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Urinary tract infection (UTI) is the second most common infection in humans, making it a global health priority. Nearly half of all women will experience a symptomatic UTI, with uropathogenic Escherichia coli (UPEC) being the major causative agent of the infection. Although there has been extensive research on UPEC virulence determinants, the importance of host-specific metabolism remains understudied. We report here that UPEC upregulates the expression of ethanolamine utilization genes during uncomplicated UTIs in humans. We further show that UPEC ethanolamine metabolism is required for effective bladder colonization in the mouse model of ascending UTI and is dispensable for bladder colonization in an immunocompromised mouse model of UTI. We demonstrate that although ethanolamine metabolism mutants do not show increased susceptibility to antimicrobial responses of neutrophils, this metabolic pathway is important for surviving the innate immune system during UTI. This study reveals a novel aspect of UPEC metabolism in the host and provides evidence for an underappreciated link between bacterial metabolism and the host immune response., (Copyright © 2018 American Society for Microbiology.)

Objectives: To determine the relationship between clinically diagnosed catheter-associated urinary tract infection (CAUTI) and standardized criteria and to assess microorganism-level differences in symptom burden in a cohort of catheterized nursing home (NH) residents., Design: Post hoc analysis of a prospective longitudinal study., Setting: Twelve NHs in southeast Michigan., Participants: NH residents with indwelling urinary catheters (n = 233; 90% white, 52% male, mean age 73.7)., Measurements: Clinical and demographic data, including CAUTI epidemiology and symptoms, were obtained at study enrollment, 14 days, and monthly thereafter for up to 1 year., Results: One hundred twenty participants with an indwelling catheter (51%) were prescribed systemic antibiotics for 182 clinically diagnosed CAUTIs. Common signs and symptoms were acute change in mental status (28%), fever (21%), and leukocytosis (13%). Forty percent of clinically diagnosed CAUTIs met Loeb's minimum criteria, 32% met National Health Safety Network (NHSN) criteria, and 50% met Loeb's minimum or NHSN criteria. CAUTIs involving Staphylococcus aureus and Enterococcus spp. were least likely to meet criteria. CAUTIs involving Klebsiella pneumoniae were most likely to meet Loeb's minimum criteria (odds ratio (OR) = 9.7, 95% confidence interval (CI) = 2.3-40.3), possibly because of an association with acute change in mental status (OR = 5.9, 95% CI = 1.8-19.4)., Conclusion: Fifty percent of clinically diagnosed CAUTIs met standardized criteria, which represents an improvement in antibiotic prescribing practices. At the microorganism level, exploratory data indicate that symptom burden may differ between microorganisms. Exploration of CAUTI signs and symptoms associated with specific microorganisms may yield beneficial information to refine existing tools to guide appropriate antibiotic treatment., (© 2016, Copyright the Authors Journal compilation © 2016, The American Geriatrics Society.)

Uropathogenic Escherichia coli (UPEC) is the primary cause of uncomplicated urinary tract infections (UTIs). Whereas most infections are isolated cases, 1 in 40 women experience recurrent UTIs. The rise in antibiotic resistance has complicated the management of chronic UTIs and necessitates new preventative strategies. Currently, no UTI vaccines are approved for use in the United States, and the development of a highly effective vaccine remains elusive. Here, we have pursued a strategy for eliciting protective immunity by vaccinating with small molecules required for pathogenesis, rather than proteins or peptides. Small iron-chelating molecules called siderophores were selected as antigens to vaccinate against UTI for this vaccine strategy. These pathogen-associated stealth siderophores evade host immune defenses and enhance bacterial virulence. Previous animal studies revealed that vaccination with siderophore receptor proteins protects against UTI. The poor solubility of these integral outer-membrane proteins in aqueous solutions limits their practical utility. Because their cognate siderophores are water soluble, we hypothesized that these bacterial-derived small molecules are prime vaccine candidates. To test this hypothesis, we immunized mice with siderophores conjugated to an immunogenic carrier protein. The siderophore-protein conjugates elicited an adaptive immune response that targeted bacterial stealth siderophores and protected against UTI. Our study has identified additional antigens suitable for a multicomponent UTI vaccine and highlights the potential use of bacterial-derived small molecules as antigens in vaccine therapies., Competing Interests: The authors declare no conflict of interest.

Escherichia coli is the leading cause of urinary tract infections (UTIs), one of the most common infections in humans. P fimbria was arguably the first proposed virulence factor for uropathogenic E. coli, based on the capacity of E. coli isolated from UTIs to adhere to exfoliated epithelial cells in higher numbers than fecal strains of E. coli. Overwhelming epidemiologic evidence has been presented for involvement of P fimbriae in colonization. It has been difficult, however, to demonstrate this requirement for uropathogenic strains in animal models of infections or in humans. In this study, a signature-tagged mutagenesis screen identified a P-fimbrial gene (papC) and 18 other genes as being among those required for full fitness of cystitis isolate E. coli F11. A P-fimbrial mutant was outcompeted by the wild-type strain in cochallenge in the murine model of ascending UTI, and this colonization defect could be complemented with the cloned pap operon. To our knowledge, this study is the first to fulfill molecular Koch's postulates in which a pathogenic strain was attenuated by mutation of pap genes and then complemented to restore fitness, confirming P fimbria as a virulence factor in a pathogenic clinical isolate., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Urinary tract infection (UTI) represents one of the most common bacterial infections in humans and uropathogenic E. coli (UPEC) is the major causative agent of UTI in people. Research on UPEC and other bacterial pathogens causing UTI has now identified the critical role of metal transport systems in the pathogenesis of UTI. Here we review the major effectors of metal transport in bacteria and host proteins that impair metal acquisition by bacterial pathogens. In particular, we describe the studies that identified iron, zinc and nickel import and copper export as key virulence and fitness determinants during UTI. Various metal transport systems and mechanisms that govern the expression of metal transport systems are also presented here. Specific examples from UPEC and other uropathogens, when available, are presented to depict the battle for metals at the host-pathogen interface during UTI.

The human genitourinary tract is a common anatomical niche for polymicrobial infection and a leading site for the development of bacteremia and sepsis. Most uncomplicated, community-acquired urinary tract infections (UTI) are caused by Escherichia coli, while another bacterium, Proteus mirabilis, is more often associated with complicated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent requirements for specific central pathways in vivo despite colonizing and occupying the same host environment. Using mutants of specific central metabolism enzymes, we determined glycolysis mutants lacking pgi, tpiA, pfkA, or pykA all have fitness defects in vivo for P. mirabilis but do not affect colonization of E. coli during UTI. Similarly, the oxidative pentose phosphate pathway is required only for P. mirabilis in vivo. In contrast, gluconeogenesis is required only for E. coli fitness in vivo. The remarkable difference in central pathway utilization between E. coli and P. mirabilis during experimental UTI was also observed for TCA cycle mutants in sdhB, fumC, and frdA. The distinct in vivo requirements between these pathogens suggest E. coli and P. mirabilis are not direct competitors within host urinary tract nutritional niche. In support of this, we found that co-infection with E. coli and P. mirabilis wild-type strains enhanced bacterial colonization and persistence of both pathogens during UTI. Our results reveal that complementary utilization of central carbon metabolism facilitates polymicrobial disease and suggests microbial activity in vivo alters the host urinary tract nutritional niche.

Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of uncomplicated urinary tract infection (UTI), manifested by inflammation of the urinary bladder, in humans and is a major global public health concern. Molecular pathogenesis of UPEC has been primarily examined using murine models of UTI. Translational research to develop novel therapeutics against this major pathogen, which is becoming increasingly antibiotic resistant, requires a thorough understanding of mechanisms involved in pathogenesis during human UTIs. Total RNA-sequencing (RNA-seq) and comparative transcriptional analysis of UTI samples to the UPEC isolates cultured in human urine and laboratory medium were used to identify novel fitness genes that were specifically expressed during human infection. Evidence for UPEC genes involved in ion transport, including copper efflux, nickel and potassium import systems, as key fitness factors in uropathogenesis were generated using an experimental model of UTI. Translational application of this study was investigated by targeting Cus, a bacterial copper efflux system. Copper supplementation in drinking water reduces E. coli colonization in the urinary bladder of mice. Additionally, our results suggest that anaerobic processes in UPEC are involved in promoting fitness during UTI in humans. In summary, RNA-seq was used to establish the transcriptional signature in UPEC during naturally occurring, community acquired UTI in women and multiple novel fitness genes used by UPEC during human infection were identified. The repertoire of UPEC genes involved in UTI presented here will facilitate further translational studies to develop innovative strategies against UTI caused by UPEC.

Competition for iron is a critical component of successful bacterial infections, but the underlying in vivo mechanisms are poorly understood. We have previously demonstrated that lipocalin 2 (LCN2) is an innate immunity protein that binds to bacterial siderophores and starves them for iron, thus representing a novel host defense mechanism to infection. In the present study we show that LCN2 is secreted by the urinary tract mucosa and protects against urinary tract infection (UTI). We found that LCN2 was expressed in the bladder, ureters, and kidneys of mice subject to UTI. LCN2 was protective with higher bacterial numbers retrieved from bladders of Lcn2-deficient mice than from wild-type mice infected with the LCN2-sensitive Escherichia coli strain H9049. Uropathogenic E. coli mutants in siderophore receptors for salmochelin, aerobactin, or yersiniabactin displayed reduced fitness in wild-type mice, but not in mice deficient of LCN2, demonstrating that LCN2 imparts a selective pressure on bacterial growth in the bladder. In a human cohort of women with recurrent E. coli UTIs, urine LCN2 levels were associated with UTI episodes and with levels of bacteriuria. The number of siderophore systems was associated with increasing bacteriuria during cystitis. Our data demonstrate that LCN2 is secreted by the urinary tract mucosa in response to uropathogenic E. coli challenge and acts in innate immune defenses as a colonization barrier that pathogens must overcome to establish infection., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Urinary tract infections (UTI) are common and represent a substantial economic and public health burden. Roughly 80% of these infections are caused by a heterogeneous group of uropathogenic Escherichia coli (UPEC) strains. Antibiotics are standard therapy for UTI, but a rise in antibiotic resistance has complicated treatment, making the development of a UTI vaccine more urgent. Iron receptors are a promising new class of vaccine targets for UTI, as UPEC require iron to colonize the iron-limited host urinary tract and genes encoding iron acquisition systems are highly expressed during infection. Previously, three of six UPEC siderophore and heme receptors were identified as vaccine candidates by intranasal immunization in a murine model of ascending UTI. To complete the assessment of iron receptors as vaccine candidates, an additional six UPEC iron receptors were evaluated. Of the six vaccine candidates tested in this study (FyuA, FitA, IroN, the gene product of the CFT073 locus c0294, and two truncated derivatives of ChuA), only FyuA provided significant protection (P = 0.0018) against UPEC colonization. Intranasal immunization induced a robust and long-lived humoral immune response. In addition, the levels of FyuA-specific serum IgG correlated with bacterial loads in the kidneys [Spearman's rank correlation coefficient ρ(14) = -0.72, P = 0.0018], providing a surrogate of protection. FyuA is the fourth UPEC iron receptor to be identified from our screens, in addition to IutA, Hma, and IreA, which were previously demonstrated to elicit protection against UPEC challenge. Together, these iron receptor antigens will facilitate the development of a broadly protective, multivalent UTI vaccine to effectively target diverse strains of UPEC.

In bacteria, laterally acquired genes are often concentrated within chromosomal regions known as genomic islands. Using a recently developed zebrafish infection model, we set out to identify unique factors encoded within genomic islands that contribute to the fitness and virulence of a reference urosepsis isolate-extraintestinal pathogenic Escherichia coli strain CFT073. By screening a series of deletion mutants, we discovered a previously uncharacterized gene, neaT, that is conditionally required by the pathogen during systemic infections. In vitro assays indicate that neaT can limit bacterial interactions with host phagocytes and alter the aggregative properties of CFT073. The neaT gene is localized within an integrated P2-like bacteriophage in CFT073, but was rarely found within other proteobacterial genomes. Sequence-based analyses revealed that neaT homologues are present, but discordantly conserved, within a phyletically diverse set of bacterial species. In CFT073, neaT appears to be unameliorated, having an exceptionally A+T-rich composition along with a notably altered codon bias. These data suggest that neaT was recently brought into the proteobacterial pan-genome from an extra-phyletic source. Interestingly, even in G+C-poor genomes, as found within the Firmicutes lineage, neaT-like genes are often unameliorated. Sequence-level features of neaT homologues challenge the common supposition that the A+T-rich nature of many recently acquired genes reflects the nucleotide composition of their genomes of origin. In total, these findings highlight the complexity of the evolutionary forces that can affect the acquisition, utilization, and assimilation of rare genes that promote the niche-dependent fitness and virulence of a bacterial pathogen.

Uropathogenic Escherichia coli (UPEC) is a leading etiological agent of bacteremia in humans. Virulence mechanisms of UPEC in the context of urinary tract infections have been subjected to extensive research. However, understanding of the fitness mechanisms used by UPEC during bacteremia and systemic infection is limited. A forward genetic screen was utilized to detect transposon insertion mutants with fitness defects during colonization of mouse spleens. An inoculum comprised of 360,000 transposon mutants in the UPEC strain CFT073, cultured from the blood of a patient with pyelonephritis, was used to inoculate mice intravenously. Transposon insertion sites in the inoculum (input) and bacteria colonizing the spleen (output) were identified using high-throughput sequencing of transposon-chromosome junctions. Using frequencies of representation of each insertion mutant in the input and output samples, 242 candidate fitness genes were identified. Co-infection experiments with each of 11 defined mutants and the wild-type strain demonstrated that 82% (9 of 11) of the tested candidate fitness genes were required for optimal fitness in a mouse model of systemic infection. Genes involved in biosynthesis of poly-N-acetyl glucosamine (pgaABCD), major and minor pilin of a type IV pilus (c2394 and c2395), oligopeptide uptake periplasmic-binding protein (oppA), sensitive to antimicrobial peptides (sapABCDF), putative outer membrane receptor (yddB), zinc metallopeptidase (pqqL), a shikimate pathway gene (c1220) and autotransporter serine proteases (pic and vat) were further characterized. Here, we report the first genome-wide identification of genes that contribute to fitness in UPEC during systemic infection in a mammalian host. These fitness factors may represent targets for developing novel therapeutics against UPEC.

Proteus mirabilis, named for the Greek god who changed shape to avoid capture, has fascinated microbiologists for more than a century with its unique swarming differentiation, Dienes line formation and potent urease activity. Transcriptome profiling during both host infection and swarming motility, coupled with the availability of the complete genome sequence for P. mirabilis, has revealed the occurrence of interbacterial competition and killing through a type VI secretion system, and the reciprocal regulation of adhesion and motility, as well as the intimate connections between metabolism, swarming and virulence. This Review addresses some of the unique and recently described aspects of P. mirabilis biology and pathogenesis, and emphasizes the potential role of this bacterium in single-species and polymicrobial urinary tract infections.

Uncomplicated urinary tract infections (UTIs) are common, with nearly half of all women experiencing at least one UTI in their lifetime. This high frequency of infection results in huge annual economic costs, decreased workforce productivity and high patient morbidity. At least 80% of these infections are caused by uropathogenic Escherichia coli (UPEC). UPEC can reside side by side with commensal strains in the gastrointestinal tract and gain access to the bladder via colonization of the urethra. Antibiotics represent the current standard treatment for UTI; however, even after treatment, patients frequently suffer from recurrent infection with the same or different strains. In addition, successful long-term treatment has been complicated by a rise in both the number of antibiotic-resistant strains and the prevalence of antibiotic-resistance mechanisms. As a result, preventative approaches to UTI, such as vaccination, have been sought. This review summarizes recent advances in UPEC vaccine development and outlines future directions for the field.

Unlabelled: The increasing antibiotic resistance of pathogenic Escherichia coli species and the absence of a pan-protective vaccine pose major health concerns. We recently identified, by subtractive reverse vaccinology, nine Escherichia coli antigens that protect mice from sepsis. In this study, we characterized one of them, ECOK1_0290, named FdeC (factor adherence E. coli) for its ability to mediate E. coli adhesion to mammalian cells and extracellular matrix. This adhesive propensity was consistent with the X-ray structure of one of the FdeC domains that shows a striking structural homology to Yersinia pseudotuberculosis invasin and enteropathogenic E. coli intimin. Confocal imaging analysis revealed that expression of FdeC on the bacterial surface is triggered by interaction of E. coli with host cells. This phenotype was also observed in bladder tissue sections derived from mice infected with an extraintestinal strain. Indeed, we observed that FdeC contributes to colonization of the bladder and kidney, with the wild-type strain outcompeting the fdeC mutant in cochallenge experiments. Finally, intranasal mucosal immunization with recombinant FdeC significantly reduced kidney colonization in mice challenged transurethrally with uropathogenic E. coli, supporting a role for FdeC in urinary tract infections., Importance: Pathogenic Escherichia coli strains are involved in a diverse spectrum of diseases, including intestinal and extraintestinal infections (urinary tract infections and sepsis). The absence of a broadly protective vaccine against all these E. coli strains is a major problem for modern society due to high costs to health care systems. Here, we describe the structural and functional properties of a recently reported protective antigen, named FdeC, and elucidated its putative role during extraintestinal pathogenic E. coli infection by using both in vitro and in vivo infection models. The conservation of FdeC among strains of different E. coli pathotypes highlights its potential as a component of a broadly protective vaccine against extraintestinal and intestinal E. coli infections.

Unlabelled: The urinary tract is one of the most frequent sites of bacterial infection in humans. Uropathogenic Escherichia coli (UPEC) strains are the leading cause of urinary tract infections (UTIs) and are responsible for greater than 80% of uncomplicated cases in adults. Infection of the urinary tract occurs in an ascending manner, with colonization of the bladder leading to possible kidney infection and bacteremia. The goal of this study was to examine the population dynamics of UPEC in vivo using a murine model of ascending UTI. To track individual UPEC lineages within a host, we constructed 10 isogenic clones of UPEC strain CFT073 by inserting unique signature tag sequences between the pstS and glmS genes at the attTn7 chromosomal site. Mice were transurethrally inoculated with a mixture containing equal numbers of unique clones. After 4 and 48 h, the tags present in the bladders, kidneys, and spleens of infected mice were enumerated using tag-specific primers and quantitative real-time PCR. The results indicated that kidney infection and bacteremia associated with UTI are most likely the result of multiple rounds of ascension and dissemination from motile UPEC subpopulations, with a distinct bottleneck existing between the kidney and bloodstream. The abundance of tagged lineages became more variable as infection progressed, especially after bacterial ascension to the upper urinary tract. Analysis of the population kinetics of UPEC during UTI revealed metapopulation dynamics, with lineages that constantly increased and decreased in abundance as they migrated from one organ to another., Importance: Urinary tract infections are some of the most common infections affecting humans, and Escherichia coli is the primary cause in most uncomplicated cases. These infections occur in an ascending manner, with bacteria traveling from the bladder to the kidneys and potentially the bloodstream. Little is known about the spatiotemporal population dynamics of uropathogenic E. coli within a host. Here we describe a novel approach for tracking lineages of isogenic tagged E. coli strains within a murine host by the use of quantitative real-time PCR. Understanding the in vivo population dynamics and the factors that shape the bacterial population may prove to be of significant value in the development of novel vaccines and drug therapies.

Bacterial growth in the host is required for pathogenesis. To successfully grow in vivo, pathogens have adapted their metabolism to replicate in specific host microenvironments. These adaptations reflect the nutritional composition of their host niches, inter-bacterial competition for carbon and energy sources, and survival in the face of bactericidal defense mechanisms. A subgroup of Escherichia coli, which cause urinary tract infection, bacteremia, sepsis, and meningitis, have adapted to grow as a harmless commensal in the nutrient-replete, carbon-rich human intestine but rapidly transition to pathogenic lifestyle in the nutritionally poorer, nitrogen-rich urinary tract. We discuss bacterial adaptations that allow extraintestinal pathogenic E. coli to establish both commensal associations and virulence as the bacterium transits between disparate microenvironments within the same individual., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Escherichia coli, a cause of ∼90% of urinary tract infections (UTI), utilizes fimbrial adhesins to colonize the uroepithelium. Pyelonephritis isolate E. coli CFT073 carries 12 fimbrial operons, 5 of which have never been studied. Using multiplex PCR, the prevalence of these 12 and 3 additional fimbrial types was determined for a collection of 303 E. coli isolates (57 human commensal, 32 animal commensal, 54 asymptomatic bacteriuria, 45 complicated UTI, 38 uncomplicated cystitis, and 77 pyelonephritis). The number of fimbrial types per E. coli isolate was distributed bimodally: those with low (3.2 ± 1.1) and those with high (8.3 ± 1.3) numbers of fimbrial types (means ± standard errors of the means). The fimbrial genes ygiL, yadN, yfcV, and c2395 were significantly more prevalent among urine isolates than human commensal isolates. The effect of deletion of Ygi and Yad fimbrial operons on growth, motility, biofilm formation, adherence to immortalized human epithelial cells, and pathogenesis in the mouse model of UTI was examined. Yad fimbriae were necessary for wild-type levels of adherence to a bladder epithelial cell line and for biofilm formation. Deletion of these fimbrial genes increased motility. Ygi fimbriae were necessary for wild-type levels of adherence to a human embryonic kidney cell line, biofilm formation, and in vivo fitness in the urine and kidneys. Complementation of each fimbrial mutant restored wild-type levels of motility, biofilm formation, adherence and, for ygi, in vivo fitness. A double deletion strain, Δygi Δyad, was attenuated in the urine, bladder, and kidneys in the mouse model, demonstrating that these fimbriae contribute to uropathogenesis.

The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In this study, microarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7 days postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across nine microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded mannose-resistant Proteus-like (MR/P) fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism enzymes, and a portion of the tricarboxylic acid (TCA) cycle enzymes. Flagella were downregulated. Ammonia assimilation gene glnA (glutamine synthetase) was repressed in vivo, while gdhA (glutamate dehydrogenase) was upregulated in vivo. Contrary to our expectations, ammonia availability due to urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant was growth deficient in minimal medium with citrate as the sole carbon source, and loss of gdhA resulted in a significant fitness defect in the mouse model of urinary tract infection. Unlike Escherichia coli, which represses gdhA and upregulates glnA in vivo and cannot utilize citrate, the data suggest that P. mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential of P. mirabilis in the urinary tract.

Uncomplicated urinary tract infections (UTI) are caused most commonly by uropathogenic Escherichia coli (UPEC). Whole-genome screening approaches, including transcriptomic, proteomic, and signature-tagged mutagenesis, have shown that UPEC highly expresses or requires genes for translational machinery, capsule, lipopolysaccharide, type 1 fimbriae, and iron acquisition systems during UTI. To identify additional genes expressed by UPEC during UTI, an immunoscreening approach termed in vivo-induced antigen technology (IVIAT) was employed to identify antigens produced during experimental infection that are not produced during in vitro culture. An inducible protein expression library, constructed from genomic DNA isolated from UPEC strain CFT073, was screened using exhaustively adsorbed pooled sera from 20 chronically infected female CBA/J mice. Using this approach, we identified 93 antigens induced by UPEC in vivo. A representative subset of these genes was tested by quantitative PCR for expression by CFT073 in vivo and during growth in human urine or LB medium in vitro; proWX, narJI, lolA, lolD, tosA (upxA), c2432, katG, ydhX, kpsS, and yddQ were poorly expressed in vitro but highly expressed in vivo. Of these, tosA, a gene encoding a predicted repeat-in-toxin family member, was expressed exclusively during UTI. Deletion of tosA in UPEC strain CFT073 resulted in significant attenuation in bladder and kidney infections during ascending UTI. By screening for in vivo-induced antigens, we identified a novel UPEC virulence factor and additional proteins that could be useful as potential vaccine targets.

Unlabelled: Uropathogenic Escherichia coli (UPEC) strains, which cause the majority of uncomplicated urinary tract infections (UTIs), carry a unique assortment of virulence or fitness genes. However, no single defining set of virulence or fitness genes has been found in all strains of UPEC, making the differentiation between UPEC and fecal commensal strains of E. coli difficult without the use of animal models of infection or phylogenetic grouping. In the present study, we consider three broad categories of virulence factors simultaneously to better define a combination of virulence factors that predicts success in the urinary tract. A total of 314 strains of E. coli, representing isolates from fecal samples, asymptomatic bacteriuria, complicated UTIs, and uncomplicated bladder and kidney infections, were assessed by multiplex PCR for the presence of 15 virulence or fitness genes encoding adhesins, toxins, and iron acquisition systems. The results confirm previous reports of gene prevalence among isolates from different clinical settings and identify several new patterns of gene associations. One gene, tosA, a putative repeat-in-toxin (RTX) homolog, is present in 11% of fecal strains but 25% of urinary isolates. Whereas tosA-positive strains carry an unusually high number (11.2) of the 15 virulence or fitness genes, tosA-negative strains have an average of only 5.4 virulence or fitness genes. The presence of tosA was predictive of successful colonization of a murine model of infection, even among fecal isolates, and can be used as a marker of pathogenic strains of UPEC within a distinct subset of the B2 lineage., Importance: Escherichia coli is the primary cause of urinary tract infections, the most common bacterial infection of humans. Virulence of a uropathogenic strain is typically defined by the clinical source of the isolate, the ability to colonize the bladder and kidneys in a murine model, the phylogenetic group of the bacterium, and virulence gene content. Here we describe a novel single gene, the repeat-in-toxin gene tosA, the presence of which predicts virulence of E. coli isolates regardless of source. Rapid identification of uropathogenic strains of E. coli may aid in the development of therapeutic and preventive therapies.

Uropathogenic Escherichia coli (UPEC), the predominant cause of uncomplicated urinary tract infection (UTI), utilizes an array of outer membrane iron receptors to facilitate siderophore and heme import from within the iron-limited urinary tract. While these systems are required for UPEC in vivo fitness and are assumed to be functionally redundant, the relative contributions of specific receptors to pathogenesis are unknown. To delineate the relative roles of distinct UPEC iron acquisition systems in UTI, isogenic mutants in UPEC strain CFT073 or 536 lacking individual receptors were competed against one another in vivo in a series of mixed infections. When combinations of up to four mutants were coinoculated using a CBA/J mouse model of ascending UTI, catecholate receptor mutants (ΔfepA, Δiha, and ΔiroN mutants) were equally fit, suggesting redundant function. However, noncatecholate siderophore receptor mutants, including the ΔiutA aerobactin receptor mutant and the ΔfyuA yersiniabactin receptor mutant, were frequently outcompeted by coinoculated mutants, indicating that these systems contribute more significantly to UPEC iron acquisition in vivo. A tissue-specific preference for heme acquisition was also observed, as a heme uptake-deficient Δhma ΔchuA double mutant was outcompeted by siderophore receptor mutants specifically during kidney colonization. The relative contribution of each receptor to UTI only partially correlated with in vivo levels of receptor gene expression, indicating that other factors likely contributed to the observed fitness differences. Overall, our results suggest that UPEC iron receptors provide both functional redundancy and niche specificity for this pathogen as it colonizes distinct sites within the urinary tract.

Murine models of urinary tract infection (UTI) have provided substantial data identifying uropathogenic E. coli (UPEC) virulence factors and assessing their expression in vivo. However, it is unclear how gene expression in these animal models compares to UPEC gene expression during UTI in humans. To address this, we used a UPEC strain CFT073-specific microarray to measure global gene expression in eight E. coli isolates monitored directly from the urine of eight women presenting at a clinic with bacteriuria. The resulting gene expression profiles were compared to those of the same E. coli isolates cultured statically to exponential phase in pooled, sterilized human urine ex vivo. Known fitness factors, including iron acquisition and peptide transport systems, were highly expressed during human UTI and support a model in which UPEC replicates rapidly in vivo. While these findings were often consistent with previous data obtained from the murine UTI model, host-specific differences were observed. Most strikingly, expression of type 1 fimbrial genes, which are among the most highly expressed genes during murine experimental UTI and encode an essential virulence factor for this experimental model, was undetectable in six of the eight E. coli strains from women with UTI. Despite the lack of type 1 fimbrial expression in the urine samples, these E. coli isolates were generally capable of expressing type 1 fimbriae in vitro and highly upregulated fimA upon experimental murine infection. The findings presented here provide insight into the metabolic and pathogenic profile of UPEC in urine from women with UTI and represent the first transcriptome analysis for any pathogenic E. coli during a naturally occurring infection in humans.

Fimbriae of the human uropathogen Proteus mirabilis are the only characterized surface proteins that contribute to its virulence by mediating adhesion and invasion of the uroepithelia. PMI2122 (AipA) and PMI2575 (TaaP) are annotated in the genome of strain HI4320 as trimeric autotransporters with "adhesin-like" and "agglutinating adhesin-like" properties, respectively. The C-terminal 62 amino acids (aa) in AipA and 76 aa in TaaP are homologous to the translocator domains of YadA from Yersinia enterocolitica and Hia from Haemophilus influenzae. Comparative protein modeling using the Hia three-dimensional structure as a template predicted that each of these domains would contain four antiparallel beta sheets and that they formed homotrimers. Recombinant AipA and TaaP were seen as ∼28 kDa and ∼78 kDa, respectively, in Escherichia coli, and each also formed high-molecular-weight homotrimers, thus supporting this model. E. coli synthesizing AipA or TaaP bound to extracellular matrix proteins with a 10- to 60-fold-higher level of affinity than the control strain. Inactivation of aipA in P. mirabilis strains significantly (P < 0.01) reduced the mutants' ability to adhere to or invade HEK293 cell monolayers, and the functions were restored upon complementation. A 51-aa-long invasin region in the AipA passenger domain was required for this function. E. coli expressing TaaP mediated autoagglutination, and a taaP mutant of P. mirabilis showed significantly (P < 0.05) more reduced aggregation than HI4320. Gly-247 in AipA and Gly-708 in TaaP were indispensable for trimerization and activity. AipA and TaaP individually offered advantages to P. mirabilis in a murine model. This is the first report characterizing trimeric autotransporters in P. mirabilis as afimbrial surface adhesins and autoagglutinins.

The urinary tract is a common site of bacterial infections; nearly half of all women experience at least one urinary tract infection (UTI) during their lifetime. These infections are classified based on the condition of the host. Uncomplicated infections affect otherwise healthy individuals and are most commonly caused by uropathogenic Escherichia coli, whereas complicated infections affect patients with underlying difficulties, such as a urinary tract abnormality or catheterization, and are commonly caused by species such as Proteus mirabilis. Virulence and fitness factors produced by both pathogens include fimbriae, toxins, flagella, iron acquisition systems, and proteins that function in immune evasion. Additional factors that contribute to infection include the formation of intracellular bacterial communities by E. coli and the production of urease by P. mirabilis, which can result in urinary stone formation. Innate immune responses are induced or mediated by pattern recognition receptors, antimicrobial peptides, and neutrophils. The adaptive immune response to UTI is less well understood. Host factors TLR4 and CXCR1 are implicated in disease outcome and susceptibility, respectively. Low levels of TLR4 are associated with asymptomatic bacteriuria while low levels of CXCR1 are associated with increased incidence of acute pyelonephritis. Current research is focused on the identification of additional virulence factors and therapeutic or prophylactic targets that might be used in the generation of vaccines against both uropathogens.

Proteus mirabilis, a Gram-negative bacterium, represents a common cause of complicated urinary tract infections in catheterized patients or those with functional or anatomical abnormalities of the urinary tract. ZnuB, the membrane component of the high-affinity zinc (Zn(2+)) transport system ZnuACB, was previously shown to be recognized by sera from infected mice. Since this system has been shown to contribute to virulence in other pathogens, its role in Proteus mirabilis was investigated by constructing a strain with an insertionally interrupted copy of znuC. The znuC::Kan mutant was more sensitive to zinc limitation than the wild type, was outcompeted by the wild type in minimal medium, displayed reduced swimming and swarming motility, and produced less flaA transcript and flagellin protein. The production of flagellin and swarming motility were restored by complementation with znuCB in trans. Swarming motility was also restored by the addition of Zn(2+) to the agar prior to inoculation; the addition of Fe(2+) to the agar also partially restored the swarming motility of the znuC::Kan strain, but the addition of Co(2+), Cu(2+), or Ni(2+) did not. ZnuC contributes to but is not required for virulence in the urinary tract; the znuC::Kan strain was outcompeted by the wild type during a cochallenge experiment but was able to colonize mice to levels similar to the wild-type level during independent challenge. Since we demonstrated a role for ZnuC in zinc transport, we hypothesize that there is limited zinc present in the urinary tract and P. mirabilis must scavenge this ion to colonize and persist in the host.

Uropathogenic Escherichia coli is the causative agent for >80% of uncomplicated urinary tract infections (UTIs). Uropathogenic E. coli strains express a number of virulence and fitness factors that allow successful colonization of the mammalian bladder. To combat this, the host has distinct mechanisms to prevent adherence to the bladder wall and to detect and kill uropathogenic E. coli in the event of colonization. In this study, we investigated the role of IL-17A, an innate-adaptive immunomodulatory cytokine, during UTI using a murine model. Splenocytes isolated from mice infected by the transurethral route robustly expressed IL-17A in response to in vitro stimulation with uropathogenic E. coli Ags. Transcript expression of IL-17A in the bladders of infected mice correlated with a role in the innate immune response to UTI, and gammadelta cells seem to be a key source of IL-17A production. Although IL-17A seems to be dispensable for the generation of a protective response to uropathogenic E. coli, its importance in innate immunity is demonstrated by a defect in acute clearance of uropathogenic E. coli in IL-17A(-/-) mice. This clearance defect is likely a result of deficient cytokine and chemokine transcripts and impaired macrophage and neutrophil influx during infection. These results show that IL-17A is a key mediator for the innate immune response to UTIs.

Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is a substantial economic and societal burden-a formidable public health issue. Symptomatic UTI causes significant discomfort in infected patients, results in lost productivity, predisposes individuals to more serious infections, and usually necessitates antibiotic therapy. There is no licensed vaccine available for prevention of UTI in humans in the United States, likely due to the challenge of targeting a relatively heterogeneous group of pathogenic strains in a unique physiological niche. Despite significant advances in the understanding of UPEC biology, mechanistic details regarding the host response to UTI and full comprehension of genetic loci that influence susceptibility require additional work. Currently, there is an appreciation for the role of classic innate immune responses-from pattern receptor recognition to recruitment of phagocytic cells-that occur during UPEC-mediated UTI. There is, however, a clear disconnect regarding how factors involved in the innate immune response to UPEC stimulate acquired immunity that facilitates enhanced clearance upon reinfection. Unraveling the molecular details of this process is vital in the development of a successful vaccine for prevention of human UTI. Here, we survey the current understanding of host responses to UPEC-mediated UTI with an eye on molecular and cellular factors whose activity may be harnessed by a vaccine that stimulates lasting and sterilizing immunity.

Uncomplicated infections of the urinary tract, caused by uropathogenic Escherichia coli, are among the most common diseases requiring medical intervention. A preventive vaccine to reduce the morbidity and fiscal burden these infections have upon the healthcare system would be beneficial. Here, we describe the results of a large-scale selection process that incorporates bioinformatic, genomic, transcriptomic, and proteomic screens to identify six vaccine candidates from the 5379 predicted proteins encoded by uropathogenic E. coli strain CFT073. The vaccine candidates, ChuA, Hma, Iha, IreA, IroN, and IutA, all belong to a functional class of molecules that is involved in iron acquisition, a process critical for pathogenesis in all microbes. Intranasal immunization of CBA/J mice with these outer membrane iron receptors elicited a systemic and mucosal immune response that included the production of antigen-specific IgM, IgG, and IgA antibodies. The cellular response to vaccination was characterized by the induction and secretion of IFN-gamma and IL-17. Of the six potential vaccine candidates, IreA, Hma, and IutA provided significant protection from experimental infection. In immunized animals, class-switching from IgM to IgG and production of antigen-specific IgA in the urine represent immunological correlates of protection from E. coli bladder colonization. These findings are an important first step toward the development of a subunit vaccine to prevent urinary tract infections and demonstrate how targeting an entire class of molecules that are collectively required for pathogenesis may represent a fundamental strategy to combat infections.

Uropathogenic Escherichia coli (UPEC) strain CFT073 contains 13 large genomic islands ranging in size from 32 kb to 123 kb. Eleven of these genomic islands were individually deleted from the genome, and nine isogenic mutants were tested for their ability to colonize the CBA/J mouse model of ascending urinary tract infection. Three genomic island mutants (Delta PAI-aspV, Delta PAI-metV, and Delta PAI-asnT) were significantly outcompeted by wild-type CFT073 in the bladders and/or kidneys following transurethral cochallenge (P

Background: Capsule expression may be important during ascending Escherichia coli urinary tract infections (UTIs)., Methods: An isogenic ksl(k2)ABCDE mutant of extraintestinal pathogenic E. coli (ExPEC) strain CFT073 that could not synthesize the K2 capsule was compared with wild-type CFT073, to determine virulence in a murine model of ascending UTI and in vitro killing assays., Results: No significant differences were observed regarding the abilities of the mutant and the wild-type CFT073 strains to colonize the murine urinary tract in single-challenge infection experiments. However, in competitive-colonization experiments, the mutant was significantly outcompeted by the wild-type strain in urine and the kidneys. The mutant strain was also more susceptible to human serum. Complementation of the mutant with a plasmid containing the ksl(k2)ABCDE genes restored capsule expression, enhanced survival in the murine urinary tract, and restored serum resistance., Conclusion: These results indicate that expression of the K2 capsule is important for the pathogenesis of UTI and provides protection against complement-mediated killing. To our knowledge, this is the first study in which the E. coli capsule has been proven to play a role in infection by use of isogenic mutants and genetic complementation.

Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium's ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes.

MR/P fimbriae of uropathogenic Proteus mirabilis undergo invertible element-mediated phase variation whereby an individual bacterium switches between expressing fimbriae (phase ON) and not expressing fimbriae (phase OFF). Under different conditions, the percentage of fimbriate bacteria within a population varies and could be dictated by either selection (growth advantage of one phase) or signaling (preferentially converting one phase to the other in response to external signals). Expression of MR/P fimbriae increases in a cell-density dependent manner in vitro and in vivo. However, rather than the increased cell density itself, this increase in fimbrial expression is due to an enrichment of fimbriate bacteria under oxygen limitation resulting from increased cell density. Our data also indicate that the persistence of MR/P fimbriate bacteria under oxygen-limiting conditions is a result of both selection (of MR/P fimbrial phase variants) and signaling (via modulation of expression of the MrpI recombinase). Furthermore, the mrpJ transcriptional regulator encoded within the mrp operon contributes to phase switching. Type 1 fimbriae of Escherichia coli, which are likewise subject to phase variation via an invertible element, also increase in expression during reduced oxygenation. These findings provide evidence to support a mechanism for persistence of fimbriate bacteria under oxygen limitation, which is relevant to disease progression within the oxygen-restricted urinary tract.

Complicated urinary tract infections (UTI) caused by Proteus mirabilis are associated with severe pathology in the bladder and kidney. To investigate the roles of two established cytotoxins, the HpmA hemolysin, a secreted cytotoxin, and proteus toxic agglutinin (Pta), a surface-associated cytotoxin, mutant analysis was used in conjunction with a mouse model of ascending UTI. Inactivation of pta, but not inactivation of hpmA, resulted in significant decreases in the bacterial loads of the mutant in kidneys (P < 0.01) and spleens (P < 0.05) compared to the bacterial loads of the wild type; the 50% infective dose (ID(50)) of an isogenic pta mutant or hpmA pta double mutant was 100-fold higher (5 x 10(8) CFU) than the ID(50) of parent strain HI4320 (5 x 10(6) CFU). Colonization by the parent strain caused severe cystitis and interstitial nephritis as determined by histopathological examination. Mice infected with the same bacterial load of the hpmA pta double mutant showed significantly reduced pathology (P < 0.01), suggesting that the additive effect of these two cytotoxins is critical during Proteus infection. Since Pta is surface associated and important for the persistence of P. mirabilis in the host, it was selected as a vaccine candidate. Mice intranasally vaccinated with a site-directed (indicated by an asterisk) (S366A) mutant purified intact toxin (Pta*) or the passenger domain Pta-alpha*, each independently conjugated with cholera toxin (CT), had significantly lower bacterial counts in their kidneys ( P = 0.001) and spleens (P = 0.002) than mice that received CT alone. The serum immunoglobulin G levels correlated with protection (P = 0.03). This is the first report describing the in vivo cytotoxicity and antigenicity of an autotransporter in P. mirabilis and its use in vaccine development.

Motility and adherence are two integral aspects of bacterial pathogenesis. Adherence, often mediated by fimbriae, permits bacteria to attach to host cells and establish infection, whereas flagellum-driven motility allows bacteria to disseminate to sites more advantageous for colonization. Both fimbriae and flagella have been proven important for virulence of uropathogenic Escherichia coli (UPEC). Reciprocal regulation is one mechanism by which bacteria may reconcile the contradictory actions of adherence and motility. PapX, a P fimbrial gene product of UPEC strain CFT073, is a functional homolog of MrpJ of Proteus mirabilis; ectopic expression of papX in P. mirabilis reduces motility. To define the connection between P fimbria expression and motility in UPEC, the role of papX in the regulation of motility of strain CFT073 was examined. Overexpression of papX decreased motility of CFT073, which correlated with both a significant reduction in flagellin protein synthesized and flagella assembled on the cell surface. Conversely, an increase in motility and flagellin production was seen in an isogenic papX deletion mutant of CFT073. Microarray and quantitative reverse transcription-PCR analysis indicated that repression of motility of CFT073 by PapX appears to occur at the transcriptional level; expression of many motility-associated genes, including flhDC, the master regulator of motility, is decreased when papX is overexpressed. Transcription of motility genes is increased in the papX mutant compared to wild type. Electrophoretic mobility gel shift analysis revealed that PapX binds to the flhD promoter. We conclude that synthesis of P fimbriae regulates flagellum synthesis to repress motility via PapX.

Proteus mirabilis, a gram-negative bacterium, is a frequent cause of complicated urinary tract infections in those with functional or anatomical abnormalities or those subject to long-term catheterization. To systematically identify surface-exposed antigens as potential vaccine candidates, proteins in the outer membrane fraction of bacteria were separated by two-dimensional gel electrophoresis and subjected to Western blotting with sera from mice experimentally infected with P. mirabilis. Protein spots reactive with sera were identified by mass spectrometry, which in conjunction with the newly completed genome sequence of P. mirabilis HI4320, was used to identify surface-exposed antigens. Culture conditions that may mimic in vivo conditions more closely than Luria broth (growth in human urine and under iron limitation and osmotic stress) were also used. Thirty-seven antigens to which a humoral response had been mounted, including 23 outer membrane proteins, were identified. These antigens are presumably expressed during urinary tract infection. Protein targets that are both actively required for virulence and antigenic may serve as protective antigens for vaccination; thus, five representative antigens were selected for use in virulence studies. Strains of P. mirabilis with mutations in three of the corresponding genes (the PMI0047 gene, rafY, and fadL) were not attenuated in the murine model of urinary tract infection. Putative iron acquisition proteins PMI0842 and PMI2596, however, both contribute to fitness in the urinary tract and thus emerge as vaccine candidates.

Proteus mirabilis is a ubiquitous bacterium associated with complicated urinary tract infection (UTI). Mutagenesis studies of the wild-type strain HI4320 in the CBA mouse model of ascending UTIs have identified attenuated mutants with transposon insertions in genes encoding the high-affinity phosphate transporter Pst (pstS, pstA). The transcription of the pst operon (pstSCAB-phoU) and other members of the phosphate regulon of Escherichia coli, including alkaline phosphatase (AP), are regulated by the two-component regulatory system PhoBR and are repressed until times of phosphate starvation. This normal suppression was relieved in pstS::Tn5 and pstA::Tn5 mutants, which constitutively produced AP regardless of growth conditions. No significant growth defects were observed in vitro for the pst mutants during the independent culture or coculture studies in rich broth, phosphate-limiting minimal salts medium, or human urine. Mutants complemented with the complete pst operon repressed AP synthesis in vitro and colonized the mouse bladder in numbers comparable to the wild-type strain HI4320. Therefore, the Pst transport system imparts a significant in vivo advantage to wild-type P. mirabilis that is not required for in vitro growth. Thus, the Pst transporter has satisfied molecular Koch's postulates as a virulence factor in the pathogenesis of urinary tract infection caused by P. mirabilis.

Catheter-associated urinary tract infections (CAUTIs) represent the most common type of nosocomial infection and are a major health concern due to the complications and frequent recurrence. These infections are often caused by Escherichia coli and Proteus mirabilis. Gram-negative bacterial species that cause CAUTIs express a number of virulence factors associated with adhesion, motility, biofilm formation, immunoavoidance, and nutrient acquisition as well as factors that cause damage to the host. These infections can be reduced by limiting catheter usage and ensuring that health care professionals correctly use closed-system Foley catheters. A number of novel approaches such as condom and suprapubic catheters, intermittent catheterization, new surfaces, catheters with antimicrobial agents, and probiotics have thus far met with limited success. While the diagnosis of symptomatic versus asymptomatic CAUTIs may be a contentious issue, it is generally agreed that once a catheterized patient is believed to have a symptomatic urinary tract infection, the catheter is removed if possible due to the high rate of relapse. Research focusing on the pathogenesis of CAUTIs will lead to a better understanding of the disease process and will subsequently lead to the development of new diagnosis, prevention, and treatment options.

Uropathogenic Escherichia coli (UPEC) cause most uncomplicated urinary tract infections (UTIs) in humans. Because UTIs are considered to occur in an ascending manner, flagellum-mediated motility has been suggested to contribute to virulence by enabling UPEC to disseminate to the upper urinary tract. Previous studies from our laboratory and others have demonstrated a modest yet important role for flagella during ascending UTI. To better understand the role of flagella in vivo, we used biophotonic imaging to monitor UPEC infection and temporospatial flagellin gene expression during ascending UTI. Using em7-lux (constitutive) and fliC-lux transcriptional fusions, we show that flagellin expression by UPEC coincides with ascension of the ureters and colonization of the kidney. The patterns of fliC luminescence observed in vitro and in vivo were also validated by comparative quantitative PCR. Because fliC expression appeared coincident during ascension, we reassessed the contribution of fliC to ascending UTI using a low-dose intraurethral model of ascending UTI. Although wild-type UPEC were able to establish infection in the bladder and kidneys by 6 hours postinoculation, fliC mutant bacteria were able to colonize the bladder but were significantly attenuated in the kidneys at this early time point. By 48 hours postinoculation, the fliC mutant bacteria were attenuated in the bladder and kidneys and were not detectable in the spleen. These data provide compelling evidence that wild-type UPEC express flagellin and presumably utilize flagellum-mediated motility during UTI to ascend to the upper urinary tract and disseminate within the host.

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.

Outer membrane proteins (OMPs) of microbial pathogens are critical components that mediate direct interactions between microbes and their surrounding environment. Consequently, the study of OMPs is integral to furthering the understanding of host-pathogen interactions and to identifying key targets for development of improved antimicrobial agents and vaccines. In this study, we used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and tandem mass spectrometry to characterize the uropathogenic Escherichia coli (UPEC) outer membrane subproteome; 30 individual OMPs present on the bacterial surface during growth in human urine were identified. Fluorescence difference gel electrophoresis was used to identify quantitative changes in levels of UPEC strain CFT073 OMPs during growth in urine; six known receptors for iron compounds were induced in this environment, i.e., ChuA, IutA, FhuA, IroN, IreA, and Iha. A seventh putative iron compound receptor, encoded by CFT073 open reading frame (ORF) c2482, was also identified and found to be induced in urine. Further, the induction of these seven iron receptors in human urine and during defined iron limitation was verified by using quantitative real-time PCR (qPCR). An eighth iron receptor, fepA, displayed similar induction levels under these conditions as measured by qPCR but was not identified by 2D-PAGE. Addition of 10 microM FeCl(2) to human urine repressed the transcription of all eight iron receptor genes. A number of fecal-commensal, intestinal pathogenic, and uropathogenic E. coli strains all displayed similar growth rates in human urine, showing that the ability to grow in urine per se is not a urovirulence trait. Thus, human urine is an iron-limiting environment and UPEC enriches its outer membrane with iron receptors to contend with this iron limitation.

Uropathogenic Escherichia coli (UPEC) strains are responsible for the majority of uncomplicated urinary tract infections, which can present clinically as cystitis or pyelonephritis. UPEC strain CFT073, isolated from the blood of a patient with acute pyelonephritis, was most cytotoxic and most virulent in mice among our strain collection. Based on the genome sequence of CFT073, microarrays were utilized in comparative genomic hybridization (CGH) analysis of a panel of uropathogenic and fecal/commensal E. coli isolates. Genomic DNA from seven UPEC (three pyelonephritis and four cystitis) isolates and three fecal/commensal strains, including K-12 MG1655, was hybridized to the CFT073 microarray. The CFT073 genome contains 5,379 genes; CGH analysis revealed that 2,820 (52.4%) of these genes were common to all 11 E. coli strains, yet only 173 UPEC-specific genes were found by CGH to be present in all UPEC strains but in none of the fecal/commensal strains. When the sequences of three additional sequenced UPEC strains (UTI89, 536, and F11) and a commensal strain (HS) were added to the analysis, 131 genes present in all UPEC strains but in no fecal/commensal strains were identified. Seven previously unrecognized genomic islands (>30 kb) were delineated by CGH in addition to the three known pathogenicity islands. These genomic islands comprise 672 kb of the 5,231-kb (12.8%) genome, demonstrating the importance of horizontal transfer for UPEC and the mosaic structure of the genome. UPEC strains contain a greater number of iron acquisition systems than do fecal/commensal strains, which is reflective of the adaptation to the iron-limiting urinary tract environment. Each strain displayed distinct differences in the number and type of known virulence factors. The large number of hypothetical genes in the CFT073 genome, especially those shown to be UPEC specific, strongly suggests that many urovirulence factors remain uncharacterized.

The prevalence and function of four chemoreceptors, Tsr, Tar, Trg, and Tap, were determined for a collection of uropathogenic, fecal-commensal, and diarrheagenic Escherichia coli strains. tar and tsr were present or functional in nearly all isolates. However, trg and tap were significantly less prevalent or functional among the uropathogenic E. coli strains (both in 6% of strains) than among fecal-commensal strains (both in > or =50% of strains) or diarrheal strains (both in > or =75% of strains) (P < 0.02).

Type 1 fimbrial phase-locked mutants of uropathogenic Escherichia coli cystitis isolate F11 were used to assess the role of the invertible element during urinary tract infection. Compared to the wild type, the phase-locked off mutant was attenuated, and constitutive production of type 1 fimbriae by the phase-locked on mutant did not provide a competitive advantage.

Uropathogenic Escherichia coli (UPEC) causes most uncomplicated urinary tract infections (UTIs) in humans. Flagellum-mediated motility and chemotaxis have been suggested to contribute to virulence by enabling UPEC to escape host immune responses and disperse to new sites within the urinary tract. To evaluate their contribution to virulence, six separate flagellar mutations were constructed in UPEC strain CFT073. The mutants constructed were shown to have four different flagellar phenotypes: fliA and fliC mutants do not produce flagella; the flgM mutant has similar levels of extracellular flagellin as the wild type but exhibits less motility than the wild type; the motAB mutant is nonmotile; and the cheW and cheY mutants are motile but nonchemotactic. Virulence was assessed by transurethral independent challenges and cochallenges of CBA mice with the wild type and each mutant. CFU/ml of urine or CFU/g bladder or kidney was determined 3 days postinoculation for the independent challenges and at 6, 16, 48, 60, and 72 h postinoculation for the cochallenges. While these mutants colonized the urinary tract during independent challenge, each of the mutants was outcompeted by the wild-type strain to various degrees at specific time points during cochallenge. Altogether, these results suggest that flagella and flagellum-mediated motility/chemotaxis may not be absolutely required for virulence but that these traits contribute to the fitness of UPEC and therefore significantly enhance the pathogenesis of UTIs caused by UPEC.

The role of the Escherichia coli iron-regulated gene homologue adhesin (Iha) in the pathogenesis of urinary tract infections (UTIs) is unknown. We performed a series of complementary analyses to confirm or refute the hypothesis that Iha is a virulence factor in uropathogenic E. coli. Fecal E. coli isolates exhibited significantly lower prevalences of iha (range, 14 to 22%) than did clinical isolates from cases of pediatric cystitis or pyelonephritis, adult pyelonephritis or urosepsis, or bacteremia (range, 38 to 74%). Recombinant Iha from E. coli pyelonephritis isolate CFT073 conferred upon nonadherent E. coli ORN172 the ability to adhere to cultured T-24 human uroepithelial cells. In a well-established mouse model of ascending UTI, CFT073 and its derivative UPEC76 (a pap [P fimbriae] mutant version of strain CFT073) each significantly outcompeted their respective iha deletion mutants in CBA/J mice 48 h after bladder challenge (P < 0.03 for urine, both kidneys, and bladders of both constructs, except for bladders of mice challenged with UPEC76 and its deletion mutant, where P = 0.11). These data suggest that Iha(CFT073) is a virulence factor and might be a target for anti-UTI interventions.

This is a follow-up to our previous study using an intranasal vaccine composed of MrpH, the tip adhesin of the MR/P fimbria, and cholera toxin to prevent urinary tract infection by Proteus mirabilis (X. Li, C. V. Lockatell, D. E. Johnson, M. C. Lane, J. W. Warren, and H. L. Mobley, Infect. Immun. 72:66-75, 2004). Here, we have expressed a cholera toxin-like chimera in which the MrpH adhesin-binding domain (residues 23 to 157) replaces the cholera toxin A1 ADP-ribosyltransferase domain. This chimera, when administered intranasally without additional adjuvant, is sufficient to induce protective immunity in mice.

Proteus mirabilis, an etiologic agent of complicated urinary tract infections, expresses mannose-resistant Proteus-like (MR/P) fimbriae whose expression is phase variable. Here we examine the role of these fimbriae in biofilm formation and colonization of the urinary tract. The majority of wild-type P. mirabilis cells in transurethrally infected mice produced MR/P fimbriae. Mutants that were phase-locked for either constitutive expression (MR/P ON) or the inability to express MR/P fimbriae (MR/P OFF) were phenotypically distinct and swarmed at different rates. The number of P. mirabilis cells adhering to bladder tissue did not appear to be affected by MR/P fimbriation. However, the pattern of adherence to the bladder surface was strikingly different. MR/P OFF colonized the lamina propria underlying exfoliated uroepithelium, while MR/P ON colonized the luminal surfaces of bladder umbrella cells and not the exfoliated regions. Wild-type P. mirabilis was usually found colonizing intact uroepithelium, but it occasionally adhered to exfoliated areas. MR/P ON formed significantly more biofilm than either P. mirabilis HI4320 (P = 0.03) or MR/P OFF (P = 0.05). MR/P OFF was able to form a biofilm similar to that of the wild type. MR/P ON formed a three-dimensional biofilm structure as early as 18 h after the initiation of the biofilm, while MR/P OFF and the wild type did not. After 7 days, however, P. mirabilis HI4320 formed a 65-mum-thick biofilm, while the thickest MR/P ON and MR/P OFF biofilms were only 12 mum thick. We concluded that MR/P fimbriae are expressed by most P. mirabilis cells infecting the urinary tract, dictate the localization of bacteria in the bladder, and contribute to biofilm formation.

A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any E. coli pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.

Proteus mirabilis, a common cause of urinary tract infections (UTI) in individuals with functional or structural abnormalities or with long-term catheterization, forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis. Known virulence factors, besides urease, are hemolysin, fimbriae, metalloproteases, and flagella. In this study we utilized the CBA mouse model of ascending UTI to evaluate the colonization of mutants of P. mirabilis HI4320 that were generated by signature-tagged mutagenesis. By performing primary screening of 2088 P. mirabilis transposon mutants, we identified 502 mutants that ranged from slightly attenuated to unrecoverable. Secondary screening of these mutants revealed that 114 transposon mutants were reproducibly attenuated. Cochallenge of 84 of these single mutants with the parent strain in the mouse model resulted in identification of 37 consistently out-competed P. mirabilis transposon mutants, 25 of which were out-competed >100-fold for colonization of the bladder and/or kidneys by the parent strain. We determined the sequence flanking the site of transposon insertion in 29 attenuated mutants and identified genes affecting motility, iron acquisition, transcriptional regulation, phosphate transport, urease activity, cell surface structure, and key metabolic pathways as requirements for P. mirabilis infection of the urinary tract. Two mutations localized to a approximately 42-kb plasmid present in the parent strain, suggesting that the plasmid is important for colonization. Isolation of disrupted genes encoding proteins with homologies to known bacterial virulence factors, especially the urease accessory protein UreF and the disulfide formation protein DsbA, showed that the CBA mouse model and mutant pools are a reliable source of attenuated mutants with mutations in virulence genes.