The solution structure of PapGII from uropathogenic Escherichia coli and its recognition of glycolipid receptors

Uropathogenic Escherichia coli (UPEC) is the primary cause of symptomatic urinary tract infection. The P-pili, a bacterial surface organelle, mediates the bacterial host–cell adhesion. The PapG adhesin has generated much interest in recent years, not only because of its clinical value, i.e. in the prevention of microbial adherence, but also because of its ability to promote virulence. Using multidimensional nuclear magnetic resonance (NMR) and deuteration we have determined the solution structure of the adhesin domain from PapGII (PapGII-198). The novel structure of PapGII-198 is composed of a large elongated jellyroll motif. Despite an automated search of the structural database failing to reveal any similar proteins, PapGII adhesin shares some structural similarities with FimH. Furthermore, interpretation of NMR-titration data has enabled us to identify the putative binding site for the globoseries of oligosaccharides. This work provides insight into UPEC pathogenesis as well as aiding the development of preventative therapies and the guidance of future mutagenesis programmes.     

Adhesion and entry of uropathogenic Escherichia coli

To effectively colonize a host animal and cause disease, many bacterial pathogens have evolved the mechanisms needed to invade and persist within host cells and tissues. Recently it was discovered that uropathogenic Escherichia coli, the primary causative agent of urinary tract infections, can invade and replicate within uroepithelial cells. This can provide E. coli with a survival advantage, allowing the microbes to better resist detection and clearance by both innate and adaptive immune defence mechanisms. Adhesive organelles, including type 1, P, and S pili along with Dr adhesins, promote both bacterial attachment to and invasion of host tissues within the urinary tract. Interactions mediated by these adhesins can also stimulate a number of host responses that can directly influence the outcome of a urinary tract infection.     

Structural requirements for the interaction of human IgA with the human polymeric Ig receptor

Transport of polymeric IgA onto mucosal surfaces to become secretory IgA is mediated by the polymeric Ig receptor (pIgR). To study the interaction of human dimeric IgA (dIgA) (the predominant form of IgA polymer) with the human pIgR (hpIgR), we generated recombinant wild-type dIgA1 and dIgA2m(1) and various mutant dIgA1 and analyzed their interaction with a recombinant human secretory component and membrane-expressed hpIgR. We found that wild-type dIgA1 and dIgA2m(1) bound to recombinant human secretory component with similar affinity and were transcytosed by the hpIgR to the same extent. Mutation of the IgA Calpha2 domain residue Cys311 to Ser reduced binding to hpIgR, possibly through disruption of noncovalent interactions between the Calpha2 domain and domain 5 of the receptor. Within the Calpha3 domain of IgA1, we found that combined mutation of residues Phe411, Val413, and Thr414, which lie close to residues previously implicated in hpIgR binding, abolished interaction with the receptor. Mutation of residue Lys377, located very close to this same region, perturbed receptor interaction. In addition, 4 aa (Pro440-Phe443), which lie on a loop at the domain interface and form part of the binding site for human FcalphaRI, appear to contribute to hpIgR binding. Lastly, use of a monomeric IgA1 mutant lacking the tailpiece revealed that the tailpiece does not occlude hpIgR-binding residues in IgA1 monomers. This directed mutagenesis approach has thus identified motifs lying principally across the upper surface of the Calpha3 domain (i.e., that closest to Calpha2) critical for human pIgR binding and transcytosis.     

Interaction of uropathogenic Escherichia coli with host uroepithelium

Investigation into the pathogenesis of Escherichia coli urinary tract infection has provided numerous insights into the mechanisms by which bacteria adhere, grow and persist in association with host tissue. Many molecular details concerning the interaction of these bacteria with their host have been elucidated, and the murine model of cystitis has generated a new paradigm by which acute and recurrent urinary tract infections may proceed. These advances could potentially result in the development of novel vaccines and therapies for this very costly disease.     

CD46 (membrane cofactor protein) acts as a human epithelial cell receptor for internalization of opsonized uropathogenic Escherichia coli

Escherichia coli is a common urinary pathogen whose uptake into epithelial cells is mediated by attachment through type 1 fimbriae. In this study, we show by using using human urinary tract epithelial cells that maximal internalization of E. coli is achieved only when bacteria are opsonized with complement. The concentrations of complement proteins in the urine rise sufficiently during infection to allow bacterial opsonization. The complement regulatory protein, CD46 (membrane cofactor protein), acts in cohort with fimbrial adhesion to promote the uptake of pathogenic E. coli. This uptake is inhibited by RNA interference to lower the expression of CD46 and by soluble CD46 that will competitively inhibit opsonized bacteria binding to cell surface CD46. We propose that efficient internalization of uropathogenic E. coli by the human urinary tract depends on cooperation between fimbrial-mediated adhesion and C3 receptor (CD46)-ligand interaction. Complement receptor-ligand interaction could pose a new target for interrupting the cycle of reinfection due to intracellular bacteria.     

Structural and sequence diversity of the pathogenicity island of uropathogenic Escherichia coli which encodes the USP protein

A total of 321 uropathogenic Escherichia coli (UPEC) strains and 12 strains of E. coli isolated from stool samples of healthy individuals, which were previously shown to be positive in colony hybridization test using the usp (encoding for the uropathogenic-specific protein) DNA probe, were examined by PCR amplification to determine the size of the usp gene and the pathogenicity island (PI). Three types of size variation were observed for the usp gene and four types for the PI. Sequencing analysis of the PIs from seven representative strains (six UPEC and one from a normal healthy individual) revealed that the usp genes can be classified into two groups, each having different sequences in the 3'-terminal region. The peptides encoded by the three open reading frames (ORFs) downstream of usp had identical 23 amino acid residues in the C-terminal region. The subregion encoding these small ORFs has a mosaic structure constituted of six segments. The positions of these segments vary from strain to strain, and in some strains, two to four segments are deleted. This indicates that rearrangements occur frequently in this region and the mosaic arrangement apparently contributes to the size variation observed in the PCR examination of the usp genes and PIs.     

Structural requirements for high efficiency endocytosis of the human transferrin receptor.

Wild-type and mutant human transferrin receptors (TR) have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. By functional studies of the mutant TRs, we have identified the tetrapeptide sequence, YXRF, in the cytoplasmic tail of the receptor as the internalization signal required for high efficiency endocytosis and shown that transplanted internalization signals from the low density lipoprotein receptor (LDLR) and the cation-independent mannose-6-phosphate receptor (Man-6-PR) are able to promote rapid internalization of the human TR. A six-residue LDLR signal, FDNPVY, is required for activity in TR, whereas a four-residue Man-6-PR signal, YSKV, is sufficient. These data indicate that internalization signals are interchangeable self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. Putative internalization signals in the cytoplasmic tails of other receptors and membrane proteins can be identified based on the sequence patterns of the LDLR, Man-6-PR, and TR signals. Two such putative four-residue internalization signals, one from the poly-Ig receptor and one from the asialoglycoprotein receptor, were tested for activity by transplantation into TR and were found to promote high efficiency internalization. These results suggest that an exposed tight turn is the conformational motif for high efficiency endocytosis.     

Covert operations of uropathogenic Escherichia coli within the urinary tract

Entry into host cells is required for many bacterial pathogens to effectively disseminate within a host, avoid immune detection and cause disease. In recent years, many ostensibly extracellular bacteria have been shown to act as opportunistic intracellular pathogens. Among these are strains of uropathogenic Escherichia coli (UPEC), the primary causative agents of urinary tract infections (UTIs). UPEC are able to transiently invade, survive and multiply within the host cells and tissues constituting the urinary tract. Invasion of host cells by UPEC is promoted independently by distinct virulence factors, including cytotoxic necrotizing factor, Afa/Dr adhesins, and type 1 pili. Here we review the diverse mechanisms and consequences of host cell invasion by UPEC, focusing also on the impact of these processes on the persistence and recurrence of UTIs.     

Isolation of generalized transducing bacteriophages for uropathogenic strains of Escherichia coli

The traditional genetic procedure for random or site-specific mutagenesis in Escherichia coli K-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenic E. coli strains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel of E. coli strains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenic E. coli strains 536, UTI89, and NU14. The highest-efficiency transducing phage, ΦEB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenic E. coli.     

Cytotoxic and hemolytic activities in uropathogenic Escherichia coli

Fifty nine Escherichia coli strains obtained from patients with upper or lower urinary tract infections (UTI) and 30 E. coli strains isolated from stools of healthy individuals were tested for hemolytic and cytotoxic activities. Forty four percent of uropathogenic E. coli (UPEC) and 3.3% of fecal E. coli were hemolytic. Among the hemolytic UPEC, 92% produced alpha-hemolysin. A cytotoxic activity was detected in culture filtrates of 71% of UPEC strains and 30% of fecal E. coli. No relationship was found between cytotoxic and hemolytic activities or between cytotoxic titers and UPEC origin (upper or lower UTI). E. coli cytotoxin has a cytocidal activity against some epithelioid cultured cell lines (Vero, HeLa and Hep-2) but was almost inactive for avian-fibroblast cells. Cytotoxin-affected cells appeared rounded, refractile and detached from the surface of the vessel. Some characteristics exhibited by the cytotoxin as the morphological response induced on cells, the increasing of cytopathic effect with time, its irreversible cytocidal activity and its heat-lability resemble the properties described for E. coli Verotoxin (VT). Adherence to uroepithelial cells is recognized as a virulence factor for UPEC. It is suggested that cell damage by cytotoxic and adhering UPEC might contribute to E. coli virulence to urinary tract.     

Instability of pathogenicity islands in uropathogenic Escherichia coli 536

The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of E. coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II536 and PAI III536 were the most unstable islands followed by PAI I536 and PAI V536, whereas PAI IV536 was stable. In addition, we found that deletion of PAI II536 and PAI III536 was induced by several environmental stimuli. Whereas excision of PAI I536, PAI II536, and PAI V536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II536 and PAI III536 were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic E. coli can be modulated by deletion of PAIs.     

Nucleotide sequence of the papA gene encoding the Pap pilus subunit of human uropathogenic Escherichia coli

The papA gene of the uropathogenic strain Escherichia coli J96, coding for the Pap pili subunit, was subjected to DNA sequencing, and found to code for an 185-amino acid-long polypeptide with a 22-amino acid-long signal peptide. Here we present the primary sequence, the hydrophilicity profile, and the predicted polypeptide secondary structure of the Pap pili subunit.     

Actin-gated intracellular growth and resurgence of uropathogenic Escherichia coli

Strains of uropathogenic Escherichia coli (UPEC) can invade terminally differentiated superficial bladder epithelial cells and subsequently multiply, forming large biofilm-like inclusions referred to as pods. In contrast, within immature bladder cells UPEC enter a more quiescent state and often fail to replicate appreciably. As immature bladder epithelial cells undergo terminal differentiation the actin cytoskeleton is radically diminished, a phenomenon that we reasoned could influence the intracellular fate of UPEC. Here we show that UPEC within undifferentiated bladder cells is trafficked into acidic compartments having key features of late endosomes and lysosomes. These UPEC-containing vacuoles are often enmeshed within a network of actin filaments, the disruption of which stimulates intravacuolar growth and efflux of UPEC in cell culture-based studies. In this in vitro model system, release of UPEC into the host cytosol further stimulates intracellular bacterial growth and the rapid development of pod-like inclusions. These inclusions, as well as those observed using an in vivo mouse model, develop in association with cytokeratin intermediate filaments that may act as scaffolding for intracellular biofilm formation. Our data suggest an aetiological basis for recurrent urinary tract infections, linking bladder cell differentiation and the accompanying redistribution of actin microfilaments with the resurgence of UPEC from quiescent intravacuolar reservoirs within the bladder epithelium.     

The influence of net surface charge on the interaction of uropathogenic Escherichia coli with human neutrophils

Escherichia coli strains, grown to suppress fimbrial expression, synthesised enhanced quantities of polysaccharide capsule, which significantly lessened their binding to heparin sepharose columns. In the presence of poly-L-lysine, these strains were strongly retained on the columns confirming their highly anionic nature. Uropathogenic strains of E. coli expressing type 1 fimbrial adhesins activated the respiratory burst, the degranulation response and the release of leukotrienes from human neutrophils (PMN) to a significantly greater extent than the same strains grown in a medium to suppress this fimbrial expression. The addition of the poly-cation poly-L-lysine, however, selectively increased neutrophil activation in response to these non-fimbriate strains. This dose-dependent effect was reversed by the addition of heparin suggesting a mechanism dependent on surface charge. The results of this study suggest that non-specific mechanisms involving the neutralisation of surface charge, in addition to specific receptor and adhesin mediated events could affect neutrophil activation at sites of infection.     

Components of SurA required for outer membrane biogenesis in uropathogenic Escherichia coli

Background

SurA is a periplasmic peptidyl-prolyl isomerase (PPIase) and chaperone of Escherichia coli and other Gram-negative bacteria. In contrast to other PPIases, SurA appears to have a distinct role in chaperoning newly synthesized porins destined for insertion into the outer membrane. Previous studies have indicated that the chaperone activity of SurA rests in its “core module” (the N- plus C-terminal domains), based on in vivo envelope phenotypes and in vitro binding and protection of non-native substrates.

Methodology/Principal Findings

In this study, we determined the components of SurA required for chaperone activity using in vivo phenotypes relevant to disease causation by uropathogenic E. coli (UPEC), namely membrane resistance to permeation by antimicrobials and maturation of the type 1 pilus usher FimD. FimD is a SurA-dependent, integral outer membrane protein through which heteropolymeric type 1 pili, which confer bladder epithelial binding and invasion capacity upon uropathogenic E. coli, are assembled and extruded. Consistent with prior results, the in vivo chaperone activity of SurA in UPEC rested primarily in the core module. However, the PPIase domains I and II were not expendable for wild-type resistance to novobiocin in broth culture. Steady-state levels of FimD were substantially restored in the UPEC surA mutant complemented with the SurA N- plus C-terminal domains. The addition of PPIase domain I augmented FimD maturation into the outer membrane, consistent with a model in which domain I enhances stability of and/or substrate binding by the core module.

Conclusions/Significance

Our results confirm the core module of E. coli SurA as a potential target for novel anti-infective development.