Uropathogenic Escherichia coli triggers oxygen-dependent apoptosis in human neutrophils through the cooperative effect of type 1 fimbriae and lipopolysaccharide

Type 1 fimbriae are the most commonly expressed virulence factor on uropathogenic Escherichia coli. In addition to promoting avid bacterial adherence to the uroepithelium and enabling colonization, type 1 fimbriae recruit neutrophils to the urinary tract as an early inflammatory response. Using clinical isolates of type 1 fimbriated E. coli and an isogenic type 1 fimbria-negative mutant (CN1016) lacking the FimH adhesin, we investigated if these strains could modulate apoptosis in human neutrophils. We found that E. coli expressing type 1 fimbriae interacted with neutrophils in a mannose- and lipopolysaccharide (LPS)-dependent manner, leading to apoptosis which was triggered by the intracellular generation of reactive oxygen species. This induced neutrophil apoptosis was abolished by blocking FimH-mediated attachment, by inhibiting NADPH oxidase activation, or by neutralizing LPS. In contrast, CN1016, which did not adhere to or activate the respiratory burst of neutrophils, delayed the spontaneous apoptosis in an LPS-dependent manner. This delayed apoptosis could be mimicked by adding purified LPS and was also observed by using fimbriated bacteria in the presence of d-mannose. These results suggest that LPS is required for E. coli to exert both pro- and antiapoptotic effects on neutrophils and that the difference in LPS presentation (i.e., with or without fimbriae) determines the outcome. The present study showed that there is a fine-tuned balance between type 1 fimbria-induced and LPS-mediated delay of apoptosis in human neutrophils, in which altered fimbrial expression on uropathogenic E. coli determines the neutrophil survival and the subsequent inflammation during urinary tract infections.     

Invasive Disease Caused by Ciprofloxacin-Resistant Uropathogenic Escherichia coli

 To evaluate the invasiveness of ciprofloxacin-resistant Escherichia coli isolated from the urinary tract, the susceptibility to ciprofloxacin of Escherichia coli strains from patients with invasive urinary tract infection was compared with that of isolates from patients with noninvasive disease. In a 14-month period, 2054 different isolates of Escherichia coli were analyzed, of which 554 (27%) were resistant to ciprofloxacin. One hundred twelve (5.4%) strains were isolated from patients with invasive disease. Resistance was significantly less frequent in isolates from patients with invasive disease (4.5%) than in isolates from patients with noninvasive disease (28.3%) (OR, 0.12; CI 95%, 0.05–0.29;P<0.001). Most ciprofloxacin-resistant strains associated with invasive disease were isolated from bacteremic patients who had recently undergone an invasive procedure involving the urinary tract. Invasive disease is caused more frequently by ciprofloxacin-susceptible strains of Escherichia coli, suggesting that resistance to ciprofloxacin may decrease the invasiveness of uropathogenic Escherichia coli.     

Uropathogenic Escherichia coli induces extrinsic and intrinsic cascades to initiate urothelial apoptosis

A murine model of urinary tract infection identified urothelial apoptosis as a key event in the pathogenesis mediated by uropathogenic Escherichia coli (UPEC), yet the mechanism of this important host response is not well characterized. We employed a culture model of UPEC-urothelium interactions to examine the biochemical events associated with urothelial apoptosis induced by the UPEC strain NU14. NU14 induced DNA cleavage within 5 h that was inhibited by the broad caspase inhibitor ZVAD, and urothelial caspase 3 activity was induced within 3 h of exposure to type 1 piliated NU14 and was dependent upon interactions mediated by the type 1 pilus adhesin FimH. Flow cytometry experiments using chloromethyl-X-rosamine and Indo-1 revealed FimH-dependent mitochondrial membrane depolarization and elevated [Ca(2+)](in), respectively, indicating activation of the intrinsic apoptotic pathway. Consistent with this possibility, overexpression of Bcl(XL) inhibited NU14 activation of caspase 3. Immunoblotting, caspase inhibitors, and caspase activity assays implicated both caspase 2 and caspase 8 in apoptosis, suggesting the involvement of the intrinsic and extrinsic apoptotic cascades. To reconcile the apparent activation of both extrinsic and intrinsic pathways, we examined Bid-green fluorescent protein localization and observed translocation from the cytosol to mitochondria in response to either NU14 or purified FimH. These data suggest that FimH acts as a tethered toxin of UPEC that activates caspase-dependent urothelial apoptosis via direct induction of the extrinsic pathway and that the intrinsic pathway is activated indirectly as a result of coupling by caspase 8-mediated Bid cleavage.     

Uropathogenic strains of Escherichia coli]

In 753 uropathogenic strains of E. coli isolated from urine of patients with urinary tract infections in two localities the authors assessed O and H antigen, mannosensitive adhesin and haemolytical capacities. In strains with assessed O antigen and some of the strains where it was not assessed the authors searched for P fimbriae and for orientation for X adhesin. O antigen was assessed using 33 antisera in 52.05% of the strains. A leading position in the group was held by group 06 (12.21% of all strains). The latter was followed by groups 02, 01, 04, 08, 018 and 021 which together accounted for more than one quarter of all strains. In a large number of O groups the authors found more than one H antigen. Mannosensitive adhesin was found, as expected, in the majority of strains (87.25%). Its absence in more than half 050 strains and in almost one third of 01 strains was striking. 37.18% of all examined strains had haemolytic capacities. 61% of all haemolytic strains belonged only to six O antigenic groups 06, 04, 02, 075, 018 and 021. P fimbriae and X adhesin were assessed in 456 strains. The presence of P fimbriae was proved in 27.41% strains, 72% of all strains with P fimbriae belonged to antigenic groups 01, 02, 04 and 06. X adhesin was detected in 8.33% strains. Of these more than one quarter were formed by strains 021. In strains of this antigenic group we did not detect, however, any P fimbriae. The achieved results do not only inform on some properties of uropathogenic strains of E. coli isolated in some parts of our territory; they indicate also that by means of the applied tests it is possible to obtain a very differentiated picture of these strains. This may be used everywhere where it is desirable to search for groups of strains with homogeneous properties or to obtain at least approximate information on their virulence.     

Uropathogenic Escherichia coli induces chronic pelvic pain

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a debilitating syndrome of unknown etiology often postulated, but not proven, to be associated with microbial infection of the prostate gland. We hypothesized that infection of the prostate by clinically relevant uropathogenic Escherichia coli (UPEC) can initiate and establish chronic pain. We utilized an E. coli strain newly isolated from a patient with CP/CPPS (strain CP1) and examined its molecular pathogenesis in cell culture and in a murine model of bacterial prostatitis. We found that CP1 is an atypical isolate distinct from most UPEC in its phylotype and virulence factor profile. CP1 adhered to, invaded, and proliferated within prostate epithelia and colonized the prostate and bladder of NOD and C57BL/6J mice. Using behavioral measures of pelvic pain, we showed that CP1 induced and sustained chronic pelvic pain in NOD mice, an attribute not exhibited by a clinical cystitis strain. Furthermore, pain was observed to persist even after bacterial clearance from genitourinary tissues. CP1 induced pelvic pain behavior exclusively in NOD mice and not in C57BL/6J mice, despite comparable levels of colonization and inflammation. Microbial infections can thus serve as initiating agents for chronic pelvic pain through mechanisms that are dependent on both the virulence of the bacterial strain and the genetic background of the host.     

Urothelial Cultures Support Intracellular Bacterial Community Formation by Uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) causes most community-acquired and nosocomial urinary tract infections (UTI). In a mouse model of UTI, UPEC invades superficial bladder cells and proliferates rapidly, forming biofilm-like structures called intracellular bacterial communities (IBCs). Using a gentamicin protection assay and fluorescence microscopy, we developed an in vitro model for studying UPEC proliferation within immortalized human urothelial cells. By pharmacologic manipulation of urothelial cells with the cholesterol-sequestering drug filipin, numbers of intracellular UPEC CFU increased 8 h and 24 h postinfection relative to untreated cultures. Enhanced UPEC intracellular proliferation required that the urothelial cells, but not the bacteria, be filipin treated prior to infection. However, neither UPEC frequency of invasion nor early intracellular trafficking events to a Lamp1-positive compartment were modulated by filipin. Upon inspection by fluorescence microscopy, cultures with enhanced UPEC intracellular proliferation exhibited large, dense bacterial aggregates within cells that resembled IBCs but were contained with Lamp1-positive vacuoles. While an isogenic fimH mutant was capable of forming these IBC-like structures, the mutant formed significantly fewer than wild-type UPEC. Similar to IBCs, expression of E. coli iron acquisition systems was upregulated by intracellular UPEC. Expression of other putative virulence factors, including hlyA, cnf1, fliC, kpsD, and the biofilm adhesin yfaL also increased, while expression of fimA decreased and that of flu did not change. These results indicate that UPEC differentially regulates virulence factors in the intracellular environment. Thus, immortalized urothelial cultures that recapitulate IBC formation in vitro represent a novel system for the molecular and biochemical characterization of the UPEC intracellular life cycle.Urinary tract infections (UTI) are the second most common infectious disease, in which uropathogenic Escherichia coli (UPEC) causes approximately 80% of community-acquired infections and 40% of nosocomial infections (51, 62, 63). UTI result in seven million clinic visits per year and cost $3.5 billion in treatment, representing a significant burden on the health care system (37). Half of all women will suffer a UTI during their lifetime, with a 25% recurrence rate within 6 months (10, 15). In 50% of these recurrent infections, the same UPEC strain causes both the primary and relapsing UTI (16, 25).Several host-pathogen interactions between urothelial cells and UPEC have been characterized, including UPEC induction of apoptosis, suppression of cytokine secretion, and invasion of urothelial cells (3, 4, 9, 11, 30, 31, 38, 40, 61). UPEC invasion can be mediated by the Dr adhesin, which binds type IV collagen and decay-accelerating factor, or by type 1 pili, which binds mannose residues by the FimH adhesin. Invasion by either mechanism can result in a persistent infection (18, 19, 41, 43, 44, 56, 57). When type 1 pilus-expressing UPEC is internalized, UPEC proliferates and differentiates into intracellular bacterial communities (IBCs), compact aggregates of intracellular bacteria with biofilm-like properties that have been characterized in a murine UTI model (1, 26, 71). IBCs are primarily identified visually by their morphology and location. IBCs are globular in shape, are tightly packed with coccoid bacteria, and typically occupy most of the host cell cytoplasm, causing urothelial cell distortion. Formation occurs 6 to 24 h postinfection, and IBCs express antigen 43 and type 1 pili and secrete a polysaccharide matrix (1, 26). Bacterial genes leuX, fimH, and surA are required for IBC formation, and several iron acquisition systems are upregulated in IBCs (22, 26, 48, 71). Similar to the murine model, IBC-like structures have been identified during human UTIs. Exfoliated cells exhibiting features resembling IBCs were found in patient urine samples, and UPEC isolates originating from asymptomic bacteriura, pyelonephritis, and cystitis patients formed IBCs in the murine UTI model (17, 52).Replicating the conditions required for IBC formation in vitro has proven to be difficult. One report described formation of IBC-like structures in a bladder carcinoma cell line after host cell permeabilization with a detergent (12). The absence of in vitro models is potentially due to the lack of cell lines that sufficiently recapitulate urothelial characteristics. Here we establish an in vitro model of IBC formation by pharmacologic manipulation of immortalized human urothelial cells. Our immortalized cell lines retain numerous characteristics of urothelial cells, including differentiation, inflammatory response, and apoptosis (3, 4, 30, 31, 39, 49, 64, 65). The IBC-like structures reported here are visually and morphologically similar to IBCs formed in the murine UTI model, occur with comparable kinetics, and similarly upregulate iron acquisition systems. Additionally, IBC formation is attenuated in a UPEC mutant lacking fimH. Thus, we have developed a model for studying UPEC IBC formation in vitro.     

携带usp基因的尿道致病性大肠杆菌强毒株诱导HeLa细胞快速早期凋亡的研究

目的 了解致病基因在尿液分离大肠杆菌中的分布情况,分析由携带usp致病基因的尿道致病性大肠杆菌介导的HeLa细胞快速凋亡.方法 采用PCR检测6种尿道致病性大肠杆菌相关致病基因在28株尿液分离的大肠杆菌的分布;通过黏附试验和锥虫蓝染色对尿液分离的大肠杆菌进行初步致病表型筛选;采用Annexin V/PI法进一步检测大肠杆菌引起HeLa细胞早期凋亡的能力;电子显微镜观察细胞凋亡特征.结果 在28株尿液分离的大肠杆菌中,检测到usp基因阳性6株.通过致病表型筛选试验筛选到2株强致病性大肠杆菌(6N和27N),其中6N菌株携带usp基因,而27N除了没有检测到usp基因,其他致病基因与6N相同.它们可以在4 h破坏大量HeLa细胞,使HeLa死亡;流式细胞仪检测凋亡结果 显示,6N菌株在1.5 h可以诱导20.75%的HeLa细胞发生早期凋亡,而27N只有1.55%的HeLa细胞发生早期凋亡.电镜结果 从形态学证实6N菌株可以快速引起HeLa细胞发生早期凋亡.结论 携带usp基因的尿道致病性大肠杆菌可以诱导HeLa发生快速早期凋亡.     

YbcL of Uropathogenic Escherichia coli Suppresses Transepithelial Neutrophil Migration

Uropathogenic Escherichia coli (UPEC) strains suppress the acute inflammatory response in the urinary tract to ensure access to the intracellular uroepithelial niche that supports the propagation of infection. Our understanding of this initial cross talk between host and pathogen is incomplete. Here we report the identification of a previously uncharacterized periplasmic protein, YbcL, encoded by UPEC that contributes to immune modulation in the urinary tract by suppressing acute neutrophil migration. In contrast to wild-type UPEC, an isogenic strain lacking ybcL expression (UTI89 ΔybcL) failed to suppress transepithelial polymorphonuclear leukocyte (PMN) migration in vitro, a defect complemented by expressing ybcL episomally. YbcL homologs are present in many E. coli genomes; expression of the YbcL variant encoded by nonpathogenic E. coli K-12 strain MG1655 (YbcL_MG) failed to complement the UTI89 ΔybcL defect, whereas expression of the UPEC YbcL variant (YbcL_UTI) in MG1655 conferred the capacity for suppressing PMN migration. This phenotypic difference was due to a single amino acid difference (V78T) between the two YbcL homologs, and a majority of clinical UPEC strains examined were found to encode the suppressive YbcL variant. Purified YbcL_UTI protein suppressed PMN migration in response to live or killed MG1655, and YbcL_UTI was detected in the supernatant during UPEC infection of bladder epithelial cells or PMNs. Lastly, early PMN influx to murine bladder tissue was augmented upon in vivo infection with UTI89 ΔybcL compared with wild-type UPEC. Our findings demonstrate a role for UPEC YbcL in suppression of the innate immune response during urinary tract infection.     

Uropathogenic properties of Escherichia coli in recurrent urinary-tract infection

Properties of Escherichia coli considered to be important in the pathogenesis of urinary-tract infection were investigated. The following properties were more common in E. coli strains isolated from urinary infections than in periurethral strains from healthy individuals: (i) O serogroups 2, 4, 6, 8, 18ab and 75; (ii) high K-antigen titre; (iii) production of haemolysin; (iv) production of fimbriae; (v) fermentation of salicin. The correlation between isolation of a strain from the urinary tract and possession of any single property was not strong; however, strains rich in a combination of these pathogenic properties were rarely isolated from the periurethral area of healthy subjects but were common in urinary infections. Nevertheless, a significant proportion of urinary strains had few pathogenic properties. Strains rich in pathogenic properties were more commonly isolated from upper urinary-tract infections than from lower-tract infections; this indicates that the properties of the invading organism may influence the localisation of infection.     

Uropathogenic Escherichia coli causes fibrotic remodelling of the epididymis

Despite antibiotic treatment, up to 40% of patients have impaired fertility after epididymitis due to serovars of Escherichia coli, a frequent pathogen. The reasons for infertility are unclear, but it may result from epididymal duct obstruction. To determine whether E. coli infection of the epididymis causes obstruction due to fibrosis, and to identify the key mediators, tissues from patients with epididymitis were assessed. Additionally, epididymitis was induced with uropathogenic E. coli (UPEC) or commensal serovars in wild‐type and MyD88^?/? mice, which are relatively unresponsive to bacterial pathogens. Epididymal organ cultures were treated with activin A and bacteria and their histology and levels of cytokines and fibrosis markers were analysed. Patients with epididymitis showed severe fibrosis of the epididymal duct. In mice, UPEC infection also caused fibrosis and ductal obstruction in the cauda epididymis. Levels of mRNA for fibrotic markers (α‐smooth muscle actin, fibronectin) and cytokines (activin A, TNFα, IL‐1α, IL‐1β, IL‐6) and total collagen levels were significantly elevated. This fibrotic response was blunted by the loss of MyD88. Activin A induced fibrosis in cultured epididymis, which was inhibited by the activin‐binding protein follistatin. In summary, bacterial epididymitis causes fibrosis and obstruction. The milder tissue damage in Myd88^?/? UPEC epididymitis highlights the importance of the host response to infection in causing epididymal damage. Elevated levels of activin A in vivo and fibrotic remodelling elicited by activin A in vitro indicate that this cytokine is a potential target for supplementary treatment to antibiotic therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Uropathogenic Escherichia coli flagella aid in efficient urinary tract colonization

In the murine model of urinary tract infections (UTI), cystitis by uropathogenic Escherichia coli (UPEC) occurs through an intimate relationship with the bladder superficial umbrella cell entailing cycles of adherence, invasion, intracellular bacterial community (IBC) formation, and dispersal (fluxing) from the intracellular environment. IBC dispersal is a key step that results in the spread of bacteria over the epithelial surface to initiate additional rounds of IBC formation. We investigated the role of flagella in mediating adherence and motility during UTI, hypothesizing that the dispersion of the IBC would be incomplete in the absence of motility, thus interrupting the IBC pathway and attenuating the infection. Using gfp reporter fusions, the expression of the flagellar class I flhDC and class III fliC genes was monitored to track key points of regulation throughout the pathogenic cascade. In vitro, growth under conditions promoting motility resulted in the robust expression of both fusions. In contrast, only the class I fusion produced significant expression throughout early stages of IBC development including the dispersion stage. Thus, unlike in vitro modeling of motility, the regulatory cascade appeared incomplete in vivo. Throughout IBC formation, nonmotile DeltafliC mutants achieved the same number of IBCs as the wild-type (wt) strain, demonstrating that flagella are neither essential nor required for first- or second-generation IBC formation. However, in competition experiments between wt and DeltafliC strains, the wt was shown to have a fitness advantage in persisting throughout the urinary tract for 2 weeks, demonstrating a subtle but measurable role for flagella in virulence.     

Conditioning of Uropathogenic Escherichia coli for Enhanced Colonization of Host

While in transit within and between hosts, uropathogenic Escherichia coli (UPEC) encounters multiple stresses, including substantial levels of nitric oxide and reactive nitrogen intermediates. Here we show that UPEC, the primary cause of urinary tract infections, can be conditioned to grow at higher rates in the presence of acidified sodium nitrite (ASN), a model system used to generate nitrosative stress. When inoculated into the bladder of a mouse, ASN-conditioned UPEC bacteria are far more likely to establish an infection than nonconditioned bacteria. Microarray analysis of ASN-conditioned bacteria suggests that several NsrR-regulated genes and other stress- and polyamine-responsive factors may be partially responsible for this effect. Compared to K-12 reference strains, most UPEC isolates have increased resistance to ASN, and this resistance can be substantially enhanced by addition of the polyamine cadaverine. Nitrosative stress, as generated by ASN, can stimulate cadaverine synthesis by UPEC, and growth of UPEC in cadaverine-supplemented broth in the absence of ASN can also promote UPEC colonization of the bladder. These results suggest that UPEC interactions with polyamines or stresses such as reactive nitrogen intermediates can in effect reprogram the bacteria, enabling them to better colonize the host.The urinary tract is normally a sterile environment, and it is both hostile and poorly accessible to most microbes. However, roughly one-half of women in the United States experience a urinary tract infection (UTI) at least once in their lifetime, and one-quarter of affected women endure recurrence (22, 25). More than 80% of UTIs are due to strains of uropathogenic Escherichia coli (UPEC), which are usually presumed to be part-time gut flora that have reached the urinary tract by ascension via the periurethral area (53). Transmission of UPEC among individuals occurs primarily by way of fecal-oral routes and, in some cases, may involve the ingestion of contaminated food products or sexual contact (15, 23, 33, 40, 41, 57). In order to survive and disseminate, UPEC must be able to adapt to multiple environments and stresses both within and outside the host.When a UPEC infection occurs, recruitment of nitric oxide (NO)-producing neutrophils to the bladder is an important line of defense (26, 48). Within hours of infection, the nitrite levels in the urine increase up to threefold, and eventually the levels of NO within the bladder are 30- to 50-fold higher than those in uninfected controls (39, 48). The high levels of NO are due in part to inducible NO synthase activity, which is upregulated within 6 h after infection (45). A role may also be played by endothelial NO synthase, which is upregulated and activated in the bladder mucosa by E. coli lipopolysaccharide (36) and by the bacteria themselves, which can produce NO with nitrite reductases under low-oxygen-tension conditions (12). NO is a precursor of a variety of reactive nitrogen intermediates (RNIs), such as peroxynitrite and nitrosothiols, which can inflict extensive damage on nucleic acids, lipids, and proteins. Thiols, amines, aromatic residues, heme groups, and iron-sulfur clusters are particularly susceptible to attack by RNIs, making many key metabolic enzymes targets (17, 18). UPEC may also encounter RNIs outside the urinary tract, possibly during passage through the upper gastrointestinal tract, where nitrate (NO₃⁻) and nitrite (NO₂⁻) levels can be very high, or on the surface of meat products, which are often treated with nitrite as a coloring agent and preservative (15, 16, 27, 29, 64).Adaptive responses that allow a bacterial population to survive one stressful condition can, in some instances, enhance its ability to handle other environmental stresses (2, 5, 31, 32, 42). This cross-protective effect may also potentiate bacterial virulence within a host. Recently, UPEC was found to have the capacity to withstand RNI levels that prevent growth of nonpathogenic E. coli K-12 strains (7, 60). RNI resistance in UPEC is controlled in part by the envelope stress response sigma factor RpoE (σ^E), the RNA chaperone Hfq, the NO-detoxifying enzyme HmpA, and polyamines (7, 38, 60). Expanding on these findings, we show here that UPEC can transiently adapt to high levels of nitrosative stress via a polyamine-linked mechanism, enabling this pathogen to grow more rapidly after subsequent exposure to RNIs and to better colonize the urinary tract in a mouse UTI model system.     

OmpA of Uropathogenic Escherichia coli Promotes Postinvasion Pathogenesis of Cystitis

Type 1 pilus directs bladder epithelial binding and invasion by uropathogenic Escherichia coli (UPEC) in the initial stage of cystitis, but the bacterial determinants of postinvasion events in the pathogenesis of cystitis are largely undetermined. We show here that the UPEC outer membrane protein A (OmpA), a monomeric, major, integral protein component of the bacterial outer membrane, functions as a critical determinant of intracellular virulence for UPEC, promoting persistent infection within bladder epithelium. Using a murine urinary tract infection (UTI) model, we demonstrate that whereas deletion of the UPEC ompA gene did not disrupt initial epithelial binding and invasion by UPEC, it did preclude completion of the intracellular bacterial community (IBC) pathway, accompanied by diminishing bacterial loads in the bladder. This defect in epithelial persistence of the ompA mutant was enhanced in competitive infections with wild-type UPEC. Microscopic examinations revealed that the ompA mutant formed significantly fewer IBCs, and those that were initiated were unable to progress past the early stages of maturation. These defects could be corrected by complementation of ompA. In addition, expression of ompA during wild-type UTI was sharply increased at time points correlated with IBC development and the arrival of host immune effector cells. Our findings establish OmpA as a key UPEC virulence factor that functions after epithelial invasion to facilitate IBC maturation and chronic bacterial persistence.The American patient population suffers each year from approximately 11 million urinary tract infections (UTIs), which are associated with an estimated $1.6 billion in clinical and indirect economic costs (13, 15). This substantial morbidity is exacerbated by the high frequency of recurrent infections despite appropriate antimicrobial therapy. Many recurrences are caused by organisms that are genotypically identical to the initial infecting strain, suggesting that in addition to reinoculation from a gastrointestinal source, recurrence might arise after incomplete eradication of the organism from the bladder (8). Uropathogenic Escherichia coli (UPEC) are the primary etiology of UTIs, causing up to 90% of community-acquired infections (45, 46). Persistence of UPEC within the urinary tracts of experimentally infected mice has been associated with their ability to invade superficial epithelial (umbrella) cells of the bladder (34, 36), establishing a niche for host colonization beyond the luminal surface to the intracellular compartment and sheltering the pathogen from host defenses. Acute cystitis is characterized by the development of intracellular bacterial communities (IBCs) in both mice and humans (3, 47). The IBCs represent clonal expansion of invaded bacteria proceeding through a complex maturation cycle (24) that facilitates the subsequent development of quiescent reservoirs or nests of UPEC within Lamp1-positive intracellular vesicles (40). These long-term resident bacteria are sequestered from antibiotic therapy and remain apparently undetected by host immune mechanisms (37).While type 1 pili and flagella are critical and contributing factors, respectively (16, 18, 53), for the early events of binding and invasion, additional factors that promote the intracellular phenotypes of UPEC are largely unidentified. We recently demonstrated that the periplasmic chaperone SurA is required for both invasion and intracellular maturation of UPEC (27). This chaperone, conserved across numerous gram-negative bacteria, facilitates transit of nascent polypeptides destined for insertion into the outer membrane (OM) as β-barrel porins (11, 31). SurA deletion abrogates production of the type 1 pilus usher FimD, thus precluding expression of type 1 pili and epithelial colonization. However, SurA-dependent proteins also contribute to postinvasion phenotypes of UPEC (19, 25). More recently, deletion of the hfq gene (encoding an RNA chaperone) was shown to impair the ability of UPEC to form IBCs, perhaps through downstream effects on membrane integrity (30). Further, it is logical to surmise that the extracellular domains of OM proteins might mediate pathogenesis by participating in interactions with the host and potentially with other bacteria in a community. Therefore, we sought to specify SurA-dependent OM proteins that contribute to the postinvasion phenotypes of UPEC during cystitis, namely, IBC maturation and intraepithelial persistence.In the present study, we explored the pathogenic role of outer membrane protein A (OmpA), a SurA-dependent, major protein component of the E. coli OM that assumes a β-barrel conformation with eight transmembrane segments and four extracellular loops (4). Multiple cellular functions have been proposed for OmpA, including maintaining cell structural integrity, serving as a phage and colicin receptor, and mediating F-factor-dependent conjugation (29). Although its role as a porin is debated (5, 7), some data suggest that OmpA may form nonspecific channels for diffusion of solutes. With regard to bacterial virulence, OmpA is necessary for successful infection by E. coli K-1, the leading gram-negative cause of neonatal septicemia and meningitis (51). E. coli K-1 ompA mutants are significantly less serum resistant (42, 52) and show decreased capacity to invade cultured human brain microvascular endothelial cells, which model the blood-brain barrier (41, 43). Other authors have speculated that OmpA represents a pathogen-associated molecular pattern that interacts with antigen-presenting cells (20-22). In UPEC the pathogenic function of OmpA has yet to be elucidated, although it was one of several OM proteins whose expression was augmented during in vitro growth of UPEC in urine (1). We demonstrate here that OmpA provides a pathogenic advantage to UPEC during establishment of its intracellular niche and promotes persistence of UPEC within the bladder following acute cystitis.     

Uropathogenic Escherichia coli Isolates from Pregnant Women in Different Countries

Urinary tract infection (UTI) is common during pregnancy and can be associated with negative outcomes for both the mother and fetus. Increased risk of infection among these patients has been attributed to physiological changes, and less focus has been placed on Escherichia coli, the most frequent causative agent. We investigated the virulence properties of isolates causing UTI in pregnant women in Sweden, Uganda, and Vietnam, as well as nonpregnant women in Sweden. Although phylogenetic group B2 was the most prevalent group, more Ugandan isolates belonged to group B1, associated with commensal strains, than isolates from other countries. Adherence to and invasion of urothelial cells, key events in the infection process, were low among group B1 isolates from pregnant Swedish women compared to those from nonpregnant patients. Similar levels of adherence and invasion were seen in isolates from pregnant women in Uganda and Vietnam. More biofilm was formed by group B2 isolates than by those belonging to group B1 and by Ugandan group B2 isolates than by those from pregnant Swedish and Vietnamese women. The antigen 43a-encoding gene, fluA_CFT073, was most prevalent among Ugandan isolates. Expression of the biofilm components, curli and cellulose, was low among all isolates. Multidrug resistance was more common among isolates from Uganda and Vietnam than among those from Swedish patients. We suggest that while bacterial virulence properties play an important role in UTI during pregnancy, physiological changes in the host may contribute more to the incidence of infection caused by less virulent E. coli.     

Virulence Plasmid Harbored by Uropathogenic Escherichia coli Functions in Acute Stages of Pathogenesis

Urinary tract infections (UTIs), the majority of which are caused by uropathogenic Escherichia coli (UPEC), afflict nearly 60% of women within their lifetimes. Studies in mice and humans have revealed that UPEC strains undergo a complex pathogenesis cycle that involves both the formation of intracellular bacterial communities (IBC) and the colonization of extracellular niches. Despite the commonality of the UPEC pathogenesis cycle, no specific urovirulence genetic profile has been determined; this is likely due to the fluid nature of the UPEC genome as the result of horizontal gene transfer and numerous genes of unknown function. UTI89 has a large extrachromosomal element termed pUTI89 with many characteristics of UPEC pathogenicity islands and that likely arose due to horizontal gene transfer. The pUTI89 plasmid has characteristics of both F plasmids and other known virulence plasmids. We sought to determine whether pUTI89 is important for virulence. Both in vitro and in vivo assays were used to examine the function of pUTI89 using plasmid-cured UTI89. No differences were observed between UTI89 and plasmid-cured UTI89 based on growth, type 1 pilus expression, or biofilm formation. However, in a mouse model of UTI, a significant decrease in bacterial invasion, CFU and IBC formation of the pUTI89-cured strain was observed at early time points postinfection compared to the wild type. Through directed deletions of specific operons on pUTI89, the cjr operon was partially implicated in this observed defect. Our findings implicate pUTI89 in the early aspects of infection.Urinary tract infections (UTIs) represent, by number, one of the most important bacterial infectious diseases in highly industrialized countries (20). Sixty-percent of all women will have at least one UTI within their lifetime (21, 57, 63). This infection results in nearly 7 million physician office visits and $3.5 billion dollars annually in the United States alone (19). It is thought that acute UTIs develop when bacteria from the fecal flora colonize the vaginal and periurethral mucosa and are subsequently introduced into the bladder by urethral ascension. Women who present with an initial episode of acute UTI have a 25 to 44% chance of developing a second and a 3% chance of experiencing three episodes within 6 months of the initial UTI (20). Recurrence occurs despite appropriate antibiotic treatment and clearance of the initial infection from the urine. A large percentage of recurrent UTI are caused by the same strain of bacteria as the initial infection (65). The high frequency of same-strain recurrences supports the notion that a UPEC reservoir may exist in the affected individual.Uropathogenic Escherichia coli (UPEC) is the leading causative agent of UTI, responsible for up to 85% of community-acquired UTI and 25% of nosocomial UTI (62). Using a well-characterized clinical UPEC isolate, UTI89, in a mouse cystitis model, it has been demonstrated that the pathogenesis of UTI involves intracellular and extracellular components (34, 35, 47). The bladder surface is covered with a urothelium composed of very large superficial umbrella cells which are coated with uroplakins that expose a terminal mannose moiety (77). Type 1 pili and its adhesin, FimH, are required for attachment and invasion, thus playing a critical role in the infection process (47, 50, 76). The FimH protein has a negatively charged pocket that accommodates a mannose unit, namely, those exposed by uroplakins on the bladder surface (28). In addition to mannose, it was shown that FimH binds α3β1 integrin subunits expressed by bladder epithelial cells mediating uptake of UPEC (18). Integrins link extracellular matrix proteins with the actin cytoskeleton and can initiate a signaling cascade resulting in bacterial internalization (46, 47). Invasion of UPEC also involves cellular lipid raft components, including uroplakin 1a, caveolin-1, and Rac-1, and internalized bacteria are retained within compartments resembling fusiform vesicles (6, 17). After invasion, bladder epithelial cells are capable of expelling UPEC, presumably as part of an innate defense (69). Another potent innate defense is the exfoliation of umbrella cells and influx of neutrophils (50). A mechanism used by UPEC to combat these defenses is to escape into the cytoplasm of superficial umbrella cells, where they are capable of rapidly replicating into biofilm-like intracellular bacterial communities (IBCs) comprised of 10⁴ to 10⁵ bacteria (1, 34). Upon IBC maturation, bacteria detach from the IBC biomass, flux out of the host cell into the bladder lumen, and spread to neighboring cells forming next-generation IBCs (34, 38). Thus, IBC formation represents a mechanism by which invasion of a single bacterium can result in rapid expansion of UPEC numbers in the urinary tract leading to disease (1, 51). Upon resolution of infection, the terminally differentiated umbrella cells are replaced through Bmp4-specific signaling, restoring the impermeable uroplakin barrier (53). In addition, bacteria can invade the transitional epithelium beneath the umbrella cells, establish a quiescent intracellular reservoir (QIR) and remain dormant until they reemerge and potentially cause a recurrence (52). Reemergence from a QIR could partially explain why recurrent UTI is often caused by a strain identical to the primary infection (65). Recent work has shown that this is a common pathway among UPEC isolates (22), and evidence of this pathway also has been observed in humans (64). The regulatory mechanisms governing IBC development and dispersal in response to environmental cues are still unknown.Despite many similarities among UPEC isolates, genomic features that are unique to UPEC have not yet been identified. Many studies have identified UPEC as a diverse class of E. coli that have evolved multiple and redundant strategies to colonize the urinary tract (75). These diverse genomes have been acquired through horizontal gene transfer (HGT). HGT generates diversity between bacterial species and, through natural selection, contributes to the evolution of bacterial species (16, 40, 66). In addition to genetic variability, newly acquired DNA can enable adaptation to life in a specialized niche through specific factors. The acquisition of DNA by HGT results in the exchange of large regions of DNA called genomic islands (GIs) between bacteria (31). GIs can be acquired in several ways: (i) inheritance of a plasmid that can remain autonomous or recombine into the chromosome, (ii) integration of a lysogenic phage into the chromosome, and/or (iii) insertion of a linear DNA fragment into the chromosome usually by transposition or recombination (14, 43, 70). Some GIs are referred to as pathogenicity islands (PAIs). PAIs contain large genomic DNA regions often greater than 20 kb that carry at least one virulence gene, are inserted within or near tRNA genes, contain direct repeats and mobility sequences, and can frequently be identified by a differing G+C content relative to the bacterial genome (24). In addition, PAIs often do not represent homogeneous pieces of DNA but can instead be mosaiclike structures generated by a multistep process involving genomic acquisition, loss, and rearrangement (25). Study of UPEC PAIs has revealed that they encode numerous virulence factors, including pili and adhesins, toxins, hemolysins, and iron uptake systems. Interestingly, these same virulence factors can be plasmid or phage encoded on intestinal E. coli isolates. Thus, UPEC PAIs could represent former plasmid-derived sequences. In addition, UPEC PAIs carry many cryptic genes, open reading frames (ORFs) of unrelated and even unknown functions, and pseudogenes. A UPEC isolate, UTI89, carries a large extrachromosomal element with many characteristics of UPEC PAIs. This plasmid, pUTI89, has a high proportion of pseudogenes generated by insertion events, ORFs homologous to proteins of nonplasmid origin, and orthologs of enteroinvasive E. coli (EIEC) proteins found on a large virulence plasmid (12). These features describe a UPEC PAI that has potentially not yet incorporated into the genome, known as a “PAI precursor” (41). pUTI89 is approximately 114 kb and, like plasmid F, contains the tra operon for conjugative transfer, as well as genes associated with plasmid replication and inheritance.Although UPEC strains can cause disease in the urinary tract, they can also exist within the human intestinal tract as part of the normal microbiota. Thus, UPEC strains are distinct from the commensal E. coli residing in the gastrointestinal tract in that they possess virulence factors enabling successful transition to and colonization of the urinary tract (8). Factors required for successful infection in addition to type 1 pili are being extensively investigated (76). While studies have demonstrated that additional adhesin systems, toxins, autotransporters, iron acquisition systems, and other factors (reviewed in reference 75) may be important in establishing infection, there is also an abundance of unknown genes labeled as hypothetical or assigned putative functions that may play a role in virulence (32). Here, we studied the role of pUTI89 in the virulence of UPEC. Regions of pUTI89 were shown to be present in the majority of a panel of clinical isolates, suggesting maintenance and transmission within the community. The effect of curing pUTI89 from strain UTI89 was investigated. We found that pUTI89 is involved in an early feature of in vivo infection, even though we could not detect any alterations in growth, type 1 pilus expression and function, or biofilm formation in vitro in the absence of pUTI89. A region on pUTI89 encoding the cjr operon found in EIEC was implicated in this early defect.     

Local Generation of Kynurenines Mediates Inhibition of Neutrophil Chemotaxis by Uropathogenic Escherichia coli

During epithelial infections, pathogenic bacteria employ an array of strategies to attenuate and evade host immune responses, including the influx of polymorphonuclear leukocytes (PMN; neutrophils). Among the most common bacterial infections in humans are those of the urinary tract, caused chiefly by uropathogenic Escherichia coli (UPEC). During the establishment of bacterial cystitis, UPEC suppresses innate responses via multiple independent strategies. We recently described UPEC attenuation of PMN trafficking to the urinary bladder through pathogen-specific local induction of indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolic enzyme previously shown to have regulatory activity only in adaptive immunity. Here, we investigated the mechanism by which IDO induction attenuates PMN migration. Local tryptophan limitation, by which IDO is known to influence T cell longevity and proliferation, was not involved in its effect on PMN trafficking. Instead, metabolites in the IDO pathway, particularly l-kynurenine, directly suppressed PMN transepithelial migration and induced an attached, spread morphology in PMN both at rest and in the presence of chemotactic stimuli. Finally, kynurenines represent known ligands of the mammalian aryl hydrocarbon receptor (AHR), and UPEC infection of Ahr^−/− mice recapitulated the derepressed PMN recruitment observed previously in Ido1^−/− mice. UPEC therefore suppresses neutrophil migration early in bacterial cystitis by eliciting an IDO-mediated increase in local production of kynurenines, which act through the AHR to impair neutrophil chemotaxis.     

Escherichia coli K1 inhibits proinflammatory cytokine induction in monocytes by preventing NF-kappaB activation

Phagocytes are well-known effectors of the innate immune system to produce proinflammatory cytokines and chemokines such as tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-8 during infections. Here, we show that infection of monocytes with wild-type Escherichia coli K1, which causes meningitis in neonates, suppresses the production of cytokines and chemokines (TNF-alpha, regulated on activation, normal T expressed and secreted, macrophage-inflammatory protein-1beta, IL-1beta, and IL-8). In contrast, infection of monocytes with a mutant E. coli, which lacks outer membrane protein A (OmpA- E. coli) resulted in robust production of cytokines and chemokines. Wild-type E. coli K1 (OmpA+ E. coli) prevented the phosphorylation and its degradation of inhibitor of kappaB, thereby blocking the translocation of nuclear factor (NF)-kappaB to the nucleus. OmpA+ E. coli-infected cells, subsequently subjected to lipopolysaccharide challenge, were crippled severely in their ability to activate NF-kappaB to induce cytokine/chemokine production. Selective inhibitors of the extracellular signal-regulated kinase (ERK) 1/2 pathway and p38 mitogen-activated protein kinase (MAPK), but not Jun N-terminal kinase, significantly reduced the activation of NF-kappaB and the production of cytokines and chemokines induced by OmpA- E. coli, indicating a role for these kinases in the NF-kappaB/cytokine pathway. It is interesting that the phosphorylation of ERK 1/2 and p38 MAPK was notably reduced in monocytes infected with OmpA+ E. coli when compared with monocytes infected with OmpA- E. coli, suggesting that the modulation of upstream events common for NF-kappaB and MAPKs by the bacterium is possible. The ability of OmpA+ E. coli K1 to inhibit the macrophage response temporarily may enable bacterial survival and growth within the host for the onset of meningitis by E. coli K1.     

Phenotypic Heterogeneity Enables Uropathogenic Escherichia coli To Evade Killing by Antibiotics and Serum Complement

Uropathogenic strains of Escherichia coli (UPEC) are the major cause of bacteremic urinary tract infections. Survival in the bloodstream is associated with different mechanisms that help to resist serum complement-mediated killing. While the phenotypic heterogeneity of bacteria has been shown to influence antibiotic tolerance, the possibility that it makes cells refractory to killing by the immune system has not been experimentally tested. In the present study we sought to determine whether the heterogeneity of bacterial cultures is relevant to bacterial targeting by the serum complement system. We monitored cell divisions in the UPEC strain CFT073 with fluorescent reporter protein. Stationary-phase cells were incubated in active or heat-inactivated human serum in the presence or absence of different antibiotics (ampicillin, norfloxacin, and amikacin), and cell division and complement protein C3 binding were measured by flow cytometry and immunofluorescence microscopy. Heterogeneity in the doubling times of CFT073 cells in serum enabled three phenotypically different subpopulations to be distinguished, all of them being recognized by the C3 component of the complement system. The population of rapidly growing cells resists serum complement-mediated lysis. The dominant subpopulation of cells with intermediate growth rate is susceptible to serum. The third population, which does not resume growth upon dilution from stationary phase, is simultaneously protected from serum complement and antibiotics.     

Uropathogenic Escherichia coli outer membrane antigens expressed during urinary tract infection

Uncomplicated urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) represents a prevalent and potentially severe infectious disease. In this study, we describe the application of an immunoproteomics approach to vaccine development that has been used successfully to identify vaccine targets in other pathogenic bacteria. Outer membranes were isolated from pyelonephritis strain E. coli CFT073 cultured under conditions that mimic the urinary tract environment, including iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells. To identify antigens that elicit a humoral response during experimental UTI, outer membrane proteins were separated by two-dimensional gel electrophoresis and probed using pooled antisera from 20 CBA/J mice chronically infected with E. coli CFT073. In total, 23 outer membrane antigens, including a novel iron compound receptor, reacted with the antisera and were identified by mass spectrometry. These antigens also included proteins with known roles in UPEC pathogenesis, such as ChuA, IroN, IreA, Iha, IutA, and FliC. These data demonstrate that an antibody response is directed against these virulence-associated factors during UTI. We also show that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent (P < 0.01) among UPEC strains than among fecal-commensal E. coli isolates. Thus, we suggest that the conserved outer membrane antigens identified in this study could be rational candidates for a UTI vaccine designed to elicit protective immunity against UPEC infection.     

Binding of Pili from Uropathogenic Escherichia coli to Membranes Secreted by Human Colonocytes and Enterocytes

PapG adhesins mediate the binding of uropathogenic Escherichia coli. Although receptors for these adhesins have not been demonstrated in intestinal epithelia, the colonic microflora includes strains of uropathogenic E. coli. We now report that surfactant-like particles secreted by the human intestine contain receptors for PapG adhesins and may provide an intestinal habitat for uropathogenic bacteria.