Optimization and qualification of an assay that demonstrates that a FimH vaccine induces functional antibody responses in women with histories of urinary tract infections.
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ABSTRACT:
SUBMITTER: Starks CM
PROVIDER: S-EPMC7872045 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Chaperone-usher pathway pili are a widespread family of extracellular, Gram-negative bacterial fibers with important roles in bacterial pathogenesis. Type 1 pili are important virulence factors in uropathogenic Escherichia coli (UPEC), which cause the majority of urinary tract infections (UTI). FimH, the type 1 adhesin, binds mannosylated glycoproteins on the surface of human and murine bladder cells, facilitating bacterial colonization, invasion, and formation of biofilm-like intracellular bacterial communities. The mannose-binding pocket of FimH is invariant among UPEC. We discovered that pathoadaptive alleles of FimH with variant residues outside the binding pocket affect FimH-mediated acute and chronic pathogenesis of two commonly studied UPEC strains, UTI89 and CFT073. In vitro binding studies revealed that, whereas all pathoadaptive variants tested displayed the same high affinity for mannose when bound by the chaperone FimC, affinities varied when FimH was incorporated into pilus tip-like, FimCGH complexes. Structural studies have shown that FimH adopts an elongated conformation when complexed with FimC, but, when incorporated into the pilus tip, FimH can adopt a compact conformation. We hypothesize that the propensity of FimH to adopt the elongated conformation in the tip corresponds to its mannose binding affinity. Interestingly, FimH variants, which maintain a high-affinity conformation in the FimCGH tip-like structure, were attenuated during chronic bladder infection, implying that FimH's ability to switch between conformations is important in pathogenesis. Our studies argue that positively selected residues modulate fitness during UTI by affecting FimH conformation and function, providing an example of evolutionary tuning of structural dynamics impacting in vivo survival.
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Project description:Gram-positive bacteria in the genus Enterococcus are a frequent cause of catheter-associated urinary tract infection (CAUTI), a disease whose treatment is increasingly challenged by multiantibiotic-resistant strains. We have recently shown that E. faecalis uses the Ebp pilus, a heteropolymeric surface fiber, to bind the host protein fibrinogen as a critical step in CAUTI pathogenesis. Fibrinogen is deposited on catheters due to catheter-induced inflammation and is recognized by the N-terminal domain of EbpA (EbpANTD), the Ebp pilus's adhesin. In a murine model, vaccination with EbpANTD confers significant protection against CAUTI. Here, we explored the mechanism of protection using passive transfer of immune sera to show that antisera blocking EbpANTD-fibrinogen interactions not only is prophylactic but also can act therapeutically to reduce bacterial titers of an existing infection. Analysis of 55 clinical CAUTI, bloodstream, and gastrointestinal isolates, including E. faecalis, E. faecium, and vancomycin-resistant enterococci (VRE), revealed a diversity of levels of EbpA expression and fibrinogen-binding efficiency in vitro Strikingly, analysis of 10 strains representative of fibrinogen-binding diversity demonstrated that, irrespective of EbpA levels, EbpANTD antibodies were universally protective. The results indicate that, despite diversity in levels of fibrinogen binding, strategies that target the disruption of EbpANTD-fibrinogen interactions have considerable promise for treatment of CAUTI. Urinary catheterization is a routine medical procedure, and it has been estimated that 30 million Foley catheters are used annually in the United States. Importantly, placement of a urinary catheter renders the patient susceptible to developing a catheter-associated urinary tract infection, accounting for 1 million cases per year. Additionally, these infections can lead to serious complications, including bloodstream infection and death. Enterococcus strains are a common cause of these infections, and management of enterococcal infections has been more difficult in recent years due to the development of antibiotic resistance and the ability of strains to disseminate, resulting in a major threat in hospital settings. In this study, we developed an antibiotic-sparing treatment that is effective against diverse enterococcal isolates, including vancomycin-resistant enterococci, during catheter-associated urinary tract infections.
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Project description:Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and alpha3beta1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections.We demonstrate that alpha-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl alpha-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl alpha-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl alpha-D-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Man alpha1,3Man beta1,4GlcNAc beta1,4GlcNAc in an extended binding site. The interactions along the alpha1,3 glycosidic bond and the first beta1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl alpha-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group.The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection.
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Project description:The present study investigated the incidence of hyperammonemia in urinary tract infections and explored the utility of urinary obstruction relief and antimicrobial administration to improve hyperammonemia.This was an observational study. Subjects were patients who were diagnosed with urinary tract infection and hospitalized between June 2008 and June 2009. We measured plasma ammonia levels on admission in patients who were clinically diagnosed with urinary tract infection and hospitalized. We assessed each patient's level of consciousness on admission using the Glasgow Coma Scale (GCS) and performed urine and blood cultures. We also assessed hearing prior to hospitalization using the Eastern Cooperative Oncology Group performance status (ECOG-PS). In cases with high ammonia levels on admission, plasma ammonia and GCS were measured 24 hours and 5-7 days later.Sixty-seven candidates were enrolled; of these, 60 cases (89.6%) with bacterial cell counts ≥10(4) CFU/mL were studied. Five cases (8.3%) presented with high plasma ammonia levels. Cases with hyperammonemia were significantly more likely to present with low GCS scores and urinary retention rate. All five cases received antimicrobial therapy with an indwelling bladder catheter to relieve urinary retention. The case 5 patient died shortly after admission due to complicated aspiration pneumonia; in the remaining cases, plasma ammonia levels were rapidly normalized and the level of consciousness improved.The occurrence of hyperammonemia in urinary tract infections is not rare. The cause of hyperammonemia is urinary retention obstruction. Therefore, along with antimicrobial administration, relief of obstruction is important for the treatment of hyperammonemia caused by this mechanism.
| S-EPMC4546155 | biostudies-other
Project description:Chronic and recurrent urinary tract infections pose a serious medical problem because there are few effective treatment options. Patients with chronic urinary tract infections are commonly treated with long-term prophylactic antibiotics that promote the development of antibiotic-resistant forms of uropathogenic Escherichia coli (UPEC), further complicating treatment. We developed small-molecular weight compounds termed mannosides that specifically inhibit the FimH type 1 pilus lectin of UPEC, which mediates bacterial colonization, invasion, and formation of recalcitrant intracellular bacterial communities in the bladder epithelium. Here, we optimized these compounds for oral bioavailability and demonstrated their fast-acting efficacy in treating chronic urinary tract infections in a preclinical murine model. These compounds also prevented infection in vivo when given prophylactically and strongly potentiated the activity of the current standard of care therapy, trimethoprim-sulfamethoxazole, against clinically resistant PBC-1 UPEC bacteria. These compounds have therapeutic efficacy after oral administration for the treatment of established urinary tract infections in vivo. Their unique mechanism of action-targeting the pilus tip adhesin FimH-circumvents the conventional requirement for drug penetration of the outer membrane, minimizing the potential for the development of resistance. The small-molecular weight compounds described herein promise to provide substantial benefit to women suffering from chronic and recurrent urinary tract infections.
| S-EPMC3694776 | biostudies-other
Project description:BackgroundUrinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) and Proteus mirabilis are the most important pathogens causing UTIs. The FimH from type 1 pili of UPEC and the MrpH from P. mirabilis play critical roles in the UTI process and have presented as ideal vaccine candidates against UTIs. There is no effective vaccine against UTI and the development of an ideal UTI vaccine is required.Materials and methodsIn this study, we planned to design a novel fusion protein of FimH from UPEC and MrpH from P. mirabilis. For this purpose, we modeled fusion protein forms computationally using the Iterative Threading Assembly Refinement (I-TASSER) server and evaluated their interactions with toll-like receptor 4 (TLR4). The best fusion protein was constructed using overlap extension polymerase chain reaction (OE-PCR) and the biological activity of fusion was evaluated by the induction of interleukin-8 (IL-8) in the HT-29 cell line.ResultsOur study indicated that based on the Protein Structure Analysis (ProSA)-web and the docking results, MrpH.FimH showed better results than did FimH.MrpH, and it was selected for construction. The results of bioassay on the HT-29 showed that FimH and MrpH.FimH induced significantly higher IL-8 responses than untreated cells or MrpH alone in the cell line tested.ConclusionsIn the present study, we designed and constructed the novel fusion protein MrpH.FimH from UPEC and P. mirabilis based on in silico methods. Our bioassay results indicate that the MrpH.FimH fusion protein is active and capable of inducing immune responses.
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Project description:The urinary tract is constantly exposed to different microorganisms that colonize the gastrointestinal tract and the urinary tract is normally well prepared to resist infections by these microorganisms. This resistance to infection is mainly accomplished by the versatility of the immune system in the urinary tract, with both innate and adaptive immune responses. With the increasing knowledge of how the immune system works in the urinary tract and also the recognition of the virulence attributes of uropathogens, several potentially effective and tailored strategies to contain or prevent urinary tract infections have emerged.
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Project description:A urinary tract infection (UTI) is a collective term for infections that involve any part of the urinary tract. It is one of the most common infections in local primary care. The incidence of UTIs in adult males aged under 50 years is low, with adult women being 30 times more likely than men to develop a UTI. Appropriate classification of UTI into simple or complicated forms guides its management and the ORENUC classification can be used. Diagnosis of a UTI is based on a focused history, with appropriate investigations depending on individual risk factors. Simple uncomplicated cystitis responds very well to oral antibiotics, but complicated UTIs may require early imaging, and referral to the emergency department or hospitalisation to prevent urosepsis may be warranted. Escherichia coli remains the predominant uropathogen in acute community-acquired uncomplicated UTIs and amoxicillin-clavulanate is useful as a first-line antibiotic. Family physicians are capable of managing most UTIs if guided by appropriate history, investigations and appropriate antibiotics to achieve good outcomes and minimise antibiotic resistance.
| S-EPMC5027397 | biostudies-literature
Project description:Proteus mirabilis is a Gram-negative bacterium and is well known for its ability to robustly swarm across surfaces in a striking bulls'-eye pattern. Clinically, this organism is most frequently a pathogen of the urinary tract, particularly in patients undergoing long-term catheterization. This review covers P. mirabilis with a focus on urinary tract infections (UTI), including disease models, vaccine development efforts, and clinical perspectives. Flagella-mediated motility, both swimming and swarming, is a central facet of this organism. The regulation of this complex process and its contribution to virulence is discussed, along with the type VI-secretion system-dependent intra-strain competition, which occurs during swarming. P. mirabilis uses a diverse set of virulence factors to access and colonize the host urinary tract, including urease and stone formation, fimbriae and other adhesins, iron and zinc acquisition, proteases and toxins, biofilm formation, and regulation of pathogenesis. While significant advances in this field have been made, challenges remain to combatting complicated UTI and deciphering P. mirabilis pathogenesis.
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