Michael S. Donnenberg

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.

The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69

Enteropathogenic Escherichia coli (EPEC) are an important aetiological agent in infant diarrhoea and the prototype for a family of pathogens exhibiting the unique virulence mechanism known as attaching and effacing (AE) (Nataro and Kaper, 1998). All genes necessary for AE are encoded on a 35 kb chromosomal pathogenicity island called the locus of enterocyte effacement (LEE), which contains genes encoding a type III secretion system, secreted proteins (Esp) and the adhesin intimin (McDaniel et al., 1995; McDaniel and Kaper, 1997). Study of the LEE will illuminate our understanding of the pathogenesis of EPEC and other AE pathogens and contribute to the growing body of knowledge about type III secretion systems and pathogenicity islands. We have recently sequenced the entire LEE of EPEC strain E2348/69 and describe below our initial analysis. Further details can be found in GenBank (accession number AF022236) and on the Molecular Microbiology Web site (http://www.blackwellscience.com/products/journals/mole.htm). The complete region was 35 624 bp with an average G þ C content of 38.36%, which is far below that of the E. coli chromosome (50.8%; Blattner et al., 1997), a pattern in keeping with many other pathogenicity islands (Hacker et al., 1997). The LEE contains 41 predicted open reading frames (ORFs) (of > 50 amino acids) arranged in at least five polycistronic operons, as predicted by the close spacing of co-directional genes. The LEE may be divided into at least three functional domains (Fig. 1): the central eae (encoding intimin), the region encoding the secreted Esp proteins and a large region encoding the type III secretion apparatus. Several LEE genes have been reported previously, and our final LEE sequence entry contains corrections to some of these previously reported genes and predicted proteins. Additionally, we have decided to adopt a standardized nomenclature (Bogdanove et al., 1996a; Yahr et al., 1997), which changes the name of several previously described genes comprising the type III secretion system of EPEC (Jarvis et al., 1995). Those genes homologous to Yersinia type III secretion (ysc) genes are referred to as esc (E. coli secretion) genes with the same suffix as the Yersinia homologue (e.g. sepA becomes escV, homologous with yscV; Table 1). Within the family of type III secretory genes, the LEE shares the highest level of predicted amino acid similarity and genetic organization with ssa genes from the SPI-2 pathogenicity island of Salmonella typhimurium (Shea et al., 1996). Genes that are not ysc homologues but are involved in type III secretion are named sep (secretion of E. coli proteins). The chaperone for the secretion of EspD is named cesD for chaperone for E. coli secreted protein D (Wainwright and Kaper, 1998). The remaining named genes, esp (E. coli secreted protein), eae (E. coli attaching and effacing) and orfU will retain their designations, and remaining ORFs are designated orf or rorf depending on the direction of transcription relative to eae. A brief description of selected LEE ORFs follows. More details can be found in Table 1, Fig. 1 and on the Molecular Microbiology home page (http://www.blackwell-science. com/products/journals/mole.htm. rOrf1 is similar to a protein of unknown function from E. coli K-12 and to a predicted lipoprotein that is encoded on the S. typhimurium virulence plasmid adjacent to rck (Heffernan et al., 1992), which has been shown to be important for virulence (Cirillo et al., 1996). rOrf2 is similar to the VirA protein of Shigella flexneri, a type III secreted protein that is involved in invasion and intercellular spreading (Uchiya et al., 1995). Secretion of rOrf2 has not been observed, and it is unclear what functions rOrf2 may have in EPEC, in which the role of invasion remains undefined. Molecular Microbiology (1998) 28(1), 1–4

The Per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)‐encoded regulator (Ler)

Enteropathogenic Escherichia coli (EPEC) is the prototype organism of a group of pathogenic Gram‐negative bacteria that cause attaching and effacing (AE) intestinal lesions. All EPEC genes necessary for the AE phenotype are encoded within a 35.6 kb pathogenicity island termed the locus of enterocyte effacement (LEE). The LEE encodes 41 predicted open reading frames (ORFs), including components of a type III secretion apparatus and secreted molecules involved in the disruption of the host cell cytoskeleton. To initiate our studies on regulation of genes within the LEE, we determined the genetic organization of the LEE, defining transcriptional units and mapping transcriptional start points. We found that components of the type III secretion system are transcribed from three polycistronic operons designated LEE1, LEE2 and LEE3. The secreted Esp molecules are part of a fourth polycistronic operon designated LEE4. Using reporter gene fusion assays, we found that the previously described plasmid‐encoded regulator (Per) activated operons LEE1, LEE2 and LEE3, and modestly increased the expression of LEE4 in EPEC. Using single‐copy lacZ fusions in K‐12‐derived strains, we determined that Per only directly activated the LEE1::lacZ fusion, and did not directly activate the other operons. Orf1 of the LEE1 operon activated the expression of single‐copy LEE2::lacZ and LEE3::lacZ fusions in trans and modestly increased the expression of LEE4::lacZ in K‐12 strains. Orf1 was therefore designated Ler, for LEE‐encoded regulator. Thus, the four polycistronic operons of the LEE that encode type III secretion components and secreted molecules are now included in the Per regulon, where Ler participates in this novel regulatory cascade in EPEC.

Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function

The mechanisms by which enteropathogenic Escherichia coli (EPEC), an important cause of diarrhea among infants in developing countries, induce symptoms are not defined. EPEC have a type III secretion system required for characteristic attaching and effacing changes that modify the cytoskeleton and apical surface of host cells. Infection of polarized intestinal epithelial cell monolayers by EPEC leads to a loss of transepithelial electrical resistance, which also requires the type III secretion system. We demonstrate here that EspF, a protein that is secreted by EPEC via the type III secretion system, is not required for quantitatively and qualitatively typical attaching and effacing lesion formation in intestinal epithelial cells. However, EspF is required in a dose-dependent fashion for the loss of transepithelial electrical resistance, for increased monolayer permeability, and for redistribution of the tight junction-associated protein occludin. Furthermore, the analysis of EPEC strains expressing EspF-adenylate cyclase fusion proteins indicates that EspF is translocated via the type III secretion system to the cytoplasm of host cells, a result confirmed by immunofluorescence microscopy. These studies suggest a novel role for EspF as an effector protein that disrupts intestinal barrier function without involvement in attaching and effacing lesion formation.

Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infant diarrhea in developing countries. Recently eaeA, a gene necessary for the characteristic intimate attachment of EPEC to epithelial cells in tissue culture, was described. We conducted a randomized, double-blind study to determine the role of the eaeA gene in human EPEC infection. 11 adult volunteers ingested 2 x 10(10) colony-forming units of O127:H6 EPEC strain E2348/69, and an equal number received the same dose of an isogenic eaeA deletion mutant constructed from E2348/69. Volunteers were monitored for the development of diarrhea, fever, and systemic and gastrointestinal complaints. Diarrhea developed in all 11 volunteers who received E2348/69 and in 4 of 11 who received the mutant (P = 0.002). Fever was more common in recipients of the wild-type strain (P = 0.024). Stool volumes were lower in recipients of the mutant. All volunteers seroconverted to E2348/69 LPS, but the geometric mean peak titers of serum IgG and IgA in recipients of the mutant were lower than those of recipients of the wild-type strain. IgA against LPS was detected in the jejunal fluid of six of six recipients of E2348/69 and 5/6 recipients of the mutant. This study unambiguously assigns a role for eaeA as an EPEC virulence gene, but the residual diarrhea seen in recipients of the mutant indicates that other factors are involved.

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.

EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infant diarrhoea. EPEC mediates several effects on host epithelial cells, including activation of signal‐transduction pathways, cytoskeletal rearrangement along with pedestal and attachingleffacing lesion formation. It has been previously shown that the EPEC eaeB (espB) gene encodes a secreted protein required for signal transduction and adherence, while eaeA encodes intimin, an EPEC membrane protein that mediates intimate adherence and contributes to focusing of cytoskeletal proteins beneath bacteria. DNA‐sequence analysis of a region between eaeA and eaeB identified a predicted open reading frame (espA) that matched the amino‐terminal sequence of a 25 kDa EPEC secreted protein. A mutant with a non‐polar insertion in espA does not secrete this protein, activate epithelial cell signal transduction or cause cytoskeletal rearrangement. These phenotypes were complemented by a cloned espA gene. The espA mutant is also defective for invasion. It is concluded that espA encodes an EPEC secreted protein that is necessary for activating epithelial signal transduction, intimate contact, and formation of attaching and effacing lesions, processes which are central to pathogenesis.

A plasmid‐encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence

Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundle‐forming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non‐invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence. We report here the mapping of the TnphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N‐terminal amino acid sequence of the major structural protein of the bundle‐forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle‐forming pilus. TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phophatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid‐encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.

Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli

Escherichia coli, a venerable workhorse for biochemical and genetic studies and for the large-scale production of recombinant proteins, is one of the most intensively studied of all organisms. The natural habitat of E. coli is the gastrointestinal tract of warm-blooded animals, and in humans, this species is the most common facultative anaerobe in the gut. Although most strains exist as harmless symbionts, there are many pathogenic E. coli strains that can cause a variety of diseases in animals and humans. In addition, from an evolutionary perspective, strains of the genus Shigella are so closely related phylogenetically that they are included in the group of organisms recognized as E. coli (1, 2). Pathogenic E. coli strains differ from those that predominate in the enteric flora of healthy individuals in that they are more likely to express virulence factors — molecules directly involved in pathogenesis but ancillary to normal metabolic functions. Expression of these virulence factors disrupts the normal host physiology and elicits disease. In addition to their role in disease processes, virulence factors presumably enable the pathogens to exploit their hosts in ways unavailable to commensal strains, and thus to spread and persist in the bacterial community. It is a mistake to think of E. coli as a homogenous species. Most genes, even those encoding conserved metabolic functions, are polymorphic, with multiple alleles found among different isolates (1). The composition of the genome of E. coli is also highly dynamic. The fully sequenced genome of the laboratory K-12 strain, whose derivatives have served an indispensable role in the laboratories of countless scientists, shows evidence of tremendous plasticity (3). It has been estimated that the K-12 lineage has experienced more than 200 lateral transfer events since it diverged from Salmonella about 100 million years ago and that 18% of its contemporary genes were obtained horizontally from other species (4). Such fluid gain and loss of genetic material are also seen in the recent comparison of the genomic sequence of a pathogenic E. coli O157:H7 with the K-12 genome. Approximately 4.1 million base pairs of “backbone” sequences are conserved between the genomes, but these stretches are punctuated by hundreds of sequences present in one strain but not in the other. The pathogenic strain contains 1.34 million base pairs of lineage-specific DNA that includes 1,387 new genes; some of these have been implicated in virulence, but many have no known function (5). The virulence factors that distinguish the various E. coli pathotypes were acquired from numerous sources, including plasmids, bacteriophages, and the genomes of other bacteria. Pathogenicity islands, relatively large (>10 kb) genetic elements that encode virulence factors and are found specifically in the genomes of pathogenic strains, frequently have base compositions that differ drastically from that of the content of the rest of the E. coli genome, indicating that they were acquired from another species. Here, we explore some of the known virulence factors that contribute to the heterogeneity of E. coli strains, and we review what is known regarding the origin and distribution of these factors.

Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract

Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature‐tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini‐Tn5 km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co‐infection experiments, 19 survival‐defective mutants were identified. These mutants were recovered in numbers 10 1 ‐ to 10 6 ‐fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn‐thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K‐12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the ‘off’ to the ‘on’ phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo . An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre‐eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.