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.     

Immune Modulation by Group B Streptococcus Influences Host Susceptibility to Urinary Tract Infection by Uropathogenic Escherichia coli

Urinary tract infection (UTI) is most often caused by uropathogenic Escherichia coli (UPEC). UPEC inoculation into the female urinary tract (UT) can occur through physical activities that expose the UT to an inherently polymicrobial periurethral, vaginal, or gastrointestinal flora. We report that a common urogenital inhabitant and opportunistic pathogen, group B Streptococcus (GBS), when present at the time of UPEC exposure, undergoes rapid UPEC-dependent exclusion from the murine urinary tract, yet it influences acute UPEC-host interactions and alters host susceptibility to persistent outcomes of bladder and kidney infection. GBS presence results in increased UPEC titers in the bladder lumen during acute infection and reduced inflammatory responses of murine macrophages to live UPEC or purified lipopolysaccharide (LPS), phenotypes that require GBS mimicry of host sialic acid residues. Taken together, these studies suggest that despite low titers, the presence of GBS at the time of polymicrobial UT exposure may be an overlooked risk factor for chronic pyelonephritis and recurrent UTI in susceptible groups, even if it is outcompeted and thus absent by the time of diagnosis.     

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.     

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.     

Murine Model of Chemotherapy-Induced Extraintestinal Pathogenic Escherichia coli Translocation

Escherichia coli is a major cause of life-threatening infections in patients with neutropenia, particularly those receiving chemotherapy for the treatment of cancer. In most cases, these infections originate from opportunistic strains living within the patient''s gastrointestinal tract which then translocate to major organ systems. There are no animal models that faithfully recapitulate these infections, and, as such, the host or bacterial factors that govern this process remain unidentified. We present here a novel model of chemotherapy-induced bacterial translocation of E. coli. Oral gavage of BALB/c mice with a clinical isolate of extraintestinal pathogenic E. coli (ExPEC) leads to stable and long-term colonization of the murine intestine. Following the induction of neutropenia with the chemotherapeutic drug cyclophosphamide, ExPEC translocates from the intestine to the lungs, liver, spleen, and kidneys with concomitant morbidity in infected animals. Translocation can also occur in mice bearing mammary tumors, even in the absence of chemotherapy. Translocation of ExPEC is also associated with an increase of the diversity of bacterial DNA detected in the blood. This is the first report of a chemotherapy-based animal model of ExPEC translocation in cancerous mice, a system that can be readily used to identify important virulence factors for this process.     

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.     

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.     

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 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.     

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.     

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.     

Environmental Modulation of Oral Treponeme Virulence in a Murine Model

This investigation examined the effects of environmental alteration on the virulence of the oral treponemes Treponema denticola and Treponema pectinovorum. The environmental effects were assessed by using a model of localized inflammatory abscesses in mice. In vitro growth of T. denticola and T. pectinovorum as a function of modification of the cysteine concentration significantly enhanced abscess formation and size. In contrast, growth of T. denticola or T. pectinovorum under iron-limiting conditions (e.g., dipyridyl chelation) had no effect on abscess induction in comparison to that when the strains were grown under normal iron conditions. In vivo modulation of the microenvironment at the focus of infection with Cytodex beads demonstrated that increasing the local inflammation had no effect on lesion induction or size. In vivo studies involved the determination of the effects of increased systemic iron availability (e.g., iron dextran or phenylhydrazine) on the induction, kinetics, and size of lesions. T. denticola induced significantly larger lesions in mice with iron pretreatment and demonstrated systemic manifestations of the infectious challenge and an accompanying spreading lesion with phenylhydrazine pretreatment (e.g., increases in circulating free hemoglobin). In contrast, T. pectinovorum virulence was minimally affected by this in vivo treatment to increase iron availability. T. denticola virulence, as evaluated by lesion size, was increased additively by in vivo iron availability, and cysteine modified growth of the microorganism. Additionally, galactosamine sensitized mice to a lethal outcome following infection with both T. denticola and T. pectinovorum, suggesting an endotoxin-like activity in these treponemes. These findings demonstrated the ability to modify the virulence capacity of T. denticola and T. pectinovorum by environmental conditions which can be evaluated by using in vivo murine models.     

Molecular Characterisation of Uropathogenic Escherichia coli Isolates at a Tertiary Care Hospital in South India

Uropathogenic Escherichia coli (UPEC) express a multitude of virulence factors (VFs) to break the inertia of the mucosal barrier of the urinary tract. The aim of the present study was undertaken to characterised the UPEC strains and to correlate carriage of specific virulence markers with different phylogroups and also to correlate these findings with clinical outcome of patients. A total of 156 non-repeated, clinically significant UPEC isolates were studied. Virulent genes were determined by two set of multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Antibiograms and patient’s clinical outcomes were collected in a structured pro forma. Of the 156 patients infected by UPEC strains with significant bacterial counts the most common predisposing factors were diabetes (45.5%) followed by carcinoma (7%). On analysis of the VF genes of the isolates, a majority of strains (140; 90%) were possessing the fimH gene followed by iutA (98; 63%), papC (76; 49%), cnf1 (46; 29.5%), hlyA (45; 29%) and neuC (8; 5%), respectively. On phylogenetic analysis, 27 (17%) isolates were belong to phylogroup A, 16 (10%) strains to Group B1, 59 (38%) were from Group B2 and 54 (35%) were from Group D. High prevalence of antibiotic resistance was observed among the isolates. The incidence of papC, cnf1 and hlyA was significantly higher (P < 0.05) among the isolates from relapse patients. Our findings indicate that virulent as well as commensal strains are capable of causing urinary tract infection. Virulence genes as well as patients-related factors are equally responsible for the development of infections and also that virulence genes may help such isolates to persist even with appropriate chemotherapy and be responsible for recurrent infections.     

Uropathogenic Escherichia coli potentiates type 1 pilus-induced apoptosis by suppressing NF-kappaB

Urinary tract infections (UTIs) are among the most common inflammatory diseases. Acute UTIs are typically caused by type 1-piliated Escherichia coli and result in urothelial apoptosis, local cytokine release, and neutrophil infiltration. To examine the urothelial apoptotic response, a human urothelial cell line was incubated with various E. coli isolates and was then characterized by flow cytometry. Uropathogenic E. coli (UPEC) induced rapid urothelial apoptosis that was strictly dependent upon interactions mediated by type 1 pili. Interestingly, nonpathogenic HB101 E. coli expressing type 1 pili induced apoptosis at approximately 50% of the level induced by UPEC, suggesting that pathogenic strains contribute to apoptosis by pilus-independent mechanisms. Consistent with this possibility, UPEC blocked activity of an NF-kappaB-dependent reporter in response to inflammatory stimuli, yet this effect was independent of functional type 1 pili and was not mediated by laboratory strains of E. coli. UPEC suppressed NF-kappaB by stabilizing IkappaBalpha, and UPEC rapidly altered cellular signaling pathways. Finally, blocking NF-kappaB activity increased the level of piliated HB101-induced apoptosis to the level of apoptosis induced by UPEC. These results suggest that UPEC blocks NF-kappaB and thereby enhances type 1 pili-induced apoptosis as a component of the uropathogenic program.     

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.     

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.