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Urinary tract infection (UTI) caused by uropathogenicEscherichia 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.

Extraintestinal pathogenicEscherichia colican successfully colonize the urinary tract of the immunocompetent host. In part, this is accomplished by dampening the host immune response. Indeed, thesisAandsisBgenes (shiA-likeinflammationsuppressor genesAandB) of uropathogenicE. colistrain CFT073, homologs of theShigella flexneriSHI-2 pathogenicity island geneshiA, suppress the host inflammatory response. A double deletion mutant (ΔsisAΔsisB) resulted in a hyperinflammatory phenotype in an experimental model of ascending urinary tract infection. The ΔsisAΔsisBmutant not only caused significantly more inflammatory foci in the kidneys of CBA/J mice (P= 0.0399), but these lesions were also histologically more severe (P= 0.0477) than lesions observed in mice infected with wild-type CFT073. This hyperinflammatory phenotype could be suppressed to wild-type levels by in vivo complementation of the ΔsisAΔsisBmutant with either thesisAorsisBgene intrans. The ΔsisAΔsisBmutant was outcompeted by wild-type CFT073 during cochallenge infection in the bladder (P= 0.0295) at 48 h postinoculation (hpi). However, during cochallenge infections, we reasoned that wild-type CFT073 could partially complement the ΔsisAΔsisBmutant. Consistent with this, the most significant colonization defect of the ΔsisAΔsisBmutant in vivo was observed during independent challenge relative to wild-type CFT073, with attenuation of the mutant observed in the bladder (P< 0.0001) and kidneys (P= 0.0003) at 6 hpi. By 24 and 48 hpi, the ΔsisAΔsisBmutant was no longer significantly attenuated in the bladder or kidneys, suggesting that thesisAandsisBgenes may be important for suppressing the host immune response during the initial stages of infection.

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.

Pathogenicity islands (PAIs) are a specific group of genomic islands that contribute to genomic variability and virulence of bacterial pathogens. Using a strain-specific comparative genomic hybridization array, we report the identification of a 94-kb PAI, designated ICE Pm1 , that is common to Proteus mirabilis , Providencia stuartii , and Morganella morganii . These organisms are highly prevalent etiologic agents of catheter-associated urinary tract infections (caUTI), the most common hospital acquired infection. ICE Pm1 carries virulence factors that are important for colonization of the urinary tract, including a known toxin ( Proteus toxic agglutinin) and the high pathogenicity island of Yersinia spp. In addition, this PAI shares homology and gene organization similar to the PAIs of other bacterial pathogens, several of which have been classified as mobile integrative and conjugative elements (ICEs). Isolates from this study were cultured from patients with caUTI and show identical sequence similarity at three loci within ICE Pm1 , suggesting its transfer between bacterial genera. Screening for the presence of ICE Pm1 among P. mirabilis colonizing isolates showed that ICE Pm1 is more prevalent in urine isolates compared to P. mirabilis strains isolated from other body sites ( P < 0.0001), further suggesting that it contributes to niche specificity and is positively selected for in the urinary tract.

Uropathogenic Escherichia coli (UPEC) cause the majority of uncomplicated urinary tract infections in humans. In the process of identifying candidate antigens for a vaccine, two methods for the identification of the UPEC surface proteome during growth in human urine were investigated. The first approach utilized a protease to ‘shave’ surface-exposed peptides from the bacterial cell surface and identify them by mass spectrometry. Although this approach has been successfully applied to a Gram-positive pathogen, the adaptation to Gram-negative UPEC resulted in cytoplasmic protein contamination. In a more direct approach, whole-cell bacteria were labeled with a biotin tag to indicate surface-exposed peptides and two-dimensional liquid chromatography-tandem mass spectrometry (2-DLC-MS/MS) was used to identify proteins isolated from the outer membrane. This method discovered 25 predicted outer membrane proteins expressed by UPEC while growing in human urine. Nine of the 25 predicted outer membrane proteins were part of iron transport systems or putative iron-regulated virulence proteins, indicating the importance of iron acquisition during growth in urine. One of the iron transport proteins identified, Hma, appears to be a promising vaccine candidate is being further investigated. The method described here presents a system to rapidly identify the outer membrane proteome of bacteria, which may prove valuable in vaccine development.

Urinary tract infection (UTI) occurs when bacteria, most commonly uropathogenic Escherichia coli (UPEC) [1], contaminate the periurethral area and traverse the urethra to colonize the bladder. Left untreated, UPEC ascend the ureters and establish a secondary infection in the kidney parenchyma. At this juncture, UPEC can elicit serious complications including renal scarring, septicemia, and even death. While young women are the most affected population, children, elderly, and hospitalized individuals are also at high risk. Up to 30% of patients experience recurrent episodes, contributing to billions of dollars spent annually to treat these infections [2]. Given the paucity of recent vaccine developments, the increasing rate of UPEC antibiotic resistance, and the need to reduce healthcare expenditures, new avenues of UTI vaccine research need to be explored. Conventional vaccinology approaches targeting bona fide virulence factors, namely FimH of type I fimbriae, have generated promising data in animal models [3] yet no vaccine is currently available in the United States. In Europe, licensed treatments consist of a complex mixture of whole killed bacteria; however, clinical data from the United States regarding recurrence and specific antibody titers does not suggest lasting immunity has been generated by this regimen [4]. Despite the advances afforded by reverse vaccinology (an in silico genomics-based approach to vaccine development [5]), it is not practical for UPEC due to a lack of high-throughput methods to test for protection. A more optimal approach to UPEC vaccine discovery would be one that is broad and unbiased yet has some aspect of selectivity to narrow the list of candidates. An example of this has been employed in pneumococcal vaccinology [6]. Applied to UPEC, it might be useful to target only PASivE candidates, UPEC proteins that are Pathogen-specific, Antigenic, Surface-exposed, and in vivo Expressed. These particular traits are attractive because they ensure that the targets are not widely expressed by commensal E. coli, are accessible to and recognized by the host immune system, and are synthesized specifically in vivo and likely important for infection and pathogenesis. To distinguish PASivE UPEC proteins, we can collectively analyze the results of genomic, proteomic, and metabolomic screens that each individually implicates proteins with one or more PASivE qualities (Table 1). The utility of large-scale genomic screens in bacterial pathogenesis and vaccine research has already been described [5]. In silico analyses, similar to those performed in reverse vaccinology studies, can also limit the candidate pool by predicting surface-exposed proteins, potential targets for secreted antibodies. While each screen has disadvantages, together they provide a comprehensive and impartial way to determine genes that encode PASivE proteins. Thus, screening for these specific candidates enriches for antigens that might provide protection based on biological relevance and creates a manageable number of genes to clone, express, and test in the murine model of ascending UTI. Table 1 Screens that can be used to identity PASivE UPEC vaccine candidates. The results generated by published and ongoing screens are particularly striking. Most of the screens primarily identified different classes of iron acquisition receptors. These outer membrane proteins are responsible for uptake of distinct siderophores or heme and are now PASivE candidates suitable for vaccine trials. This result supports the notion that similar to many other organisms, iron is crucial for UPEC to survive in its respective host niche, and the genomic dedication to functional redundancy supports the fact that the mammalian urinary tract is iron-limited. Indeed, mice vaccinated with the denatured IroN siderophore receptor are protected from transurethral UPEC challenge [7], a proof-of-concept for testing other PASivE proteins indicated by “omics” screens. Other potential targets identified by these studies include an array of metabolic transporters, putative adhesins, and hypothetical proteins that may represent novel virulence factors. The testing phase presents a new challenge for UPEC vaccinology. The field should advance towards a subunit vaccine directed against PASivE proteins. Additionally, being a mucosal pathogen, the vaccine must be administered with an appropriate adjuvant by a route and schedule that stimulates long-term immunity and not tolerance against UPEC. To this end, animal trials must be used to address these issues and also distinguish immunological correlates of protection and details regarding the molecular and cellular factors that play a role in the adaptive immune response to UTI. Screening for PASivE candidates requires the pathogen be culturable and have an animal model of infection, is limited to the identification of protein antigens, and is benefited greatly by a sequenced and annotated genome. Moreover, while lacking the breadth of coverage and total objectivity of reverse vaccinology, an “omics”-based screening initiative for PASivE proteins is particularly useful to produce a list of probable vaccine candidates. This scheme represents a rational approach for vaccine design to curb the social, economic, and personal burden caused by UPEC-mediated UTI. This approach can easily be translated to identify vaccine candidates for other significant human pathogens which are known to cause mucosal and systemic disease.

The urinary tract is among the most common sites of bacterial infection, and Escherichia coli is by far the most common species infecting this site. Individuals at high risk for symptomatic urinary tract infection (UTI) include neonates, preschool girls, sexually active women, and elderly women and men. E. coli that cause the majority of UTIs are thought to represent only a subset of the strains that colonize the colon. E. coli strains that cause UTIs are termed uropathogenic E. coli (UPEC). In general, UPEC strains differ from commensal E. coli strains in that the former possess extragenetic material, often on pathogenicity-associated islands (PAIs), which code for gene products that may contribute to bacterial pathogenesis. Some of these genes allow UPEC to express determinants that are proposed to play roles in disease. These factors include hemolysins, secreted proteins, specific lipopolysaccharide and capsule types, iron acquisition systems, and fimbrial adhesions. The current dogma of bacterial pathogenesis identifies adherence, colonization, avoidance of host defenses, and damage to host tissues as events vital for achieving bacterial virulence. These considerations, along with analysis of the E. coli CFT073, UTI89, and 536 genomes and efforts to identify novel virulence genes should advance the field significantly and allow for the development of a comprehensive model of pathogenesis for uropathogenic E. coli .Further study of the adaptive immune response to UTI will be especially critical to refine our understanding and treatment of recurrent infections and to develop vaccines.

Proteus mirabilis, a common agent of nosocomially acquired and catheter-associated bacteriuria, can cause acute pyelonephritis. In ascending infections, bacteria colonize the bladder and ascend the ureters to the proximal tubules of the kidney. We postulate that Proteus species uses the HpmA hemolysin and urease to elicit tissue damage that allows entry of these bacteria into the kidney. To study this interaction, strains of Proteus mirabilis and P. vulgaris and their isogenic hemolysin-negative (hpmA) or isogenic urease-negative (ureC) constructs were overlaid onto cultures of human renal proximal tubular epithelial cells (HRPTEC) isolated from kidneys obtained by immediate autopsy. Cytotoxicity was measured by release of soluble lactate dehydrogenase (LDH). Two strains of P. mirabilis inoculated at 10(6) CFU caused a release of 80% of total LDH after 6 h, whereas pyelonephritogenic hemolytic Escherichia coli CFT073 released only 25% at 6 h (P less than 0.012). Ten P. mirabilis isolates and five P. vulgaris isolates were all hemolytic and cytotoxic and produced urease which was induced by urea. The HpmA hemolysin is apparently responsible for the majority of cytotoxicity in vitro since the hemolysin-negative (hpmA) mutants of P. mirabilis and P. vulgaris were significantly less cytotoxic than wild-type strains. P. mirabilis WPM111 (hemolysin negative) was used to test the effect of urease-catalyzed urea hydrolysis on HRPTEC viability. In the presence of 50 mM urea, WPM111 caused the release of 42% of LDH versus 1% at 6 h in the absence of substrate (P = 0.003). We conclude that the HpmA hemolysin of Proteus species acts as a potent cytotoxin against HRPTEC. In addition, urease apparently contributes to this process when substrate urea is available.

A 1.9-kb cloned fragment of chromosomal DNA randomly selected from a Helicobacter pylori cloned library was evaluated as a potential probe. The probe detected 19 of 19 H. pylori strains and yielded a specificity of 98.7% when tested against 306 other bacterial strains representing 32 different species. False-positive results with non-H. pylori strains were due to the presence of contaminating vector sequences. A polymerase chain reaction (PCR) assay was developed by using 20-base oligonucleotide primers homologous to a portion of the 1.9-kb fragment. The PCR assay amplified a 203-nucleotide-pair product which was analyzed by agarose gel electrophoresis and Southern hybridization by using a third 20-base 32P-labeled oligonucleotide complementary to a region of DNA between the primers. The PCR assay was 100% sensitive, detecting all 35 H. pylori strains tested, and did not amplify sequences in several closely related species. The assay was sensitive for as little as one copy of the cloned plasmid DNA or 100 H. pylori bacterial cells. To evaluate the PCR assay for clinical samples, gastric biopsy and aspirate specimens were tested by PCR, and the results were compared with those of microbiologic culture and histologic examination. In fresh biopsy specimens, H. pylori sequences were detected by PCR in 13 of 14 (93%) positive tissues and 0 of 19 negative tissues. In gastric aspirate specimens, 11 of 13 (85%) positive tissues were positive by PCR. H. pylori DNA was detected in 1 of 14 aspirate specimens negative by culture, histology, and PCR of the accompanying biopsy tissue. PCR is a rapid, accurate, and sensitive method for the detection of H. pylori.

Urease (urea amidohydrolase, EC 3.5.1.5) catalyzes the hydrolysis of urea to yield ammonia and carbon dioxide. Research on this enzyme has gained momentum since the discovery of Helicobacter pylori as a causative agent of human gastritis. The remarkably high urease activity of each organism has served as the basis of diagnostic tests for the presence of the organism in the urease biopsy test and urea breath test. Urease undoubtedly plays a central role in H. pylori pathogenesis. Hydrolysis of urea with generation of ammonia may enable survival of this acid-sensitive organism in the gastric mucosa. Ammonia generated by urea hydrolysis may also produce severe cytotoxic effects within gastric epithelium. The enzyme also elicits a strong immune response during acute infection, suggesting that this abundant antigen is readily available to the immune system. An increase in serum IgG titer is predictive of ongoing infection. Much progress has been made with regard to the molecular biology of urease. The high molecular weight protein (estimated by several investigators to be 300-520 kDa) has been purified, revealing two distinct subunits of 29.5 kDa and 66 kDa, a unique subunit structure as compared with other microbial ureases. However, amino acid sequences are nevertheless well conserved when compared with other bacterial ureases and that of the jack bean, Canavalia ensiformis. Furthermore, genes encoding urease of H. pylori have been cloned, sequenced, and amplified by the polymerase chain reaction.

Urinary catheters are indwelling medical devices used in both the nosocomial and nursing home settings for relief of urinary retention and incontinence. Due to the frequent- and sometimes unnecessary-use of indwelling catheters in these settings (circa 21–50% of patients), many individuals are placed at risk from sequelae associated with catheter placement. The most notable complication associated with indwelling urinary catheters is the development of nosocomial urinary tract infections (UTIs) known as catheter-associated UTIs (CAUTIs). Over one million CAUTI cases per annum are recorded in the United States; this represents greater than 40% of all nosocomial infections. Both the presence of the catheter itself and the nosocomial setting predispose individuals to CAUTIs. CAUTIS may be caused by a number of etiological agents; the most common of these being Escherichia coli and Proteus mirabilis. Microorganisms isolated less commonly from CAUTIs include Staphylococcus aureus, Klebsiella spp., Pseudomonas spp. and Enterobacter spp. Regardless of the genus, pathogens wishing to survive within the environment of the catheterized urinary tract must possess essential virulence factors including, most importantly, those related to adherence to the catheter itself. This chapter seeks to set out in detail the nature of these virulence factors.

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.

SUMMARY 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.

Urease of Helicobacter pylori (formerly Campylobacter pylori) is believed to represent a critical virulence determinant for this species. Ammonia generated by hydrolysis of urea may protect the acid-sensitive bacterium as it colonizes human gastric mucosa. An H. pylori strain, cultured from a gastric biopsy of a patient with complaints of abdominal pain and a history of peptic ulcer disease, was isolated on selective medium and cultured in Mueller-Hinton broth supplemented with 4% fetal calf serum. Whole cells were ruptured by French pressure cell lysis, and soluble protein was chromatographed on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 resins. Purified urease represented 6% of the soluble protein of crude extract, was estimated to have a native molecular size of 550 kilodaltons (kDa), and was composed of two distinct subunits of apparent molecular sizes of 66 and 29.5 kDa. On the basis of subunit size, a 1:1 subunit ratio as measured by scanning densitometry of Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels, and estimated native molecular size, the data are consistent with a stoichiometry of (29.5 kDa-66 kDa)6 for the structure of the native enzyme. Km for urea was estimated at 0.2 mM. By N-terminal analysis, the 29.5-kDa subunit of H. pylori urease was found to share significant amino acid sequence similarity with the smallest of three subunits of the Proteus mirabilis and Morganella morganii ureases, as well as to the amino terminus of the unique jack bean subunit. The 66-kDa subunit also shared up to 80% similarity with the largest of three subunits of P. mirabilis, M. morganii, and Klebsiella aerogenes ureases and to internal sequences (amino acids 271 to 285) of the jack bean urease subunit. Thus, the amino acid sequence is conserved among ureases with one, two, and three distinct subunits, suggesting a common ancestral urease gene. Also, urease subunits of M. morganii and jack bean were specifically recognized by antisera raised against the 66-kDa subunit of H. pylori urease, demonstrating that at least some antigenic determinants were conserved among ureases from different species.

Proteus mirabilis, a common cause of urinary tract infection, can lead to serious complications including pyelonephritis. Adherence factors, urease, and hemolysin may be virulence determinants. These factors were compared for bacteria cultured from 16 patients with acute pyelonephritis and 35 with catheter-associated bacteriuria and for 20 fecal isolates. Pyelonephritis isolates were more likely (P less than .05) to express the mannose-resistant/Proteus-like (MR/P) hemagglutinin in the absence of mannose-resistant/Klebsiella-like (MR/K) hemagglutinin than were catheter-associated or fecal isolates. Pyelonephritis isolates produced urease activity of 63 +/- 27 (mean +/- SD) mumol of NH3/min/mg of protein, not significantly different from catheter-associated or fecal isolates. Hybridization of Southern blots of P. mirabilis chromosomal DNA with two urease gene probes demonstrated that urease gene sequences were conserved in all isolates. Geometric mean of reciprocal hemolytic titers for pyelonephritis isolates was 27.9; for urinary catheter isolates, 18.0; and for fecal isolates, 55.7 (not significantly different, P greater than .1). Although in vivo expression of urease and hemolysin may not be reliable indexes of virulence, MR/P hemagglutination in the absence of MR/K hemagglutination may be necessary for development of pyelonephritis.

These studies were undertaken to define conditions under which Helicobacter (formerly Campylobacter) pylori and viable human gastric mucosa could coexist in tissue culture with the ultimate goal of de

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 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., Author Summary The human urinary tract is a leading source for polymicrobial infections and for the development of bacteremia and sepsis. Treating these potentially dangerous infections have recently become more challenging due to the appearance of uropathogenic strains that are resistant to the many of the most commonly prescribed antibiotics. The majority of urinary tract infections (UTI) are caused by Escherichia coli, while another bacterium, Proteus mirabilis, is more likely to cause catheter-associated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent nutritional requirements despite growing in the same host environment. This result indicates that E. coli and P. mirabilis do not directly compete for nutrients during UTI. Indeed, we found that persistence of both pathogens is enhanced when they co-colonize the host. This work represents an important step toward understanding the basic nutritional requirements for two major pathogens that cause UTI and shows how mixed infections can change these requirements. Understanding how bacteria grow during infections is fundamental to ultimately uncover new ways to combat increasingly drug-resistant bacterial infections.

The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli , is a member of the SPATE (serine protease autotransporters of Enterobacteriaceae ) family and, as such, has serine protease activity and causes cytopathic effects on various cell types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second (S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or both serines reduced protease activity to background levels ( P < 0.001); a single mutation in the second serine reduced activity by 60% compared to wild type ( P < 0.001). After reversion of the S256I mutation to wild type (I256S), we confirmed S 256 as the catalytically active serine. None of these mutations affected secretion of the mature passenger domain or release into the supernatant. The S256I mutation, however, abrogated the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized by rounding and elongation, respectively, and a high level of cell detachment. Moreover, S 256 is essential for Sat to mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated proteins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins in vitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside.

The growth of the gastric pathogen Helicobacter pylori in the absence of serum remains challenging, and nutritional requirements have only partially been defined, while almost nothing is known about nutritional requirements of other Helicobacter spp. Although previous data showed that H. pylori grows in the chemically defined medium F-12, but not in other tissue culture media examined, the specific components responsible for growth were not entirely understood. Here we describe the optimization of amino acids, metals, and sodium chloride for H. pylori . Iron, zinc, and magnesium were critical for growth; copper was not required. Optimization of sodium chloride was further beneficial. Nutritional requirements and antibiotic resistance patterns of several other Helicobacter spp. revealed that all except H. felis grew in serum-free, unsupplemented F-12. All Helicobacter spp. were resistant to at least six antimicrobial agents when cultured in the presence of serum. However, in the absence of serum, H. pylori , H. mustelae , and H. muridarum became sensitive to polymyxin B and/or trimethoprim. Much of the data were obtained using a convenient ATP assay to quantify growth. H. pylori has surprisingly few absolute requirements for growth: 9 amino acids, sodium and potassium chloride, thiamine, iron, zinc, magnesium, hypoxanthine, and pyruvate. These data suggest that H. pylori and other Helicobacter spp. are not as fastidious as previously thought. The data also suggest that chemically defined media described herein could yield the growth of a wide range of Helicobacter spp., allowing a more detailed characterization of Helicobacter physiology and interactions with host cells.

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 is the most common etiological agent of urinary tract infections. Bacteria can often express multiple adhesins during infection in order to favor attachment to specific niches within the urinary tract. We have recently demonstrated that type 1 fimbria, a phase-variable virulence factor involved in adherence, was the most highly expressed adhesin during urinary tract infection. Here, we examine whether the expression of type 1 fimbriae can affect the expression of other adhesins. Type 1 fimbrial phase-locked mutants of E. coli strain CFT073, which harbors genes for numerous adhesins, were employed in this study. CFT073-specific DNA microarray analysis of these strains demonstrates that the expression of type 1 fimbriae coordinately affects the expression of P fimbriae in an inverse manner. This represents evidence for direct communication between genes relating to pathogenesis, perhaps to aid the sequential occupation of different urinary tract tissues. While the role of type 1 fimbriae during infection has been clear, the role of P fimbriae must be further defined to assert the relevance of coordinated regulation in vivo. Therefore, we examined the ability of P fimbrial isogenic mutants, constructed in a type 1 fimbrial-negative background, to compete in the murine urinary tract over a period of 168 h. No differences in the colonization of these mutants were observed. However, comparison of these results with previous studies suggests that inversely coordinated expression of adhesin gene clusters does occur in vivo. Interestingly, the mutant that was incapable of expressing either type 1 or P fimbriae compensated by synthesizing F1C fimbriae.

Background. The gastric pathogen Helicobacter pylori produces urease in amounts up to 10% of its cell protein. This enzyme, which catalyzes the hydrolysis of urea to ammonia and carbon dioxide, protects the bacterium from gastric acid. Urease, a nickel metalloenzyme, requires active uptake of nickel ions from the environment to maintain its activity. NixA is a nickel transport protein that resides in the cytoplasmic membrane. Mutation of nixA significantly reduces but does not abolish urease activity, strongly suggesting the presence of a second transporter. We postulated that the dipeptide permease (dpp) genes that are homologous to the nik operon of Escherichia coli could be a second nickel transporter. The predicted Dpp polypeptides DppA, DppC, and DppD of H. pylori share approximately 40%, 53%, and 56% amino acid sequence identity with their respective E. coli homologs. Methods. A mutation in dppA, constructed by insertional inactivation with a chloramphenicol resistance cassette, was introduced by allelic exchange into H. pylori strain 26695. Results. When compared to the parental strain, urease activity was not decreased in a dppA mutant. Conclusions. DppA does not contribute to the synthesis of catalytically active urease in H. pylori 26695 and is likely not a nickel importer in H. pylori

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 2,088 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 ∼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.

Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.

Proteus mirabilis, a cause of catheter-associated urinary tract infection, relies on several virulence factors to colonize the urinary tract. Among these, urease contributes to the development of urinary stones resulting from the increase in local pH due to urease-mediated hydrolysis of urea to NH3and CO2. UreR, an AraC-like transcriptional activator, activates transcription of the genes encoding the urease subunits and accessory proteins (ureDABCEFG) in the presence of urea. UreR also initiates transcription of its own gene in a urea-inducible manner by binding to the intergenic region betweenureRandureD. The intergenic region contains poly(A) tracts that appear to be the target of H-NS. It has been shown thatEscherichia coliandP. mirabilisH-NS acts to repress transcription ofureRin anE. colimodel system. It was hypothesized that H-NS represses urease gene expression in the absence of UreR and urea by binding to the intergenic region. To demonstrate this theP. mirabilis hnsgene was cloned and the 15·6 kDa H-NS was overexpressed and purified as amyc-His tail fusion. Using a gel shift assay, purified H-NS-myc-His bound preferentially to a 609 bp DNA fragment containing the entireureR-ureDintergenic region. H-NS and UreR were able to displace each other from theureR-ureDintergenic region. Circular permutation analysis revealed that the intergenic region is bent. Moreover, H-NS recognizes this curvature, binds the DNA fragment and induces further bending of the DNA as shown by a circular ligation assay. The effects of H-NS, urea and temperature (25 vs 37 °C) on urease expression were shown inE. colicontaining anhnsknockout andP. mirabiliswhere expression was increased at 37 °C. Increased transcription from pureRwas seen in theE. coli hnsknockout when temperature was increased from 25 to 37 °C. These findings suggest H-NS and UreR differentially regulate urease in a negative and positive manner, respectively.

Helicobacter pylori strains have been shown to display considerable heterogeneity with respect to DNA sequence. Diverse restriction fragment length polymorphism (RFLP) patterns are generated among strains by restriction endonuclease digestion of whole chromosomal DNA (1-3), digestion of specific polymerase chain reaction (PCR) products (4), or arbitrary primer-PCR and random amplified polymorphic DNA-PCR (5-7). These techniques demonstrate numerous distinct and reproducible patterns that can be used to differentiate strains. Sequence analyses of specific genes have confirmed that there are frequent nucleotide substitutions among strains, usually in the third position of codons, which may not result in amino acid substitution within the corresponding polypeptides (8). This type of heterogeneity is found in all H. pylori strains, whether they are considered virulent or nonvirulent.

We present the complete genome sequence of uropathogenic Escherichia coli , strain CFT073. A three-way genome comparison of the CFT073, enterohemorrhagic E. coli EDL933, and laboratory strain MG1655 reveals that, amazingly, only 39.2% of their combined (nonredundant) set of proteins actually are common to all three strains. The pathogen genomes are as different from each other as each pathogen is from the benign strain. The difference in disease potential between O157:H7 and CFT073 is reflected in the absence of genes for type III secretion system or phage- and plasmid-encoded toxins found in some classes of diarrheagenic E. coli . The CFT073 genome is particularly rich in genes that encode potential fimbrial adhesins, autotransporters, iron-sequestration systems, and phase-switch recombinases. Striking differences exist between the large pathogenicity islands of CFT073 and two other well-studied uropathogenic E. coli strains, J96 and 536. Comparisons indicate that extraintestinal pathogenic E. coli arose independently from multiple clonal lineages. The different E. coli pathotypes have maintained a remarkable synteny of common, vertically evolved genes, whereas many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain.

Helicobacter pylori and Proteus mirabilis ureases are nickel-requiring metallo-enzymes that hydrolyse urea to NH3 and CO2. In both H. pylori and in an Escherichia coli model of H. pylori urease activity, a high affinity nickel transporter, NixA, is required for optimal urease activity, whereas the urea-dependent UreR positive transcriptional activator governs optimal urease expression in P. mirabilis. The H. pylori flbA gene is a flagellar biosynthesis and regulatory gene that modulates urease activity in the E. coli model of H. pylori urease activity. All flbA mutants of eight strains of H. pylori were non-motile and five had a strain-dependent alteration in urease activity. The flbA gene decreased urease activity 15-fold when expressed in E. coli containing the H. pylori urease locus and the nixA gene; this was reversed by disruption of flbA. The flbA gene decreased nixA transcription. flbA also decreased urease activity three-fold in E. coli containing the P. mirabilis urease locus in a urea- and UreR-dependent fashion. Here the flbA gene repressed the P. mirabilis urease promoter. Thus, FlbA decreased urease activity of both H. pylori and P. mirabilis, but through distinct mechanisms. H. pylori wild-type strain SS1 colonised gerbils at a mean of 5.4 x 10(6) cfu/g of antrum and caused chronic gastritis and lesions in the antrum. In contrast, the flbA mutant did not colonise five of six gerbils and caused no lesions, indicating that motility mediated by flbA was required for colonisation. Because FlbA regulates flagellar biosynthesis and secretion, as well as forming a structural component of the flagellar secretion apparatus, two seemingly unrelated virulence attributes, motility and urease, may be coupled in H. pylori and P. mirabilis and possibly also in other motile, ureolytic bacteria.

The secreted autotransporter toxin (Sat) of uropathogenic Escherichia coli exhibits cytopathic activity upon incubation with HEp-2 cells. We further investigated the effects of Sat on cell lines more relevant to the urinary tract, namely, those derived from bladder and kidney epithelium. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (CRL-1749) and kidney (CRL-1573) cell lines. This activity has been associated with only a few other known toxins. Following transurethral infection of CBA mice with a sat mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type E. coli CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type E. coli CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat, a vacuolating cytotoxin expressed by uropathogenic E. coli CFT073, elicits defined damage to kidney epithelium during upper urinary tract infection and thus contributes to pathogenesis of urinary tract infection.

Proteus mirabilis is a common cause of urinary tract infection (UTI) in individuals with structural abnormalities or long-term catheterization. The expression of mannose-resistant/Proteus-like (MR/P) fimbria is phase variable because of the inversion of a 251 bp DNA fragment that carries the promoter for the mrp operon. Previous studies have shown that mrpI, which is transcribed divergently from the mrp operon, encodes a recombinase capable of switching the orientation of this invertible element. In this study, we constructed isogenic mrpI null mutants from a clinical isolate of P. mirabilis, HI4320. A polymerase chain reaction (PCR)-based invertible element assay revealed that the isogenic mrpI null mutants were locked in one phase, either expressing (locked on) MR/P fimbriae or not (locked off), which indicated that MrpI was the sole recombinase that regulated the phase variation of MR/P fimbria. The locked-on and locked-off mutants were evaluated for virulence in the CBA mouse model of ascending UTI by co-challenges with each other and with the wild-type strain. Results from these experiments demonstrated conclusively that the MR/P fimbria was a critical bladder colonization factor of uropathogenic P. mirabilis and also suggested that the ability to switch off the expression of MR/P fimbria might be important for kidney colonization.

Proteus mirabilis is a documented cause of urinary tract infection (UTI) in the complicated urinary tract. Urease-mediated urea hydrolysis is responsible for both virulence of the organism and the ability to cause urolithiasis. A urease-negative mutant of P. mirabilis is unable to initiate stone formation and colonizes the kidney at a significantly lower rate. The considerable pathology caused by P. mirabilis warrants the development of a vaccine. We have initiated the advancement of vaccine studies and have determined that the MR/P fimbria, a surface adhesin of P. mirabilis, is a promising vaccine candidate. Successful vaccination would be expected both to prevent colonization by P. mirabilis and urolithiasis.

Recent genomic analyses of Escherichia coli O157:H7 strain EDL933 revealed two loci encoding urease gene homologues ( ureDABCEFG ), which are absent in nonpathogenic E. coli strain K-12. This report demonstrates that the cloned EDL933 ure gene cluster is capable of synthesizing urease in an E. coli DH5α background. However, when the gene fragment is transformed back into the native EDL933 background, the enzymatic activity of the cloned determinants is undetectable. We speculate that an unidentified trans -acting factor in enterohemorrhagic E. coli (EHEC) is responsible for this regulation of ure expression. In addition, Fur-like recognition sites are present in three independent O157:H7 isolates upstream of ureD and ureA . Enzymatic assays confirmed a difference in urease expression of cloned EHEC ure clusters in E. coli MC3100Δ fur . Likewise, interruption of fur in O157:H7 isolate IN1 significantly diminished urease activity. We propose that, similar to the function of Fur in regulating the acid response of Salmonella enterica serovar Typhimurium, it modulates urease expression in EHEC, perhaps contributing to the acid tolerance of the organism.

Helicobacter pylori mutants devoid of urease activity fail to colonize the gastric mucosa of mice; however, the effect of decreased levels of urease on colonization has not been examined. The nixA gene, required for full urease activity, encodes a cytoplasmic membrane nickel transporter that imports nickel ions and leads to incorporation of nickel ions into apourease. A nixA mutant of the Sydney strain of H. pylori (SS1) was constructed by disruption of the nixA gene with a kanamycin resistance cassette. This mutant retained only half the urease activity of the wild-type (wild-type) SS1 strain. C57BL/6j ( n = 75) and BALB/c ( n = 75) mice were inoculated independently with the wild-type or the nixA strain. The level and distribution of colonization were assessed by bacterial colony counts and histological grading at 4, 12, and 24 weeks postinfection. Colonization levels of the nixA strain in BALB/c mice were significantly lower compared with SS1 ( P = 0.005), while colonization in C57BL/6j mice was similar for both the wild-type and mutant strains. Subtle differences in colonization of the different regions of the stomach, determined by microscopic grading, were observed between wild-type SS1 and the nixA strain in BALB/c mice. On the contrary, when C57BL/6j ( n = 35) and BALB/c ( n = 35) mice were coinfected with the wild-type and nixA strains simultaneously, the nixA mutant failed to colonize and was outcompeted by the wild-type SS1 strain, which established normal levels of colonization. These results demonstrate the importance of the nixA gene for increasing the fitness of H. pylori for gastric colonization. Since nixA is required for full urease activity, the decreased fitness of the nixA mutant is likely due to reduced urease activity; however, pleiotropic effects of the mutation cannot be completely ruled out.

Publisher Summary The urinary tract is a common site of bacterial infection. Individuals at high risk for symptomatic urinary tract infection (UTI) include neonates, pre-school girls, sexually active women, and the elderly. In addition, UTI is the most frequent cause of infections among nursing home residents and the most common documented source of bacteraemia in the elderly. Escherichia coli (E. coli) is the most common cause of community-acquired infection in the individuals with a normal urinary tract. Nevertheless, other important microorganisms for which much less information is available also cause UTI. This chapter focuses on these less commonly isolated but important organisms. While E. coli is the cause of most infections in the normal urinary tract, many other bacterial species, including other genera of the Enterobacteriaceae as well as Gram-positive organisms, are able to colonize both the normal and the structurally compromised urinary tract. Of these organisms, most is known about P. mirabilis, although other species share common themes of pathogenicity, including adherence, production of urease, and a limited ability to invade host epithelium.

Bacterial pili and fimbriae are hairlike structures extending from the cell surface that are involved in conjugation, adherence and twitching motility. Keywords: pili; fimbriae; conjugation; adhesins; phase variation

The antimicrobial effect of nitric oxide (NO) is an essential part of innate immunity. The vigorous host response to the human gastric pathogenHelicobacter pylorifails to eradicate the organism, despite up-regulation of inducible NO synthase (iNOS) in the gastric mucosa. Here we report that wild-type strains ofH. pyloriinhibit NO production by activated macrophages at physiologic concentrations ofl-arginine, the common substrate for iNOS and arginase. Inactivation of the generocF, encoding constitutively expressed arginase inH. pylori, restored high-output NO production by macrophages. By using HPLC analysis, we show thatl-arginine is effectively consumed in the culture medium by wild-type but not arginase-deficientH. pylori. The substantially higher levels of NO generated by macrophages cocultured withrocF-deficientH. pyloriresulted in efficient killing of the bacteria, whereas wild-typeH. pyloriexhibited no loss of survival under these conditions. Killing of the arginase-deficientH. pyloriwas NO-dependent, because peritoneal macrophages from iNOS−/−mice failed to affect the survival of therocFmutant. Thus, bacterial arginase allowsH. pylorito evade the immune response by down-regulating eukaryotic NO production.

Proteus mirabilis infects the urinary tract of humans and is most commonly responsible for causing disease in individuals with structural abnormalities of the urinary tract or in patients who undergo long-term catheterization (16). Cystitis, acute pyelonephritis, and urinary stone formation are all possible consequences of P. mirabilis infection (17). P. mirabilis produces a urea-inducible urease, a high-molecular-weight, multimeric, cytoplasmic nickel metalloenzyme. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide (18). During the course of infection, the production of ammonia by urea hydrolysis raises the pH in the local environment, subsequently precipitating polyvalent ions that are normally soluble in urine. The result is the formation of urinary stones. The elevated pH also creates an environment that is more favorable for growth of this species (4). Increased ammonia production can also lead to acute inflammation with possible tissue necrosis (18). The P. mirabilis urease gene cluster is found in single copy on the chromosome and consists of eight contiguous genes, ureRDABCEFG (12, 19, 24). The ureA (UreA, 11 kDa), ureB (UreB, 12 kDa), and ureC (UreC, 61 kDa) genes encode the structural polypeptides required for the assembly of a catalytically inactive urease apoenzyme (18). The accessory genes, ureD (UreD, 31 kDa), ureE (UreE, 18 kDa), ureF (UreF, 23 kDa), and ureG (UreG, 22 kDa), encode proteins required for insertion of nickel ions into the metalloenzyme resulting in catalytically active urease (18). The urease gene cluster is regulated by the gene product of ureR (UreR, 33 kDa). P. mirabilis UreR and the plasmid-encoded UreR found in Escherichia coli are positive transcriptional activators of the urease genes. The two proteins share 70% amino acid identity (6) and are functionally interchangeable in the activation of transcription from the ureR (pureR) and ureD (pureD) promoters in both the P. mirabilis and plasmid-encoded urease gene clusters (6). The UreR binding sites of both promoters have the consensus sequence T(A/G)(T/C)(A/T)(T/G)(C/T)T(A/T)(T/A)ATTG (25). Both UreR proteins have been shown to activate transcription from pureD in the presence of urea (11, 6). In addition, UreR regulates its own transcription in the presence of urea from pureR in the direction opposite the rest of the gene cluster (6). In the absence of urea induction, H-NS represses ureR expression (3). Because UreR activates transcription in a urea-inducible manner, it is hypothesized that UreR binds urea; however, this has not been directly demonstrated. UreR is a member of the AraC family of transcriptional regulators and contains a putative helix-turn-helix in addition to an AraC signature sequence (5, 19). The AraC signature sequence, found within all AraC family members, is a second helix-turn-helix that is hypothesized to also bind DNA (7). Moreover, UreR also contains three conserved leucine residues (Leu147, Leu148, and Leu158) in the same relative location with the same spatial distance relative to each other as in AraC (Leu150, Leu151, and Leu161). These leucine residues are critical for AraC dimerization (23), and we therefore also hypothesize that UreR dimerizes via this mechanism. In the presence of arabinose, AraC uses these three critical leucines for dimerization via an antiparallel coiled-coil in a “knobs-into-holes” manner, as elucidated by X-ray crystallographic studies (23). This coiled-coil is also the primary dimerization face in the absence of arabinose, shown by both size exclusion chromatography and sedimentation velocity analytical ultracentrifugation of an AraC mutant with mutations in Leu150, Leu151, Asn154, and Leu161 (15). A secondary dimerization face in the β barrel of AraC is evident; however, it does not appear to represent the primary means of dimer interaction (15). AraC contains two separate and independent domains, each with a distinct function, namely, dimerization and DNA binding; UreR is predicted to have similar domains with similar functions. Previously, chimeric proteins containing the two domains of AraC to characterize each of the domain's functions were synthesized (2). The predicted AraC DNA-binding domain was fused to C/EBP, a known eukaryotic transcriptional activator that dimerizes via a leucine zipper. The C/EBP-AraC fusion was found to bind to pBAD and activate transcription (2). The hypothesized AraC dimerization domain was fused to the LexA DNA-binding domain. This fusion was predicted to mimic full-length LexA and demonstrated the need for dimerization in order to repress transcription of genes normally turned off by LexA. A chromosomal transcriptional fusion of psulA to lacZ was repressed in the presence of the AraC-LexA fusion protein. This strategy was used to identify both domains of AraC (2). In this study, we constructed fusion proteins to identify putative domains of UreR and assign dimerization and DNA-binding functions to each of the domains as well as identifying key amino acid residues involved in dimerization and urea induction.

Exchange of genetic material between bacterial species is mediated by the basic processes of conjugation, transduction and transformation. This exchange is fundamental for bacterial evolution and adaptation in a dynamic environment. Keywords: conjugation; genetic exchange; integrons; pathogenicity islands; transduction; transformation

Escherichia coli is the primary cause of uncomplicated infections of the urinary tract including cystitis. More serious infections, characterized as acute pyelonephritis, can also develop. Type 1 fimbriae of E. coli contribute to virulence in the urinary tract; however, only recently has the expression of the type 1 fimbriae been investigated in vivo using molecular techniques. Transcription of type 1 fimbrial genes is controlled by a promoter that resides on a 314-bp invertible element capable of two orientations. One places the promoter in the ON orientation, allowing for transcription; the other places the promoter in the OFF orientation, preventing transcription. A PCR-based assay was developed to measure the orientation of the invertible element during an experimental urinary tract infection in mice. Using this assay, it was found that the percentage of the population ON in urine samples correlated with the respective CFU per gram of bladder ( P = 0.0006) but not with CFU per gram of kidney ( P > 0.069). Cystitis isolates present in the urine of mice during the course of infection had a higher percentage of their invertible elements in the ON orientation than did pyelonephritis isolates (85 and 34%, respectively, at 24 h; P < 0.0001). In general, cystitis isolates, unlike pyelonephritis isolates, were more likely to maintain their invertible elements in the ON orientation for the entire period of infection. E. coli cells expressing type 1 fimbriae, expelled in urine, were shown by scanning electron microscopy to be densely packed on the surface of uroepithelial cells. These results suggest that expression of type 1 fimbriae is more critical for cystitis strains than for pyelonephritis strains in the early stages of an infection during bladder colonization.

Background.Helicobacter pylori survives transient exposure to extreme acid prior to adherence and growth on the gastric epithelium at neutral pH. Materials and Methods. The effect of pH stress on protein profiles of H. pylori was observed using two-dimensional gel electrophoresis (2-D gels). H. pylori 26695 was grown microaerobically in tryptone-yeast extract broth, 3% fetal bovine serum. Growth in acid alkalinized the medium, whereas growth in base caused acidification. For 2-D gel analysis of protein profiles, cultures were grown in media buffered at pH 5.7 and at pH 7.5. Results. Under all pH conditions, the most abundant proteins observed were the urease structural subunit UreB and the chaperonin GroEL. Growth in acid significantly increased the abundance of UreB. Thus, urease expression is not completely constitutive, as reported previously, but shows regulation by pH. Another protein observed only at low pH was identified as mammalian apolipoprotein A-I, possibly taken up by H. pylori from bovine serum in the growth medium. This finding, if confirmed, suggests that uptake of high-density lipoprotein from the human host may facilitate acquisition of cholesterol, required for formation of the unique cholesteryl glucosides in the membrane of H. pylori. In growth above pH 7, three stress proteins were induced: GroES (HspA), GroEL (HspB), and the antioxidant AhpC homolog TsaA. In addition, N-terminal sequence analysis identified five additional proteins that had not previously been reported on 2-D gels of H. pylori (FMN, SodB, TrxB, TsaA, and Tsr). Conclusions. In summary, our 2-D gel study reveals expression of several proteins dependent on growth pH.

The urinary tract is among the most common sites of bacterial infection, and Escherichia coli is by far the most common cause of community-acquired infection in individuals with a normal urinary tract. In addition, E. coli is the predominant cause of uncomplicated cystitis and acute pyelonephritis. Of women going to physicians for a urinary tract infection (UTI), 95% do so for symptoms of cystitis. It is estimated that 40% of adult women will suffer symptoms of cystitis during their lifetime, and E. coli will be identified as the etiologic agent in 75%-80% of these cases.

Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions amongP. mirabilisaccessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo,P. mirabilisUreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

This chapter reviews current understanding of how pylori persistently colonize the human gastric mucosa and discuss factors that determine whether infections progress to clinically evident disease or remain asymptomatic. Helicobacter pylori are extraordinary among bacteria in their ability to colonize the human gastric mucosa, an inherently inhospitable acidic environment, despite the development of host immune and inflammatory responses. To the available knowledge, no other bacteria, with the possible exception of the closely related H. heilmanii , can establish long-term residence in the human gastric mucosa. More than half of the world's human population is persisently colonized with H. pylori , usually without disease. However, in about 10% of cases, colonization, probably in combination with other critical risk factors, leads to serious illnesses, ranging from peptic ulcers to two different forms of gastric cancer. H . pylori occupy an interesting position in the history of medicine and biomedical research..

Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.

colonizes the gastricmucosa of humans and can causelifelong gastritis if left untreated.Although infection can be asymp-tomatic, it can also manifest clini-cally as peptic or duodenal ulcerand can result in the developmentof gastric cancer. One of the bac-terium’s most prominent featuresis the production of high levels ofurease, an enzyme that hydrolyzesurea to CO