (HlyA) is a major cause of E. coli virulence. It lyses erythrocytes by a colloid osmotic shock due to the formation of hydrophilic pores in the cell wall. The size of these channels can be estimated using osmotic protectant of increasing dimensions. To show that the formation of pores does not depend critically on the osmotic swelling we prepared resealed human erythrocyte ghosts loaded with a fluorescent marker. When attacked by HlyA the internal marker was released, indicating the formation of toxin channels so large as to let it through. The channels can be directly demonstrated also in purely lipidic model systems such as planar membranes and unilamellar vesicles, which lack any putative protein receptor. HlyA has been recognised as a member of a large family of exotoxins elaborated by Gram-negative organisms including Proteus, Bordetella, Morganella, Pasteurella and Actinobacillus. These toxins have quite different target cell specificity and in many cases are leukocidal. When tried on planar membranes however, even specific leukotoxins open channels not dissimilar from those formed by HlyA, suggesting this might be a common step in their action. Comparison of the hydrophobic properties of six members of the toxin family indicates the presence of a conserved cluster of ten contiguous amphipathic helixes, located in the N-terminal half of the molecule, which might be involved in channel formation.