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.     

Uropathogenic Escherichia coli AL511 requires flagellum to enter renal collecting duct cells

Escherichia coli is the leading cause of urinary tract infections, but the mechanisms governing renal colonization by this bacterium remain poorly understood. We investigated the ability of 13 E. coli strains isolated from the urine of patients with pyelonephritis and cystitis and normal stools to invade collecting duct cells, which constitute the first epithelium encountered by bacteria ascending from the bladder. The AL511 clinical isolate adhered to mouse collecting duct mpkCCD_cl4 cells, used as a model of renal cell invasion, and was able to enter and persist within these cells. Previous studies have shown that bacterial flagella play an important role in host urinary tract colonization, but the role of flagella in the interaction of E. coli with renal epithelial cells remains unclear. An analysis of the ability of E. coli AL511 mutants to invade renal cells showed that flagellin played a key role in bacterial entry. Both flagellum filament assembly and the motor proteins MotA and MotB appeared to be required for E. coli AL511 uptake into collecting duct cells. These findings indicate that pyelonephritis-associated E. coli strains may invade renal collecting duct cells and that flagellin may act as an invasin in this process.     

Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors

Bacterial adherence to and invasion of eukaryotic cells are important mechanisms of pathogenicity. Most Gram-positive bacteria interact with the components of the host extracellular matrix (ECM) to adhere to, colonize and invade cells and tissues. The bacterial proteins that bind to components of the ECM harbour signal sequences for their secretion and mechanisms of anchoring to the host cell surface. However, in recent years, some cell-surface adhesins and invasins of Gram-positive bacteria have been described that do not possess a signal sequence or a membrane anchor. These proteins are secreted by an as-yet-unknown mechanism and are probably localized on the bacterial surface by reassociation. These anchorless but surface-located adhesins and invasins represent a new class of virulence factors.     

Dynamin2- and endothelial nitric oxide synthase-regulated invasion of bladder epithelial cells by uropathogenic Escherichia coli

Invasion of bladder epithelial cells by uropathogenic Escherichia coli (UPEC) contributes to antibiotic-resistant and recurrent urinary tract infections (UTIs), but this process is incompletely understood. In this paper, we provide evidence that the large guanosine triphosphatase dynamin2 and its partner, endothelial nitric oxide (NO) synthase (NOS [eNOS]), mediate bacterial entry. Overexpression of dynamin2 or treatment with the NO donor S-nitrosothiols increases, whereas targeted reduction of endogenous dynamin2 or eNOS expression with ribonucleic acid interference impairs, bacterial invasion. Exposure of mouse bladder to small molecule NOS inhibitors abrogates infection of the uroepithelium by E. coli, and, concordantly, bacteria more efficiently invade uroepithelia isolated from wild-type compared with eNOS(-/-) mice. E. coli internalization promotes rapid phosphorylation of host cell eNOS and NO generation, and dynamin2 S-nitrosylation, a posttranslational modification required for the bacterial entry, also increases during E. coli invasion. These findings suggest that UPEC escape urinary flushing and immune cell surveillance by means of eNOS-dependent dynamin2 S-nitrosylation and invasion of host cells to cause recurrent UTIs.     

Tamm-Horsfall protein binds to type 1 fimbriated Escherichia coli and prevents E. coli from binding to uroplakin Ia and Ib receptors

The adherence of uropathogenic Escherichia coli to the urothelial surface, a critical first step in the pathogenesis of urinary tract infection (UTI), is controlled by three key elements: E. coli adhesins, host receptors, and host defense mechanisms. Although much has been learned about E. coli adhesins and their urothelial receptors, little is known about the role of host defense in the adherence process. Here we show that Tamm-Horsfall protein (THP) is the principal urinary protein that binds specifically to type 1 fimbriated E. coli, the main cause of UTI. The binding was highly specific and saturable and could be inhibited by d-mannose and abolished by endoglycosidase H treatment of THP, suggesting that the binding is mediated by the high-mannose moieties of THP. It is species-conserved, occurring in both human and mouse THPs. In addition, the binding to THP was much greater with an E. coli strain bearing a phenotypic variant of the type 1 fimbrial FimH adhesin characteristic of those prevalent in UTI isolates compared with the one prevalent in isolates from the large intestine of healthy individuals. Finally, a physiological concentration of THP completely abolished the binding of type 1 fimbriated E. coli to uroplakins Ia and Ib, two putative urothelial receptors for type 1 fimbriae. These results establish, on a functional level, that THP contains conserved high-mannose moieties capable of specific interaction with type 1 fimbriae and strongly suggest that this major urinary glycoprotein is a key urinary anti-adherence factor serving to prevent type 1 fimbriated E. coli from binding to the urothelial receptors.     

The solution structure of the invasive tip complex from Afa/Dr fibrils

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.     

Escherichia coli physiology and metabolism dictates adaptation to diverse host microenvironments

Bacterial growth in the host is required for pathogenesis. To successfully grow in vivo, pathogens have adapted their metabolism to replicate in specific host microenvironments. These adaptations reflect the nutritional composition of their host niches, inter-bacterial competition for carbon and energy sources, and survival in the face of bactericidal defense mechanisms. A subgroup of Escherichia coli, which cause urinary tract infection, bacteremia, sepsis, and meningitis, have adapted to grow as a harmless commensal in the nutrient-replete, carbon-rich human intestine but rapidly transition to pathogenic lifestyle in the nutritionally poorer, nitrogen-rich urinary tract. We discuss bacterial adaptations that allow extraintestinal pathogenic E. coli to establish both commensal associations and virulence as the bacterium transits between disparate microenvironments within the same individual.     

Clathrin, AP-2, and the NPXY-binding subset of alternate endocytic adaptors facilitate FimH-mediated bacterial invasion of host cells

The FimH adhesin, localized at the distal tips of type 1 pili, binds mannose-containing glycoprotein receptors like alpha3beta1 integrins and stimulates bacterial entry into target host cells. Strains of uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections, utilize FimH to invade bladder epithelial cells. Here we set out to define the mechanism by which UPEC enters host cells by investigating four of the major entry routes known to be exploited by invasive pathogens: caveolae, clathrin, macropinocytosis and secretory lysosomes. Using pharmacological inhibitors in combination with RNA interference against specific endocytic pathway components, mutant host cell lines and a mouse infection model system, we found that type 1 pili-dependent bacterial invasion of host cells occurs via a cholesterol- and dynamin-dependent phagocytosis-like mechanism. This process did not require caveolae or secretory lysosomes, but was modulated by calcium levels, clathrin, and cooperative input from the primary clathrin adaptor AP-2 and a subset of alternate adaptors comprised of Numb, ARH and Dab2. These alternate clathrin adaptors recognize NPXY motifs, as found within the cytosolic tail of beta1 integrin, suggesting a functional link between the engagement of integrin receptors by FimH and the clathrin-dependent uptake of type 1-piliated bacteria.     

Integrin-mediated host cell invasion by type 1-piliated uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, typically express filamentous adhesive organelles called type 1 pili that mediate both bacterial attachment to and invasion of bladder urothelial cells. Several host proteins have previously been identified as receptors for type 1 pili, but none have been conclusively shown to promote UPEC entry into host bladder cells. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, we have identified beta1 and alpha3 integrins as key host receptors for UPEC. FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, beta1 and alpha3 integrin receptors co-localize with invading type 1-piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by beta1 and alpha3 integrin-specific antibodies and by disruption of the beta1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of beta1 integrin that alter integrin signaling also variably affect UPEC entry into host cells, by either attenuating or boosting invasion frequencies. Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signaling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that beta1 and alpha3 integrins are functionally important receptors for type 1 pili-expressing bacteria within the urinary tract and possibly at other sites within the host.     

Escherichia coli bind to urinary bladder epithelium through nonspecific sialic acid mediated adherence

The first step in the bacterial colonization and infection of uropathogenic Escherichia coli is adherence to uroepithelium. Over 80% of all urinary tract infections are caused by E. coli. Uropathogenic E. coli express several adherence factors including type 1 and P fimbriae, which mediate attachment to the uroepithelium through specific binding to different glycoconjugate receptors. We showed that P and type 1 fimbriae are not the sole adhesins on uropathogenic E. coli and sialic acid also mediates nonspecific bacterial adherence of uropathogenic E. coli and urinary bladder epithelium.     

Precise and rapid assessment of Escherichia coli adherence to vaginal epithelial cells by flow cytometry

BACKGROUND: In the pathogenesis of Escherichia coli urinary tract infections (UTIs) in women, infecting bacteria adhere to vaginal and periurethral epithelial cells prior to ascending to the bladder and causing infection. Complex interactions among specific bacterial adhesins and various host factors appear to influence adherence of E. coli to mucosal surfaces such as the urogenital epithelium. To conduct population-based studies assessing host epithelial cell determinants that influence bacterial attachment, a method of measuring bacterial adherence utilizing clinically derived epithelial cell samples is needed. METHODS: We developed and standardized an efficient, accurate, high-throughput method for analyzing the adherence of uropathogenic E. coli to clinical samples containing a large number of exfoliated vaginal epithelial cells (VEC). Three wild-type E. coli strains isolated from women with UTI (IA2 expressing pap-encoded, class II fimbriae only; F24 expressing pap-encoded, class II and type 1 fimbriae; and F20, without pap-encoded or type I fimbriae) were transformed with gfpmut3, encoding green fluorescent protein, incubated with VECs, and analyzed by flow cytometry. RESULTS: Enumeration of the binding of each E. coli strain to 10,000 VECs showed reproducible, highly significant strain-dependent differences in adherence to VECs. Differential analysis of the relative contributions of type 1 pili and P fimbrial-mediated binding to the adherence phenotype was performed. It demonstrated that IA2 binding was dependent entirely on P fimbriae, whereas F24 binding was dependent on both P and type 1 fimbriae. CONCLUSIONS: This method has great potential for use in high-throughput analyses of clinically derived epithelial cell samples and will be valuable in population-based investigations of host-parasite interactions in UTI utilizing VECs collected from specific patient groups.     

Host collagen signal induces antigen I/II adhesin and invasin gene expression in oral Streptococcus gordonii

Microbial interactions with host molecules, and programmed responses to host environmental stimuli, are critical for colonization and initiation of pathogenesis. Bacteria of the genus Streptococcus are primary colonizers of the human mouth. They express multiple cell-surface adhesins that bind salivary components and other oral bacteria and enable the development of polymicrobial biofilms associated with tooth decay and periodontal disease. However, the mechanisms by which streptococci invade dentine to infect the tooth pulp and periapical tissues are poorly understood. Here we show that production of the antigen I/II (AgI/II) family polypeptide adhesin and invasin SspA in Streptococcus gordonii is specifically upregulated in response to a collagen type I signal, minimally the tri-peptide Gly-Pro-Xaa (where Xaa is hydroxyproline or alanine). Increased AgI/II polypeptide expression promotes bacterial adhesion and extended growth of streptococcal cell chains along collagen type I fibrils that are characteristically found within dentinal tubules. These observations define a new model of host matrix signal-induced tissue penetration by bacteria and open the way for novel therapy opportunities for oral invasive diseases.     

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.     

Type 1, P and S fimbriae, and afimbrial adhesin I are not essential for uropathogenic Escherichia coli to adhere to and invade bladder epithelial cells

Fimbrial (type 1, P, and S) and afimbrial adhesins, the unique virulence traits of uropathogenic Escherichia coli (UPEC), are well recognized for their role in the initial step of uropathogenesis. In this study, we investigated whether these adhesins are dispensable for UPEC in adherence and invasion of uroepithelial cells by using E. coli isolates (n=40) from cystitis patients and T-24 cells, the bladder carcinoma cell line. We found all isolates adherent to T-24 cells within 15 min of infection. In invasion assay, all isolates could invade T-24 cells to a variable degree; 22.5% of them were found highly invasive. About 33% of isolates that do not have any recognized adhesins were as invasive as other isolates. The amplitude of invasiveness was also independent of the adhesins. In conclusion, this study demonstrates that type 1 fimbriae, P fimbriae, S fimbriae, and afimbrial adhesin I are not required for UPEC to adhere to and invade uroepithelial cells.     

A fluorescent antibody staining technique to detect bacterial adherence to urinary tract epithelial cells

A fluorescent antibody technique has been devised to assess specifically the adherence of Escherichia coli in vitro to uroepithelial cells from healthy women and bacterial adherence in vivo to cells from women with symptomatic urinary tract infection. Similar values can be obtained using methylene blue as the bacterial stain, but this depends on the experience of the observer. The results indicate that E. coli adherence to uroepithelial cells is a factor in the infection process. We suggest that uroepithelial cells from patients with symptoms of a urinary tract infection whose urine has a low bacterial count (less than 10(3) cells/ml) could be examined for the presence of adherent uropathogens, which may be indicative of an infection. Although the fluorescent staining technique possibly would be expensive, the results would be specific and reliable. Other diagnostic and research applications suggest themselves as in studies of bacterial colonization of mucosal tissues or plastic catheters, where conventional light microscopy and radiolabelling methods are not effective.     

Fermentative degradation of acetone by an enrichment culture in membrane-separated culture devices and in cell suspensions

Abstract We have recently demonstrated that cultured human intestinal HT-29 and Caco-2 cell lines express receptors for the F1845 fimbrial adhesin harbored by the diarrheagenic C1845 Escherichia coli (Kernéis et al., Infect. Immun. 59 (1991) 4013–4018). This adhesinn belongs to a family of adhesins including the Dr hemagglutinin and the afimbrial adhesin AFA-1 harbored by uropathogenic E. coli . Here we investigated the cell association of laboratory E. coli strains expressing the Dr hemagglutinin and the afimbrial adhesin AFA-I with human cultured enterocyte-like or mucosecreting cells. We observed that the E. coli strains bearing these adhesins adhere both to human intestinal undifferentiated and differentiated fluid-transporting cells, and to mucus-secreting cells. This result strongly suggests a high capacity of intestinal colonization for the uropathogenic E. coli harboring adhesive factors belonging to the Dr adhesin family. These results further corroborate the intestinal colonization by uropathogenic E. coli of the Dr family related to the fecal-perineal-urethral hypothesis of urinary tract infection pathogenesis.     

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting “bacteriomimetic” materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.     

Ferritinophagy drives uropathogenic Escherichia coli persistence in bladder epithelial cells

Autophagy is a cellular recycling pathway, which in many cases, protects host cells from infections by degrading pathogens. However, uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections (UTIs), persist within the urinary tract epithelium (urothelium) by forming reservoirs within autophagosomes. Iron is a critical nutrient for both host and pathogen, and regulation of iron availability is a key host defense against pathogens. Iron homeostasis depends on the shuttling of iron-bound ferritin to the lysosome for recycling, a process termed ferritinophagy (a form of selective autophagy). Here, we demonstrate for the first time that UPEC shuttles with ferritin-bound iron into the autophagosomal and lysosomal compartments within the urothelium. Iron overload in urothelial cells induces ferritinophagy in an NCOA4-dependent manner causing increased iron availability for UPEC, triggering bacterial overproliferation and host cell death. Addition of even moderate levels of iron is sufficient to increase and prolong bacterial burden. Furthermore, we show that lysosomal damage due to iron overload is the specific mechanism causing host cell death. Significantly, we demonstrate that host cell death and bacterial burden can be reversed by inhibition of autophagy or inhibition of iron-regulatory proteins, or chelation of iron. Together, our findings suggest that UPEC persist in host cells by taking advantage of ferritinophagy. Thus, modulation of iron levels in the bladder may provide a therapeutic avenue to controlling UPEC persistence, epithelial cell death, and recurrent UTIs.     

Host-specificity of uropathogenic Escherichia coli depends on differences in binding specificity to Gal alpha 1-4Gal-containing isoreceptors.

Four G adhesins, cloned from uropathogenic Escherichia coli strains, were examined for binding to glycolipids and various eukaryotic cells. PapGAD110 and PapGIA2 showed virtually identical binding patterns to Gal alpha 1-4Gal-containing glycolipids, while PapGJ96 differed slightly and PrsGJ96 markedly with respect to the effect of neighbouring groups on the binding. Their hemagglutination patterns confirmed the existence of three receptor-binding specificities. While the PapG adhesins bound to uroepithelial cells from man (T24) but not to those from the dog (MDCK II), the reverse was true of PrsG. These binding patterns were largely explained by the absence or presence of appropriate glycolipid isoreceptors, although the inability of the PapG adhesins to bind MDCK II cells was attributed to an inappropriate presentation of their receptor epitopes. The high prevalence of PrsG-like specificities observed among wild-type dog uropathogenic E. coli isolates, together with the determined isoreceptor composition of human and dog kidney target tissues, suggest variation in receptor specificity as a mechanism for shifting host specificity, and that this variation has evolved in response to the topography of the host cellular receptors. The receptor-binding half proposed for the predicted amino acid sequences of the four G adhesins and the corresponding adhesin of one of the dog E. coli isolates varied considerably among the three receptor-binding groups of adhesins, but only little within each group.     

Uropathogenic Escherichia coli induces serum amyloid a in mice following urinary tract and systemic inoculation

Serum amyloid A (SAA) is an acute phase protein involved in the homeostasis of inflammatory responses and appears to be a vital host defense component with protective anti-infective properties. SAA expression remains poorly defined in many tissues, including the urinary tract which often faces bacterial challenge. Urinary tract infections (UTIs) are usually caused by strains of uropathogenic Escherichia coli (UPEC) and frequently occur among otherwise healthy individuals, many of whom experience bouts of recurrent and relapsing infections despite the use of antibiotics. To date, whether SAA is present in the infected urothelium and whether or not the induction of SAA can protect the host against UPEC is unclear. Here we show, using mouse models coupled with immunofluorescence microscopy and quantitative RT-PCR, that delivery of UPEC either directly into the urinary tract via catheterization or systemically via intraperitoneal injection triggers the expression of SAA. As measured by ELISA, serum levels of SAA1/2 were also transiently elevated in response to UTI, but circulating SAA3 levels were only up-regulated substantially following intraperitoneal inoculation of UPEC. In in vitro assays, physiological relevant levels of SAA1/2 did not affect the growth or viability of UPEC, but were able to block biofilm formation by the uropathogens. We suggest that SAA functions as a critical host defense against UTIs, preventing the formation of biofilms both upon and within the urothelium and possibly providing clinicians with a sensitive serological marker for UTI.