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Type IV pili (T4P) are retractable multifunctional nanofibers present on the surface of numerous bacterial and archaeal species. Their importance to microbiology is difficult to overstate. The scientific journey leading to our current understanding of T4P structure and function has included many innovative research milestones. Although multiple T4P reviews over the years have emphasized recent advances, we find that current reports often omit many of the landmark discoveries in this field. Here, we attempt to highlight chronologically the most important work on T4P, from the discovery of pili to the application of sophisticated contemporary methods, which has brought us to our current state of knowledge. As there remains much to learn about the complex machine that assembles and retracts T4P, we hope that this review will increase the interest of current researchers and inspire innovative progress.

Escherichia coli can be a harmless commensal organism or cause a range of diseases in humans, including diarrhea, urinary tract infections, meningitis, sepsis, and skin and soft tissue infections. Here, we describe the genome of an isolate that was associated with necrotizing fasciitis and the decompensation of previously undiagnosed cirrhosis.

The BioFire FilmArray® Gastrointestinal panel is a multiplex PCR assay widely used to determine the etiology of infectious gastroenteritis directly from stool specimens. Recently a positive BioFire result for fecal enteropathogenic Escherichia coli (EPEC) was reported by a clinical microbiology laboratory for an adult patient with diarrhea and bacteremia. Since EPEC infrequently infects adults and rarely causes bacteremia, we isolated fecal E. coli and characterized the patient's blood and fecal E. coli isolates. Draft genome sequencing using a combination of methods indicated that the blood and fecal strains are virtually identical, are from sequence type 963 (phylogroup D) and exhibit neither the virulence genes characteristic of EPEC and extraintestinal pathogenic E. coli (ExPEC) nor classic EPEC-associated phenotypes. These findings support a gut source for the patient's bacteremia but exclude EPEC as the causative organism, and suggest that results of multiplex PCR assays from complex samples can be misleading, and should be interpreted with caution when they are discordant with clinical information. BioProject accession numbers for strains MVAST5574 and MVAST5635 genomes are PRJNA611789 and PRJNA611804, respectively.

Enteropathogenic Escherichia coli (EPEC) remains a significant cause of infant diarrheal illness and associated morbidity and mortality in developing countries. EPEC strains are characterized by their ability to colonize the small intestines of their hosts by a multistep program involving initial loose attachment to intestinal epithelial cells followed by an intimate adhesion phase. The initial loose interaction of typical EPEC with host intestinal cells is mediated by bundle-forming pili (BFP). BFP are type 4b pili (T4bP) based on structural and functional properties shared with T4bP expressed by other bacteria. The major structural subunit of BFP is called bundlin, a T4b pilin expressed from the bfpA gene in the BFP operon, which contains three additional genes that encode the pilin-like proteins BfpI, BfpJ, and BfpK. In this study, we show that, in the absence of the BFP retraction ATPase (BfpF), BfpI, BfpJ, and BfpK are dispensable for BFP biogenesis. We also demonstrate that these three minor pilins are incorporated along with bundlin into the BFP filament and contribute to its structural integrity and host cell adhesive properties. The results confirm that previous findings in T4aP systems can be extended to a model T4bP such as BFP. IMPORTANCE Bundle-forming pili contribute to the host colonization strategy of enteropathogenic Escherichia coli . The studies described here investigate the role for three minor pilin subunits in the structure and function of BFP in EPEC. The studies also suggest that these subunits could be antigens for vaccine development.

P-glycoprotein (P-gp), a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family of transporter molecules, is responsible for maintaining low intracellular concentrations of a variety of extracellular compounds and xenobiotics, and for transport of various intracellular molecules. Many drugs are P-gp substrates and intracellular concentrations of these agents may be critical for drug action. Experience in oncology indicates that repeated exposure to P-gp substrate cytotoxic drugs leads to the selection of drug-resistant tumor cells that overexpress P-gp. Since immunosuppressive agents such as cyclosporine, tacrolimus, sirolimus and corticosteroids are substrates for P-gp and since T-cells also express P-gp, it is conceivable that an analogous mechanism exits for therapy-resistant graft rejection. As will be discussed in this article, P-gp may interfere with the response to immunosuppressive therapy through several distinct mechanisms, and as such may represent an attractive therapeutic target.

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.

Enteropathogenic Escherichia coli (EPEC) and other pathogenic bacteria use dynamic type IV pili to adhere to the host. Here we show that the capacity of the EPEC type IV pili to retract is required for the breakdown of the host epithelial tight-junction barrier, efficient actin-pedestal formation, and translocation of effectors via the type III secretion system., Enteropathogenic Escherichia coli (EPEC) is an important human pathogen that causes acute infantile diarrhea. The type IV bundle-forming pili (BFP) of typical EPEC strains are dynamic fibrillar organelles that can extend out and retract into the bacterium. The bfpF gene encodes for BfpF, a protein that promotes pili retraction. The BFP are involved in bacterial autoaggregation and in mediating the initial adherence of the bacterium with its host cell. Importantly, BFP retraction is implicated in virulence in experimental human infection. How pili retraction contributes to EPEC pathogenesis at the cellular level remains largely obscure, however. In this study, an effort has been made to address this question using engineered EPEC strains with induced BFP retraction capacity. We show that the retraction is important for tight-junction disruption and, to a lesser extent, actin-rich pedestal formation by promoting efficient translocation of bacterial protein effectors into the host cells. A model is proposed whereby BFP retraction permits closer apposition between the bacterial and the host cell surfaces, thus enabling timely and effective introduction of bacterial effectors into the host cell via the type III secretion apparatus. Our studies hence suggest novel insights into the involvement of pili retraction in EPEC pathogenesis.

Summary Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe-like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N-terminal segment of EspD, encompassing residues 1–171, contains two amphipathic domains spanning residues 24–41 and 66–83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His6-EspD1–171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His6-EspD1–171 undergoes a pH depended conformational change that increases the α-helix content of this protein approximately sevenfold. This change coincides with insertion of the region circumscribing Trp47 into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His6-EspD1–171 forms a homodimer that is postulated to promote EspD–EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66–83 gene showed that this protein was secreted but non-functional.

Type 3 secretion systems (T3SSs) are critical for the virulence of numerous deadly Gram-negative pathogens. T3SS translocator proteins are required for effector proteins to traverse the host cell membrane and perturb cell function. Translocator proteins include two hydrophobic proteins, represented in enteropathogenic Escherichia coli (EPEC) by EspB and EspD, which are thought to interact and form a pore in the host membrane. Here we adapted a sequence motif recognized by a host kinase to demonstrate that residues on the carboxyl-terminal side of the EspB transmembrane domain are localized to the host cell cytoplasm. Using functional internal polyhistidine tags, we confirm an interaction between EspD and EspB, and we demonstrate, for the first time, an interaction between EspD and the hydrophilic translocator protein EspA. Using a panel of espB insertion mutations, we describe two regions on either side of a putative transmembrane domain that are required for the binding of EspB to EspD. Finally, we demonstrate that EspB variants incapable of binding EspD fail to adopt the proper host cell membrane topology. These results provide new insights into interactions between translocator proteins critical for virulence.

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant morbidity and mortality due to diarrhoea in developing countries. The pathogenesis of EPEC is dependent on a coordinated multi-step process culminating in the intimate adherence of the organisms to the host's intestinal mucosa. During the initial stages of the EPEC colonization process, the fimbrial adhesin, bundle-forming pili (BFP), plays an integral role. We previously reported that the major BFP structural subunit, bundlin, displays lectin-like properties, which enables BFP to initially tether EPEC to N-acetyllactosamine (LacNAc) glycan receptors on host cell surfaces. We also reported that incubating EPEC with synthetic LacNAc-bearing neoglycoconjugates not only inhibits their adherence to host cells, but also induces BFP retraction and subsequent degradation of the bundlin subunits. Herein, we demonstrate that the periplasmic serine protease, DegP, is required for degrading bundlin during this process. We also show that DegP appears to act as a bundlin chaperone during BFP assembly and that LacNAc-BSA-induced BFP retraction is followed by transcriptional upregulation of the BFP operon and downregulation of the locus of enterocyte effacement operons in EPEC.

The Cpx envelope stress response is induced by the misfolding of periplasmic proteins and restores envelope homeostasis by upregulating several periplasmic protein folding and degrading factors. The Cpx response also regulates the expression of a variety of envelope-spanning protein complexes, including flagella, secretion systems and pili, which play an important role in pathogenesis. In a previous study, we inactivated the Cpx response in enteropathogenic Escherichia coli (EPEC), a causative agent of infant diarrhoea, and observed decreased expression of its major adhesin, the bundle-forming pilus (BFP). Here, we examined the mechanism underlying this BFP expression defect, and found that this phenotype can be attributed to insufficient expression of periplasmic folding factors, such as DsbA, DegP and CpxP. Hence, a low level of Cpx pathway activity promotes BFP synthesis by upregulating factors important for folding of BFP component proteins. Conversely, we found that full induction of the Cpx response inhibits BFP expression, mainly by repressing transcription of the bfp gene cluster. In combination with a previous report examining EPEC type III secretion, our results demonstrate that the Cpx response co-ordinates the repression of cell-surface structures during periods of envelope stress.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhoea in developing countries. The aim of this study was to describe the allelic diversity of critical EPEC virulence genes and their association with clinical characteristics. One hundred and twenty EPEC strains isolated from a cohort diarrhoea study in Peruvian children were characterized for the allele type of eae (intimin), bfpA (bundlin pilin protein of bundle-forming pilus) and perA (plasmid encoded regulator) genes by PCR-RFLP. Atypical EPEC strains (eae+, bfp−) were the most common pathotype in diarrhoea (54/74, 73 %) and control samples from children without diarrhoea (40/46, 87 %). Overall, there were 13 eae alleles; the most common were beta (34/120, 28 %), theta (24/120, 20 %), kappa (14/120, 12 %) and mu (8/120, 7 %). There were five bfpA alleles; the most common were beta1/7 (10/26), alpha3 (7/26) and beta5 (3/26). There were three perA alleles: beta (8/16), alpha (7/16) and gamma (1/16). The strains belonged to 36 distinct serogroups; O55 was the most frequent. The gamma-intimin allele was more frequently found in diarrhoea episodes of longer duration (>7 days) than those of shorter duration (3/26, 12 % vs 0/48, 0 %, PP

Enteropathogenic Escherichia coli (EPEC) are diarrhoeagenic E. coli, and are a significant cause of gastrointestinal illness among young children in developing countries. Typical EPEC are identified by the presence of the bundle-forming pilus encoded by a virulence plasmid, which has been linked to an increased severity of illness, while atypical EPEC lack this feature. Comparative genomics of 70 total EPEC from lethal (LI), non-lethal symptomatic (NSI) or asymptomatic (AI) cases of diarrhoeal illness in children enrolled in the Global Enteric Multicenter Study was used to investigate the genomic differences in EPEC isolates obtained from individuals with various clinical outcomes. A comparison of the genomes of isolates from different clinical outcomes identified genes that were significantly more prevalent in EPEC isolates of symptomatic and lethal outcomes than in EPEC isolates of asymptomatic outcomes. These EPEC isolates exhibited previously unappreciated phylogenomic diversity and combinations of virulence factors. These comparative results highlight the diversity of the pathogen, as well as the complexity of the EPEC virulence factor repertoire. Comparative genomics of 70 lethal, non-lethal symptomatic and asymptomatic enteropathogenic Escherichia coli (EPEC) isolates identifies the virulence-associated genes that are significantly more prevalent in symptomatic and lethal infections.

Clostridium difficile is the leading cause of nosocomial infections in the United States, adding billions of dollars per year to health care costs. A vaccine targeted against the bacterium would be extremely beneficial in decreasing the morbidity and mortality caused by C. difficile-associated disease; a vaccine directed against a colonization factor would hinder the spread of the bacterium as well as prevent disease. Type IV pili (T4Ps) are extracellular appendages composed of protein monomers called pilins. They are involved in adhesion and colonization in a wide variety of bacteria and archaea, and are putative colonization factors in C. difficile. We hypothesized that vaccinating mice with pilins would lead to generation of anti-pilin antibodies, and would protect against C. difficile challenge. We found that immunizing C57Bl/6 mice with various pilins, whether combined or as individual proteins, led to low antipilin antibody titers and no protection upon C. difficile challenge. Passive transfer of anti-pilin antibodies led to high serum anti-pilin IgG titers, but to undetectable fecal anti-pilin IgG titers and did not protect against challenge. The low antibody titers observed in these experiments may be due to the particular strain of mice used. Further experiments, possibly with a different animal model of C. difficile infection, are needed to determine if an anti-T4P vaccine would be protective against C. difficile infection.

The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is an important virulence factor. We examined the role of divergent alleles of bfpA encoding bundlin, the BFP pilin protein, in pilus biogenesis, pilus interactions, and immune responses. We found that the BFP biogenesis machine from an EPEC strain that expresses one bundlin type is capable of assembling all other bundlin types. Furthermore, we found that EPEC strains expressing divergent bundlin types are capable of forming mixed autoaggregates, suggesting that different pilin types can intertwine. However, we found that there was a marked difference between alleles in immunogenicity in both rabbits and mice of a peptide derived from a region of bundlin undergoing apparent diversifying selection. In addition, despite a high degree of cross-reactivity between divergent bundlin proteins, in both mice and rabbits responses appeared to be stronger against the homologous pilin protein than against the heterologous protein. This result was verified using sera from a volunteer study, which demonstrated that the human antibody responses after an initial challenge with live EPEC were stronger against the homologous bundlin protein than against a divergent bundlin protein. However, a repeat challenge induced equivalent responses. These results are consistent with the hypothesis that human immune responses against bundlin exert selective pressure on bfpA sequence divergence.

Burkholderia mallei is the cause of glanders and a proven biological weapon. We identified and purified the type IV pilin protein of this organism to study its potential as a subunit vaccine. We found that purified pilin was highly immunogenic. Furthermore, mice infected via sublethal aerosol challenge developed significant increases in titers of antibody against the pilin, suggesting that it is expressed in vivo. Nevertheless, we found no evidence that high-titer antipilin antisera provided passive protection against a sublethal or lethal aerosol challenge and no evidence of protection afforded by active immunization with purified pilin. These results contrast with the utility of type IV pilin subunit vaccines against other infectious diseases and highlight the need for further efforts to identify protective responses against this pathogen.

A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli., A novel isolate of Escherichia coli with features of extraintestinal and enteropathogenic strains was isolated from the blood of a man with multiorgan dysfunction following 3 weeks of diarrhea., Graphical Abstract Figure. A novel isolate of Escherichia coli with features of extraintestinal and enteropathogenic strains was isolated from the blood of a man with multiorgan dysfunction following 3 weeks of diarrhea.

Type IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenic Escherichia coli (EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, from Pseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer., IMPORTANCE Secretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins.

The phoU gene is the last cistron in the pstSCAB–phoU operon and functions as a negative regulator of the Pho regulon. The authors previously identified a phoU mutant of extraintestinal pathogenic Escherichia coli strain CFT073 and demonstrated that this mutant was attenuated for survival in the murine model of ascending urinary tract infection. It is hypothesized that the PhoU protein might serve as a urovirulence factor by indirectly affecting the expression of virulence-related genes. In this study, the phoU mutant was further characterized and PhoU was confirmed as a virulence factor. Western blot analysis demonstrated that insertion of the transposon in the phoU gene disrupted the expression of PhoU. The phoU mutant had derepressed alkaline phosphatase activity under phosphate-excess and -limiting conditions. In single-challenge murine ascending urinary tract infection experiments, quantitative cultures of urine, bladder and kidney revealed no significant differences between the phoU mutant strain and the wild-type strain CFT073. However, in competitive colonization experiments, the phoU mutant strain was significantly out-competed by the wild-type strain in the kidneys and urine and recovered in lower amount in the bladder. Complementation of the phoU mutant with a plasmid containing the wild-type phoU gene restored the expression of PhoU and alkaline phosphate activity to wild-type levels and no significant difference in colonization was observed between the phoU mutant containing the complementing plasmid and wild-type in competitive colonization experiments. In human urine, the phoU mutant and wild-type grew comparably when inoculated independently, indicating that the attenuation observed was not due to a general growth defect. However, as observed in vivo, the wild-type out-competed the phoU mutant in competition growth experiments in human urine. These data indicate that PhoU contributes to efficient colonization of the murine urinary tract and add PhoU to a short list of confirmed urovirulence factors.

Enteropathogenic Escherichia coli (EPEC) strains continue to cause severe and sometimes fatal infantile diarrhea, particularly in Africa. Increased efforts at diagnosis, defining the clinical spectrum of disease, understanding pathogenic mechanisms, and delineating immune responses are desperately needed to develop new strategies to combat EPEC.

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 pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.

Summary Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.

Type IV pili (Tfps) are filamentous surface appendages expressed by Gram-negative microorganisms and play numerous roles in bacterial cell biology. Tfp biogenesis machineries are highly conserved and resemble protein secretion and DNA uptake systems. Although components of Tfp biogenesis systems have been identified, it is not known how they interact to form these machineries. Using the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli as a model Tfp system, we provide evidence of a cytoplasmic membrane subassembly of the Tfp assembly machine composed of putative cytoplasmic nucleotide-binding and cytoplasmic membrane proteins. A combination of genetic, biochemical and biophysical approaches revealed interactions among putative cytoplasmic nucleotide-binding proteins BfpD and BfpF and cytoplasmic membrane proteins BfpC and BfpE of the BFP biogenesis machine. The polytopic membrane protein BfpE appears to be a central component of this subassembly as it interacts with BfpC, BfpD and BfpF. We report that BFP biogenesis probably requires interactions among BfpC, BfpD and BfpE, whereas BFP retraction requires interaction of the PilT-like putative ATPase BfpF with a conserved domain of BfpE. BfpE is the first protein that is not a member of the PilT family to be implicated in Tfp retraction. Furthermore, we found that the putative ATPases BfpD and BfpF play antagonistic roles in BFP biogenesis and retraction, respectively, by interacting with distinct domains of the BFP biogenesis machine.

Escherichia coli is the leading cause of urinary tract infections (UTIs), one of the most common infections in humans. P fimbria was arguably the first proposed virulence factor for uropathogenic E. coli, based on the capacity of E. coli isolated from UTIs to adhere to exfoliated epithelial cells in higher numbers than fecal strains of E. coli. Overwhelming epidemiologic evidence has been presented for involvement of P fimbriae in colonization. It has been difficult, however, to demonstrate this requirement for uropathogenic strains in animal models of infections or in humans. In this study, a signature-tagged mutagenesis screen identified a P-fimbrial gene (papC) and 18 other genes as being among those required for full fitness of cystitis isolate E. coli F11. A P-fimbrial mutant was outcompeted by the wild-type strain in cochallenge in the murine model of ascending UTI, and this colonization defect could be complemented with the cloned pap operon. To our knowledge, this study is the first to fulfill molecular Koch's postulates in which a pathogenic strain was attenuated by mutation of pap genes and then complemented to restore fitness, confirming P fimbria as a virulence factor in a pathogenic clinical isolate.

Among the microbial flora that naturally inhabit the human gastrointestinal tract, Escherichia coli is the most abundant facultative anaerobe. While members of the family Enterobacteriaceae are natural inhabitants of the gastrointestinal tract and are abundant in the environment, there are particular predisposing factors and circumstances that allow both intestinal and extraintestinal infections to occur in community and nosocomial settings. In developing countries of Asia, Latin America, and Africa, where diarrhea is one of the most lethal threats to children under the age of five, enteropathogenic E. coli (EPEC) is a major cause of infantile diarrhea. A study describing early predictors of adverse outcomes following neonatal bacterial meningitis found that elevated total protein in the initial cerebrospinal fluid (CSF) analysis, as well as a CSF-to-blood glucose ratio of < 0.5, corresponded with an adverse outcome. The current definition of postinfectious irritable bowel syndrome (IBS) according to the Rome II criteria, which most studies follow, is the new onset of symptoms developing acutely after an illness characterized by two or more of the following: fever, vomiting, acute diarrhea, or positive stool culture. There are two main types of Shiga toxins, and Shiga toxin-producing E. coli (STEC) (O157 and non-O157 alike) that produce both Stx2 and Stx1, or Stx2 alone, are more likely to be associated with HUS than those that produce Stx1 only. Prebiotic oligosaccharides may also have similar applications for EPEC. Clearly, new and innovative approaches will be required to reduce the burden of E. coli infections and their sequelae.

Enteropathogenic Escherichia coli (EPEC) primarily infects children in developing countries and causes diarrhea that can be deadly. EPEC pathogenesis occurs through type III secretion system (T3SS)-mediated injection of effectors into intestinal epithelial cells (IECs); these effectors alter actin dynamics, modulate the immune response, and disrupt tight junction (TJ) integrity. The resulting compromised barrier function and increased gastrointestinal (GI) permeability may be responsible for the clinical symptoms of infection. Type I interferon (IFN) mediates anti-inflammatory activities and serves essential functions in intestinal immunity and homeostasis; however, its role in the immune response to enteric pathogens, such as EPEC, and its impact on IEC barrier function have not been examined. Here, we report that IFN-β is induced following EPEC infection and regulates IEC TJ proteins to maintain barrier function. The EPEC T3SS effector NleD counteracts this protective activity by inhibiting IFN-β induction and enhancing tumor necrosis factor alpha to promote barrier disruption. The endoribonuclease RNase L is a key mediator of IFN induction and action that promotes TJ protein expression and IEC barrier integrity. EPEC infection inhibits RNase L in a T3SS-dependent manner, providing a mechanism by which EPEC evades IFN-induced antibacterial activities. This work identifies novel roles for IFN-β and RNase L in IEC barrier functions that are targeted by EPEC effectors to escape host defense mechanisms and promote virulence. The IFN-RNase L axis thus represents a potential therapeutic target for enteric infections and GI diseases involving compromised barrier function.

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in children in developing countries. Many EPEC genes involved in virulence are contained within the locus of enterocyte effacement (LEE), a large pathogenicity island. One of the genes at the far righthand end of the LEE encodes EspF, an EPEC secreted protein of unknown function. EspF, like the other Esps, is a substrate for secretion by the type III secretory system. Previous studies found that an espF mutant behaved as wild type in assays of adherence, invasion, actin condensation and tyrosine phosphorylation. As EPEC can kill host cells, we tested esp gene mutants for host cell killing ability. The espF mutant was deficient in host cell killing despite having normal adherence. The addition of purified EspF to tissue culture medium did not cause any damage to host cells, but expression of espF in COS or HeLa cells caused cell death. The mode of cell death in cells transfected with espF appeared to be pure apoptosis. EspF appears to be an effector of host cell death in epithelial cells; its proline-rich structure suggests that it may act by binding to SH3 domains or EVH1 domains of host cell signalling proteins.

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.

Typical enteropathogenic Escherichia coli (EPEC) strains produce bundle-forming pili (BFP), type IVB fimbriae that have been implicated in EPEC virulence, antigenicity, autoaggregation, and localized adherence to epithelial cells (LA). BFP are polymers of bundlin, a pilin protein that is encoded by the bfpA gene found on a large EPEC plasmid. Striking sequence variation has previously been observed among type IV pilin genes of other gram-negative bacterial pathogens (e.g., Pseudomonas and Neisseria spp.). In contrast, the established sequences of bfpA genes from two distantly related prototype EPEC strains vary by only a single base pair. To determine whether bundlin sequences vary more extensively, we used PCR to amplify the bfpA genes from 19 EPEC strains chosen for their various serotypes and sites and years of isolation. Eight different bfpA alleles were identified by sequencing of the PCR products. These alleles can be classified into two major groups. The α group contains three alleles derived from strains carrying O55, O86, O111, O119, O127, or O128 somatic antigens. The β group contains five alleles derived from strains carrying O55, O110, O128ab, O142, or nontypeable antigens. Sequence comparisons show that bundlin has highly conserved and variable regions, with most of the variation occurring in the C-terminal two-thirds of the protein. The results of multilocus enzyme electrophoresis support the hypothesis that bfpA sequences have spread horizontally across distantly related clonal lineages. Strains with divergent bundlin sequences express bundlin protein, produce BFP, and carry out autoaggregation and LA. However, four strains lack most or all of these phenotypes despite having an intact bfpA gene. These results have important implications for our understanding of bundlin structure, transmission of the bfp gene cluster among EPEC strains, and the role of bundlin variation in the evasion of host immune system responses.

Enteric bacteria use a limited array of macromolecular systems to implement diverse pathogenic strategies. The cellular targets of several enteric virulence factors have recently been identified. The themes that have emerged from these studies include the exploitation of molecules that regulate the actin cytoskeleton and the activation of apoptotic pathways to serve the pathogen.

Enteropathogenic Escherichia coli (EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic Δ espB mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the Δ espB mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the Δ espB mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.

Cytotoxic necrotizing factor-1 (CNF1) is commonly found in Escherichia coli isolates from patients with urinary tract infection (UTI). To determine whether CNF1 is an important UTI virulence factor we compared the ability of a clinical E. coli UTI isolate and a CNF1-negative mutant of that isolate to colonize and induce histological changes in the urinary tract in a murine model of ascending UTI. We found no evidence that the mutant strain was attenuated.

Enteropathogenic Escherichia coli expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells. A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of E. coli . We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA. Here we report the construction and analysis of nonpolar mutations in six genes of the bfp cluster, bfpG , bfpB , bfpC , bfpD , bfpP , and bfpH , as well as the further analysis of a previously described bfpA mutant strain that is unable to express bundlin, the pilin protein. We found that mutations in bfpB , which encodes an outer membrane protein; bfpD , which encodes a putative nucleotide-binding protein; and bfpG and bfpC , which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA. The mutation in bfpP , the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA. The mutation in bfpH , which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA. For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes. We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing both inner and outer membrane proteins does not require any accessory proteins. These studies indicate that many bfp gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly.

The mechanisms by which bacteria resist cell-mediated immune responses to cause chronic infections are largely unknown. We report the identification of a large gene present in enteropathogenic strains of Escherichia coli (EPEC) that encodes a toxin that specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma interferon production in response to a variety of stimuli. Lymphostatin, the product of this gene, is predicted to be 366 kDa and shares significant homology with the catalytic domains of the large clostridial cytotoxins. A mutant EPEC strain that has a disruption in this gene lacks the ability to inhibit lymphokine production and lymphocyte proliferation. Enterohemorrhagic E. coli strains of serotype O157:H7 possess a similar gene located on a large plasmid. Loss of the plasmid is associated with loss of the ability to inhibit IL-2 expression while transfer of the plasmid to a nonpathogenic strain of E. coli is associated with gain of this activity. Among 89 strains of E. coli and related bacteria tested, lifA sequences were detected exclusively in strains capable of attaching and effacing activity. Lymphostatin represents a new class of large bacterial toxins that blocks lymphocyte activation.

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.

Enteropathogenic Escherichia coli (EPEC) may be considered a paradigm for a multistage interaction between pathogen and host cell. EPEC strains produce a type IV pilus that is associated with initial adherence to host cells, and these strains possess a type III secretion apparatus that is necessary for transducing signals to host cells. Secretion of three Esp proteins is required for activation of a phosphotyrosine-containing receptor that allows EPEC to bind intimately to host cells via the bacterial outer membrane protein intimin. Intimately attached bacteria rest upon a pedestal composed of host cytoskeletal proteins in an arrangement recognized as the attaching and effacing phenotype. The precise molecular interactions that lead to these dramatic alterations in the host cell cytoskeleton remain to be elucidated.

The pathophysiology of enteropathogenic Escherichia coli (EPEC) diarrhea remains uncertain. In vitro, EPEC stimulates a rapid increase in short-circuit current ( I sc ) across Caco-2 cell monolayers coincident with intimate attaching and effacing (A/E) bacterial adhesion. This study has examined the roles of specific EPEC virulence proteins in this I sc response. EPEC genes encoding EspA, EspB, and EspD, essential for signal transduction in host cells and A/E activity, were also required for modulation of Caco-2 electrolyte transport.

The EspB protein of enteropathogenic Escherichia coli (EPEC) is exported via a type III secretion apparatus. EspB is critical for signaling the host cell and for the development of the attaching and effacing lesion characteristic of EPEC infection. We used cellular fractionation and confocal laser scanning microscopy to determine the cellular location of EspB during infection of HeLa cells. Both methods indicated that EspB is targeted to the cytoplasm of infected cells. Using mutants, we found that EspB targeting to the host cell cytoplasm requires the type III secretion apparatus and the secreted proteins EspA and EspD, but not intimin. These results provide insights into the function of the type III secretion apparatus of EPEC and the functions of the Esp proteins.

The natural habitat of many members of the family Enterobacteriaceae is the intestinal tract of humans and animals. The group includes many genera, such as Escherichia, Salmonella, Shigella, Klebsiella , and Serratia . Some Enterobacteriaceae are part of normal flora and incidentally cause disease, while others, including Salmonella and Shigella , are regularly pathogenic for humans. The Enterobacteriaceae , particularly Escherichia coli , are responsible for much of the childhood diarrheal illnesses seen in the developing world. The emerging resistance of the Enterobacteriaceae , particularly Klebsiella , to various classes of antibiotics, makes them increasingly challenging infections to treat.

Enteropathogenic Escherichia coli strains are able to signal host cells, cause dramatic cytoskeletal rearrangements, and adhere intimately to the cell surface in a process known as the attaching and effacing effect. A pathogenicity island of 35 kb known as the locus of enterocyte effacement (LEE) is necessary and sufficient for this effect. The LEE encodes an outer membrane adhesin called intimin, a type III secretion apparatus, and the EspA and EspB secreted proteins. The DNA sequence of the region between espA and espB revealed a new gene, espD. The product of espD was demonstrated by using a T7 expression system. We constructed a nonpolar mutation in espD and found that the mutant is incapable of the signal transduction events that lead to activation of the putative intimin receptor in host cells and that the mutant fails to induce the attaching and effacing effect. These phenotypes were restored to the mutant by complementation with a plasmid containing the cloned espD locus. We demonstrated by immunoblotting and microsequencing that the EspD protein is secreted via the type III apparatus. Thus, we describe a novel locus encoding a secreted protein that is required for attaching and effacing activity.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Gram-positive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface.

Timely diagnosis of foodborne infection can be critical not only for the patient, but also for the larger community because of the potential to interrupt further spread. This article presents the diagnostic approach to patients with foodborne illness, discussing epidemiologic clues of various foodborne pathogens and their distinguishing clinical features of diagnostic importance. Also discussed are situations whereby stool cultures should be ordered; other helpful stool tests; nonculture methods of identifying organisms and their applicability in clinical settings; the role of pulsed-field gel electrophoresis in typing organisms; and large-scale sharing of data to aid in identification of large outbreaks.