Jennifer N. Walker

Urinary tract infections: epidemiology, mechanisms of infection and treatment options

Urinary tract infections (UTIs) are a severe public health problem and are caused by a range of pathogens, but most commonly by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus. High recurrence rates and increasing antimicrobial resistance among uropathogens threaten to greatly increase the economic burden of these infections. In this Review, we discuss how basic science studies are elucidating the molecular details of the crosstalk that occurs at the host–pathogen interface, as well as the consequences of these interactions for the pathophysiology of UTIs. We also describe current efforts to translate this knowledge into new clinical treatments for UTIs.

Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation

Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1-dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo. Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds.

Quantitative metabolomics reveals an epigenetic blueprint for iron acquisition in uropathogenic Escherichia coli

Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies.

Positive selection identifies an in vivo role for FimH during urinary tract infection in addition to mannose binding

FimH, the type 1 pilus adhesin of uropathogenic Escherichia coli (UPEC), contains a receptor-binding domain with an acidic binding pocket specific for mannose. The fim operon, and thus type 1 pilus production, is under transcriptional control via phase variation of an invertible promoter element. FimH is critical during urinary tract infection for mediating colonization and invasion of the bladder epithelium and establishment of intracellular bacterial communities (IBCs). In silico analysis of FimH gene sequences from 279 E. coli strains identified specific amino acids evolving under positive selection outside of its mannose-binding pocket. Mutating two of these residues (A27V/V163A) had no effect on phase variation, pilus assembly, or mannose binding in vitro. However, compared to wild-type, this double mutant strain exhibited a 10,000-fold reduction in mouse bladder colonization 24 h after inoculation and was unable to form IBCs even though it bound normally to mannosylated receptors in the urothelium. In contrast, the single A62S mutation altered phase variation, reducing the proportion of piliated cells, reduced mannose binding 8-fold, and decreased bladder colonization 30-fold in vivo compared to wild-type. A phase-locked ON A62S mutant restored virulence to wild-type levels even though in vitro mannose binding remained impaired. Thus, positive selection analysis of FimH has separated mannose binding from in vivo fitness, suggesting that IBC formation is critical for successful infection of the mammalian bladder, providing support for more general use of in silico positive selection analysis to define the molecular underpinnings of bacterial pathogenesis.

Utilization of an Intracellular Bacterial Community Pathway in Klebsiella pneumoniae Urinary Tract Infection and the Effects of FimK on Type 1 Pilus Expression

ABSTRACT Klebsiella pneumoniae is an important cause of urinary tract infection (UTI), but little is known about its pathogenesis in vivo. The pathogenesis of the K. pneumoniae cystitis isolate TOP52 was compared to that of the uropathogenic Escherichia coli (UPEC) isolate UTI89 in a murine cystitis model. Bladder and kidney titers of TOP52 were lower than those of UTI89 at early time points but similar at later time points. TOP52, like UTI89, formed biofilm-like intracellular bacterial communities (IBCs) within the murine bladder, albeit at significantly lower levels than UTI89. Additionally, filamentation of TOP52 was observed, a process critical for UTI89 evasion of neutrophil phagocytosis and persistence in the bladder. Thus, the IBC pathway is not specific to UPEC alone. We investigated if differences in type 1 pilus expression may explain TOP52s early defect in vivo. The type 1 pilus operon is controlled by recombinase-mediated (fimE, fimB, and fimX) phase variation of an invertible promoter element. We found that K. pneumoniae carries an extra gene of unknown function at the 3′ end of its type 1 operon, fimK, and the genome lacks the recombinase fimX. A deletion mutant of fimK was constructed, and TOP52 ΔfimK had higher titers and formed more IBCs in the murine cystitis model than wild type. The loss of fimK or expression of E. coli fimX from a plasmid in TOP52 resulted in a larger phase-ON population and higher expression levels of type 1 pili and gave TOP52 the ability to form type 1-dependent biofilms. Complementation with pfimK decreased type 1 pilus expression and biofilm formation of TOP52 ΔfimK and decreased UTI89 biofilm formation. Thus, K. pneumoniae appears programmed for minimal expression of type 1 pili, which may explain, in part, why K. pneumoniae is a less prevalent etiologic agent of UTI than UPEC.

Bone Morphogenetic Protein 4 Signaling Regulates Epithelial Renewal in the Urinary Tract in Response to Uropathogenic Infection

The transitional epithelium of the bladder normally turns over slowly but upon injury undergoes rapid regeneration fueled by basal uroepithelial stem and/or early progenitor cells (USCs). Little is known about the mechanisms underlying the injury response. We investigate the mechanism of bladder epithelial regeneration in response to infection with uropathogenic E. coli (UPEC). Infection resulted in rapid sloughing of superficial cells, a marked inflammatory response, and a substantial spike in basal cell proliferation. In mice with induced urothelial ablation of a member of the TGF-beta receptor superfamily, bone morphogenetic protein (Bmp)-4 receptor, infection led to aberrant urothelial renewal resulting from a block in USC differentiation into superficial cells. Chemical injury also caused sloughing but no inflammation or USC activation. Together, our study indicates that UPEC infection but not chemical injury activates the USC niche, and Bmp signaling is required for regulation of the USC response to infection.

LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitis.

Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity‐associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili‐mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI IIUTI89 disrupts the adjacent leuX tRNA locus. Loss of wild‐type leuX‐encoded tRNA5Leu significantly delayed, but did not eliminate, FimB recombinase‐mediated phase variation of type 1 pili. FimX, an additional FimB‐like, leuX‐independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non‐piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI IIUTI89 genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI IIUTI89 during the establishment of acute cystitis is maintenance of wild‐type leuX, and not PAI IIUTI89 gene content.

Molecular Variations in Klebsiella pneumoniae and Escherichia coli FimH Affect Function and Pathogenesis in the Urinary Tract

ABSTRACT Type 1 pili mediate binding, invasion, and biofilm formation of uropathogenic Escherichia coli (UPEC) in the host urothelium during urinary tract infection (UTI) via the adhesin FimH. In this study, we characterized the molecular basis of functional differences between FimH of the UPEC isolate UTI89 and the Klebsiella pneumoniae cystitis isolate TOP52. Type 1 pili characteristically mediate mannose-sensitive hemagglutination of guinea pig erythrocytes. Although the adhesin domain of K. pneumoniae TOP52 FimH (FimH52) is highly homologous to that of E. coli, with an identical mannose binding pocket and surrounding hydrophobic ridge, it lacks the ability to agglutinate guinea pig erythrocytes. In addition, FimH-dependent biofilm formation in K. pneumoniae is inhibited by heptyl mannose, but not methyl mannose, suggesting the need for contacts outside of the mannose binding pocket. The binding specificity differences observed for FimH52 resulted in significant functional differences seen in the pathogenesis of K. pneumoniae UTI compared to E. coli UTI. Infections in a murine model of UTI demonstrated that although the K. pneumoniae strain TOP52 required FimH52 for invasion and IBC formation in the bladder, FimH52 was not essential for early colonization. This work reveals that a limited amount of sequence variation between the FimH of E. coli and K. pneumoniae results in significant differences in function and ability to colonize the urinary tract.

The Staphylococcus aureus ArlRS two-component system is a novel regulator of agglutination and pathogenesis.

Staphylococcus aureus is a prominent bacterial pathogen that is known to agglutinate in the presence of human plasma to form stable clumps. There is increasing evidence that agglutination aids S. aureus pathogenesis, but the mechanisms of this process remain to be fully elucidated. To better define this process, we developed both tube based and flow cytometry methods to monitor clumping in the presence of extracellular matrix proteins. We discovered that the ArlRS two-component system regulates the agglutination mechanism during exposure to human plasma or fibrinogen. Using divergent S. aureus strains, we demonstrated that arlRS mutants are unable to agglutinate, and this phenotype can be complemented. We found that the ebh gene, encoding the Giant Staphylococcal Surface Protein (GSSP), was up-regulated in an arlRS mutant. By introducing an ebh complete deletion into an arlRS mutant, agglutination was restored. To assess whether GSSP is the primary effector, a constitutive promoter was inserted upstream of the ebh gene on the chromosome in a wildtype strain, which prevented clump formation and demonstrated that GSSP has a negative impact on the agglutination mechanism. Due to the parallels of agglutination with infective endocarditis development, we assessed the phenotype of an arlRS mutant in a rabbit combined model of sepsis and endocarditis. In this model the arlRS mutant displayed a large defect in vegetation formation and pathogenesis, and this phenotype was partially restored by removing GSSP. Altogether, we have discovered that the ArlRS system controls a novel mechanism through which S. aureus regulates agglutination and pathogenesis.

Antibody-Based Therapy for Enterococcal Catheter-Associated Urinary Tract Infections

ABSTRACT 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. IMPORTANCE 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. 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.