Preventing urinary tract infection: progress toward an effective Escherichia coli vaccine

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.     

ATG16L1 deficiency in macrophages drives clearance of uropathogenic E. coli in an IL-1β-dependent manner

Urinary tract infections (UTIs) are frequent, commonly recurrent, and costly. Deficiency in a key autophagy protein, ATG16L1, protects mice from infection with the predominant bacterial cause of UTIs, Uropathogenic E. coli (UPEC). Here, we report that loss of ATG16L1 in macrophages accounts for this protective phenotype. Compared with wild-type macrophages, macrophages deficient in ATG16L1 exhibit increased uptake of UPEC and enhanced secretion of interleukin-1β (IL-1β). The increased IL-1β production is dependent upon activation of the NLRP3 inflammasome and caspase-1. IL-1β secretion was also enhanced during UPEC infection of ATG16L1-deficient mice in vivo, and inhibition of IL-1β signaling abrogates the ATG16L1-dependent protection from UTIs. Our results argue that ATG16L1 normally suppresses a host-protective IL-1β response to UPEC by macrophages.     

Escherichia coli from urine of female patients with urinary tract infections is competent for intracellular bacterial community formation

Nearly 50% of women experience at least one urinary tract infection (UTI) in their lifetime. Studies with mice have revealed that uropathogenic Escherichia coli (UPEC) isolates invade superficial umbrella cells that line the bladder, allowing them to find a safe haven and subvert clearance by innate host responses. Rapid intracellular replication results in the formation of distinctive intracellular bacterial communities (IBCs). In this study, we evaluated whether UPEC strains cultured from the urine of women and classified as causing acute cystitis, recurrent cystitis, asymptomatic bacteriuria, or pyelonephritis could progress through the IBC cascade in a well-characterized mouse model of cystitis. Of 18 UPEC isolates collected from women, 15 formed IBCs. Variations in the size, number, and kinetics of IBC formation were observed with strains isolated from women with different clinical syndromes. Two of the three isolates that did not form IBCs when inoculated alone were able to do so when coinoculated with an isolate that was capable of generating IBCs. The mixed infections dramatically altered the behavior of the coinfecting bacteria relative to their behavior in a single infection. The study also showed that mice with five different genetic backgrounds can support IBC formation. Although UPEC isolates differ genetically in their virulence factors, the majority of UPEC isolates from different types of UTI proceed through the IBC pathway, confirming the generality of IBCs in UTI pathogenesis in mice.     

The glycobiology of uropathogenic E. coli infection: the sweet and bitter role of sugars in urinary tract immunity

Urinary tract infections (UTI) are among the most prevalent infectious diseases and the most common cause of nosocomial infections, worldwide. Uropathogenic E. coli (UPEC) are responsible for approximately 80% of all UTI, which most commonly affect the bladder. UPEC colonize the urinary tract by ascension of the urethra, followed by cell invasion, and proliferation inside and outside urothelial cells, thereby causing symptomatic infections and quiescent intracellular reservoirs that may lead to recurrence. Sugars, or glycans, are key molecules for host–pathogen interactions, and UTI are no exception. Surface glycans regulate many of the events associated with UPEC adhesion and infection, as well as induction of the host immune response. While the bacterial protein FimH binds mannose‐containing host glycoproteins to initiate infection and UPEC‐secreted polysaccharides block immune mechanisms to favour intracellular replication, host glycans on the urothelial surface and on secreted glycoproteins prevent or limit infection by inhibiting UPEC adhesion. Given the importance of glycans during UTI, here we review the glycobiology of UPEC infection to highlight fundamental sugar‐mediated processes of immunological interest for their potential clinical applications. Interdisciplinary approaches incorporating glycomics and infection biology may help to develop novel non‐antibiotic‐based therapeutic strategies for bacterial infections as the spread of antimicrobial‐resistant uropathogens is currently threatening modern healthcare systems.     

Effect of trimethoprim-sulfamethoxazole on recurrent bacteriuria and bacterial persistence in mice infected with uropathogenic Escherichia coli

One of the more perplexing aspects of urinary tract infections (UTIs) is their high propensity to recur. It has been proposed that recurrent infections are a result of the reintroduction of bacteria from the gastrointestinal tract (GIT) to the urinary tract (UT); however, since a significant subset of recurrent UTIs are caused by an identical bacterial strain, it has been challenging to formally prove this hypothesis for same-strain recurrences by using epidemiologic approaches. We present data here obtained by using a mouse model of UTIs in which it was shown that 36% (5 of 14) of mice infected with uropathogenic Escherichia coli (UPEC) will have at least one bacteriuric recurrence, with 21% (3 of 14) having more than one recurrence during a 6-week period after an acute UTI. Intraurethrally infected mice develop UPEC reservoirs in both their feces and their bladders. Ten days of trimethoprim-sulfamethoxazole (SXT) therapy reduces urinary recurrences and eradicates fecal colonization, whereas 3 days of SXT treatment has no effect over a twenty-eight-day observation period despite clearing fecal colonization acutely. Interestingly, SXT is unable to eradicate bacteria from the bladder reservoir even after a 10-day treatment regimen, thus demonstrating that the bladder reservoir can persist even in the face of long-term antibiotic therapy.     

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

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

Population dynamics and niche distribution of uropathogenic Escherichia coli during acute and chronic urinary tract infection

Urinary tract infections (UTIs) have complex dynamics, with uropathogenic Escherichia coli (UPEC), the major causative agent, capable of colonization from the urethra to the kidneys in both extracellular and intracellular niches while also producing chronic persistent infections and frequent recurrent disease. In mouse and human bladders, UPEC invades the superficial epithelium, and some bacteria enter the cytoplasm to rapidly replicate into intracellular bacterial communities (IBCs) comprised of ∼10⁴ bacteria each. Through IBC formation, UPEC expands in numbers while subverting aspects of the innate immune response. Within 12 h of murine bladder infection, half of the bacteria are intracellular, with 3 to 700 IBCs formed. Using mixed infections with green fluorescent protein (GFP) and wild-type (WT) UPEC, we discovered that each IBC is clonally derived from a single bacterium. Genetically tagged UPEC and a multiplex PCR assay were employed to investigate the distribution of UPEC throughout urinary tract niches over time. In the first 24 h postinfection (hpi), the fraction of tags dramatically decreased in the bladder and kidney, while the number of CFU increased. The percentage of tags detected at 6 hpi correlated to the number of IBCs produced, which closely matched a calculated multinomial distribution based on IBC clonality. The fraction of tags remaining thereafter depended on UTI outcome, which ranged from resolution of infection with or without quiescent intracellular reservoirs (QIRs) to the development of chronic cystitis as defined by persistent bacteriuria. Significantly more tags remained in mice that developed chronic cystitis, arguing that during the acute stages of infection, a higher number of IBCs precedes chronic cystitis than precedes QIR formation.     

Strain- and host species-specific inflammasome activation,IL-1β release,and cell death in macrophages infected with uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans.     

Estrogenic action on innate defense mechanisms in the urinary tract

Clinical data suggest an impact of estrogen on the pathogenesis of urinary tract infections (UTI). In particular, women after menopause often suffer from recurrent UTI, characterized by at least three acute UTI episodes within a year. Aside from bacterial factors promoting persistence within the urinary bladder, the low estrogen levels induce structural and chemical changes in the urogenital tract which facilitate UTI. Increased residual urine volume and changes in the vaginal microflora are well documented risk factors. Local supplementation with estrogen can at least partly reverse these changes. Treatment allows the re-establishment of a lactobacilli-dominated vaginal microflora and improves epithelial differentiation and integrity in the urogenital tract. This estrogenic effect on the epithelium is marked by an increased production of antimicrobial peptides and a tighter intercellular connection, preventing bacteria from reaching cells where they can hide and later emerge and cause a new infection. Estrogen in the dosages and applications used to date is considered safe for the endometrium in the majority of women. Based on the actions and safety of estrogen, local supplementation thus offers a treatment option for postmenopausal women suffering from recurrent UTI.     

Estrogen receptors in human bladder cells regulate innate cytokine responses to differentially modulate uropathogenic E. coli colonization

The bladder epithelial cells elicit robust innate immune responses against urinary tract infections (UTIs) for preventing the bacterial colonization. Physiological fluctuations in circulating estrogen levels in women increase the susceptibility to UTI pathogenesis, often resulting in adverse health outcomes. Dr adhesin bearing Escherichia coli (Dr E. coli) cause recurrent UTIs in menopausal women and acute pyelonephritis in pregnant women. Dr E. coli bind to epithelial cells via host innate immune receptor CD55, under hormonal influence. The role of estrogens or estrogen receptors (ERs) in regulating the innate immune responses in the bladder are poorly understood. In the current study, we investigated the role of ERα, ERβ and GPR30 in modulating the innate immune responses against Dr E. coli induced UTI using human bladder epithelial carcinoma 5637 cells (HBEC). Both ERα and ERβ agonist treatment in bladder cells induced a protection against Dr E. coli invasion via upregulation of TNFα and downregulation of CD55 and IL10, and these effects were reversed by action of ERα and ERβ antagoinsts. In contrast, the agonist-mediated activation of GPR30 led to an increased bacterial colonization due to suppression of innate immune factors in the bladder cells, and these effects were reversed by the antagonist-mediated suppression of GPR30. Further, siRNA-mediated ERα knockdown in the bladder cells reversed the protection against bacterial invasion observed in the ERα positive bladder cells, by modulating the gene expression of TNFα, CD55 and IL10, thus confirming the protective role of ERα. We demonstrate for the first time a protective role of nuclear ERs, ERα and ERβ but not of membrane ER, GPR30 against Dr E. coli invasion in HBEC 5637 cells. These findings have many clinical implications and suggest that ERs may serve as potential drug targets towards developing novel therapeutics for regulating local innate immunity and treating UTIs.     

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

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

Modulation of host innate immune response in the bladder by uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC), the most frequent cause of urinary tract infection (UTI), is associated with an inflammatory response which includes the induction of cytokine/chemokine secretion by urothelial cells and neutrophil recruitment to the bladder. Recent studies indicate, however, that UPEC can evade the early activation of urothelial innate immune response in vitro. In this study, we report that infection with the prototypic UPEC strain NU14 suppresses tumor necrosis factor alpha (TNF-alpha)-mediated interleukin-8 (CXCL-8) and interleukin-6 (CXCL-6) secretion from urothelial cell cultures compared to infection with a type 1 piliated E. coli K-12 strain. Furthermore, examination of a panel of clinical E. coli isolates revealed that 15 of 17 strains also possessed the ability to suppress cytokine secretion. In a murine model of UTI, NU14 infection resulted in diminished levels of mRNAs encoding keratinocyte-derived chemokine, macrophage inflammatory peptide 2, and CXCL-6 in the bladder relative to infection with an E. coli K-12 strain. Furthermore, reduced stimulation of inflammatory chemokine production during NU14 infection correlated with decreased levels of bladder and urine myeloperoxidase and increased bacterial colonization. These data indicate that a broad phylogenetic range of clinical E. coli isolates, including UPEC, may evade the activation of innate immune response in the urinary tract, thereby providing a pathogenic advantage.     

Time Course and Host Responses to Escherichia coli Urinary Tract Infection in Genetically Distinct Mouse Strains

Recurrent urinary tract infections (UTIs) are a significant clinical problem for many women; however, host susceptibility factors have not been completely defined. The mouse model of induced UTI provides an experimental environment in which to identify specific host characteristics that are important in initial bacterial colonization of the urinary tract and in resolution of an infection. This study examined initial susceptibility, bacterial clearance, and host defense mechanisms during induction and resolution of Escherichia coli UTIs in genetically distinct strains of mice. Of the ten inbred strains tested, six (BALB/c, C3H/HeN, C57BL/6, DBA.1, DBA.2, and AKR) showed progressive resolution of bladder infections over a 14-day period. A constant, low-level bladder infection was observed in SWR and SJL mice. High bladder infection levels persisted over the 14-day study period in C3H/HeJ and C3H/OuJ mice. Kidney infection levels generally correlated with bladder infection levels, especially in C3H/HeJ and C3H/OuJ mice, the two most susceptible strains, in which infections became more severe with time after challenge. The degree of inflammation in bladder and kidneys, as well as antibody-forming cell responses, positively correlated with infection intensity in all strains except C3H/HeJ, which had minimal inflammation despite high infection levels. These results demonstrate two important aspects of host defense against UTI. First, the innate immune response to an infection in the bladder or kidneys consists primarily of local inflammation, which is followed by an adaptive response characterized in part by an antibody response to the infecting bacteria. Second, a UTI will be spontaneously resolved in most cases; however, in mice with specific genetic backgrounds, a UTI can persist for an extended length of time. The latter result strongly suggests that the presence or absence of specific host genes will determine how effectively an E. coli UTI will be resolved.     

Mannose-binding lectin in pre-menopausal women with recurrent urinary tract infections

Mannose-binding lectin (MBL) comprises an oligomeric serum protein that is a member of the collectin class of the C-type lectin super-family. Its deficiency is genetically determined and confers predisposition to recurrent infections as well as increased infection severity. This correlation has been demonstrated in recurrent furunculosis caused by Staphylococcus aureus, and in pneumococcal and Candida infections. The present study aimed to determine whether there is a correlation between MBL serum levels and recurrent urinary tact infections (UTI) in pre-menopausal women. The present aged-matched double-blind controlled study was conducted in 100 pre-menopausal adult women: 50 who suffered from recurrent UTI and 50 without UTI. The MBL concentration was measured in a single serum sample from each patient using an enzyme-linked immunosorbent assay. MBL serum levels [median (range)] were 2500 (4–12 000) ng/mL and 2105 (4–22 800) ng/mL for the research and control groups, respectively. The results from the two groups were compared and were not statistically different (p 0.4). According to these results, MBL serum levels are not associated with an increased risk for recurrent UTI in pre-menopausal women.     

The IrgA homologue adhesin Iha is an Escherichia coli virulence factor in murine urinary tract infection

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.     

T-even-related bacteriophages as candidates for treatment of Escherichia coli urinary tract infections

Multidrug-resistant uropathogenic Escherichia coli (UPEC) is increasing gradually on a worldwide scale. We therefore examined the possibility of bacteriophage (phage) therapy for urinary tract infections (UTIs) caused by the UPEC strains as an alternative to chemotherapy. In addition to the well-known T4 phage, KEP10, which was newly isolated, was used as a therapeutic phage candidate. KEP10 showed a broader bacteriolytic spectrum (67%) for UPEC strains than T4 (14%). Morphological and genetic analyses showed that KEP10 resembles phage T4. Phages T4 and KEP10 injected into the peritoneal cavity of mice were distributed immediately to all organs examined and maintained a high titer for at least 24 h. They were stable in the urine of both mice and humans for 24 h at 37°C. Administration of these phages into the peritoneal cavity caused a marked decrease in the mortality of mice inoculated transurethrally with a UPEC strain, whereas most of the control mice died within a few days of bacterial infection. Inoculation with phage alone produced no adverse effects attributable to the phage per se. The present study experimentally demonstrated the therapeutic potential of phage for E. coli-induced UTIs, and T-even-related phages may be suitable candidates with which to treat them.     

Intranasal immunization with fusion protein MrpH·FimH and MPL adjuvant confers protection against urinary tract infections caused by uropathogenic Escherichia coli and Proteus mirabilis

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) and Proteus mirabilis are among the most common infections in the world. Currently there are no vaccines available to confer protection against UTI in humans. In this study, the immune responses and protection of FimH of UPEC with MrpH antigen of P. mirabilis in different vaccine formulations with and without MPL adjuvant were assessed. Mice intranasally immunized with the novel fusion protein MrpH·FimH induced a significant increase in IgG and IgA in serum, nasal wash, vaginal wash, and urine samples. Mice immunized with fusion MrpH·FimH also showed a significant boost in cellular immunity. Addition of MPL as the adjuvant enhanced FimH and MrpH specific humoral and cellular responses in both systemic and mucosal samples. Vaccination with MrpH·FimH alone or in combination with MPL showed the highest efficiency in clearing bladder and kidney infections in mice challenged with UPEC and P. mirabilis. These findings may indicate that the protection observed correlates with the systemic, mucosal and cellular immune responses induced by vaccination with these preparations. Our data suggest MrpH·FimH fusion protein with or without MPL as adjuvant could be potential vaccine candidates for elimination of UPEC and P. mirabilis. These data altogether are promising and these formulations are good candidates for elimination of UPEC and P. mirabilis.