(p)ppGpp and CodY Promote Enterococcus faecalis Virulence in a Murine Model of Catheter-Associated Urinary Tract Infection.

In Firmicutes, the nutrient-sensing regulators (p)ppGpp, the effector molecule of the stringent response, and CodY work in tandem to maintain bacterial fitness during infection. Here, we tested (p)ppGpp and codY mutant strains of Enterococcus faecalis in a catheter-associated urinary tract infection (CAUTI) mouse model and used global transcriptional analysis to investigate the relationship of (p)ppGpp and CodY. The absence of (p)ppGpp or single inactivation of codY led to lower bacterial loads in catheterized bladders and diminished biofilm formation on fibrinogen-coated surfaces under in vitro and in vivo conditions. Single inactivation of the bifunctional (p)ppGpp synthetase/hydrolase rel did not affect virulence, supporting previous evidence that the association of (p)ppGpp with enterococcal virulence is not dependent on the activation of the stringent response. Inactivation of codY in the (p)ppGpp0 strain restored E. faecalis virulence in the CAUTI model as well as the ability to form biofilms in vitro Transcriptome analysis revealed that inactivation of codY restores, for the most part, the dysregulated metabolism of (p)ppGpp0 cells. While a clear linkage between (p)ppGpp and CodY with expression of virulence factors could not be established, targeted transcriptional analysis indicates that a possible association between (p)ppGpp and c-di-AMP signaling pathways in response to the conditions found in the bladder may play a role in enterococcal CAUTI. Collectively, data from this study identify the (p)ppGpp-CodY network as an important contributor to enterococcal virulence in catheterized mouse bladder and support that basal (p)ppGpp pools and CodY promote virulence through maintenance of a balanced metabolism under adverse conditions.IMPORTANCE Catheter-associated urinary tract infections (CAUTIs) are one of the most frequent types of infection found in the hospital setting that can develop into serious and potentially fatal bloodstream infections. One of the infectious agents that frequently causes complicated CAUTI is the bacterium Enterococcus faecalis, a leading cause of hospital-acquired infections that are often difficult to treat due to the exceptional multidrug resistance of some isolates. Understanding the mechanisms by which E. faecalis causes CAUTI will aid in the discovery of new druggable targets to treat these infections. In this study, we report the importance of two nutrient-sensing bacterial regulators, named (p)ppGpp and CodY, for the ability of E. faecalis to infect the catheterized bladder of mice.

Prospective cohort study of microbial and inflammatory events immediately preceding Escherichia coli recurrent urinary tract infection in women.

BACKGROUND: A prospective cohort study was conducted to characterize the temporal sequence of microbial and inflammatory events immediately preceding Escherichia coli recurrent urinary tract infection (rUTI). METHODS: Women with acute cystitis and a history of UTI within the previous year self-collected periurethral and urine samples daily and recorded measurements of urine leukocyte esterase, symptoms, and sexual intercourse daily for 3 months. rUTI strains were characterized by pulsed-field gel electrophoresis and genomic virulence profiling. Urinary cytokine levels were measured. RESULTS: There were 38 E. coli rUTIs in 29 of 104 women. The prevalence of periurethral rUTI strain carriage increased from 46% to 90% during the 14 days immediately preceding rUTI, with similar increases in same-strain bacteriuria (from 7% to 69%), leukocyte esterase (from 31% to 64%), and symptoms (from 3% to 43%), most notably 2-3 days before rUTI (P<.05 for all comparisons). Intercourse with periurethral carriage of the rUTI strain also increased before rUTI (P=.008). Recurrent UTIs preceded by bacteriuria, pyuria, and symptoms were caused by strains less likely to have P fimbriae than other rUTI strains (P=.002). CONCLUSIONS: Among women with frequent rUTIs, the prevalences of periurethral rUTI strain carriage, bacteriuria, pyuria, and intercourse dramatically increase over the days preceding rUTI. A better understanding of the pathogenesis of rUTI will lead to better prevention strategies.

C-Terminal properties are important for ring-fused 2-pyridones that interfere with the chaperone function in uropathogenic E. coli.

Virulence-associated organelles, termed pili or fimbriae, are assembled via the highly conserved chaperone-usher pathway in a vast number of pathogenic bacteria. Substituted bicyclic 2-pyridones, pilicides, inhibit pilus formation, possibly by interfering with the active site residues Arg8 and Lys112 of chaperones in uropathogenic E. coli. In this article we describe the synthesis and evaluation of nine analogues of a biologically active pilicide. Derivatization was performed with respect to its C-terminal features and the affinities for the chaperone PapD were studied with 1H relaxation-edited NMR spectroscopy. It could be concluded that the carboxylic acid functionality and also its spatial location was important for binding. In all cases, binding was significantly reduced or even abolished when the carboxylic acid was replaced by other substituents. In addition, in vivo results from a hemagglutination assay are presented where the derivatives have been evaluated for their ability to inhibit pilus formation in uropathogenic E. coli.

Structural basis of the interaction of the pyelonephritic E. coli adhesin to its human kidney receptor.

PapG is the adhesin at the tip of the P pilus that mediates attachment of uropathogenic Escherichia coli to the uroepithelium of the human kidney. The human specific allele of PapG binds to globoside (GbO4), which consists of the tetrasaccharide GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc linked to ceramide. Here, we present the crystal structures of a binary complex of the PapG receptor binding domain bound to GbO4 as well as the unbound form of the adhesin. The biological importance of each of the residues involved in binding was investigated by site-directed mutagenesis. These studies provide a molecular snapshot of a host-pathogen interaction that determines the tropism of uropathogenic E. coli for the human kidney and is critical to the pathogenesis of pyelonephritis.

Establishment of a persistent Escherichia coli reservoir during the acute phase of a bladder infection.

The vast majority of urinary tract infections are caused by strains of uropathogenic Escherichia coli that encode filamentous adhesive organelles called type 1 pili. These structures mediate both bacterial attachment to and invasion of bladder epithelial cells. However, the mechanism by which type 1 pilus-mediated bacterial invasion contributes to the pathogenesis of a urinary tract infection is unknown. Here we show that type 1-piliated uropathogens can invade the superficial epithelial cells that line the lumenal surface of the bladder and subsequently replicate, forming massive foci of intracellular E. coli termed bacterial factories. In response to infection, superficial bladder cells exfoliate and are removed with the flow of urine. To avoid clearance by exfoliation, intracellular uropathogens can reemerge and eventually establish a persistent, quiescent bacterial reservoir within the bladder mucosa that may serve as a source for recurrent acute infections. These observations suggest that urinary tract infections are more chronic and invasive than generally assumed.

Dynamic interactions between host and pathogen during acute urinary tract infections.

Urinary tract infections (UTIs) have traditionally been viewed as acute and often self-limiting infections caused predominantly by noninvasive Escherichia coli. However, this concept has been challenged by recent findings demonstrating that an acute bladder infection results from a complex series of host-pathogen interactions that can lead to bacterial invasion and persistence and that ultimately can determine the course of the infectious disease. The ability of E. coli to gain a foothold in the bladder is greatly facilitated by type 1 pilus-mediated attachment to and invasion of bladder epithelial cells. Invasion allows uropathogenic strains of E. coli to exploit the intracellular environment by replicating within these epithelial cells while evading a multitude of host defenses. An intracellular location also provides them a safe haven from many common antibiotic therapies. However, attachment and invasion also activates a cascade of innate host defenses, leading to the death and exfoliation of bladder cells and the production of inflammatory mediators. The ability of uropathogenic E. coli to flux out of cells and colonize surrounding cells provides them a mechanism to subvert these defense mechanisms and persist in the bladder epithelium for weeks following the acute infection. The persistence of E. coli in bladder tissue may be relevant to more chronic diseases of the urinary tract such as recurrent UTIs and interstitial cystitis.

The impact of glaucoma on the quality of life of patients in Norway. I. Results from a self-administered questionnaire.

PURPOSE: To evaluate the feelings and experiences of patients living with glaucoma. METHODS: A questionnaire was delivered to patients willing to take part, at a regular visit to their ophthalmologist, for filling in anonymously at home. A total of 589 questionnaires were returned. RESULTS: More than 80% reported negative emotions on learning that they had glaucoma, one-third were afraid of going blind. Half the patients had no visual problem at all, 14% complained of poor or very poor vision. This proportion increased with age. One-fourth of the patients on topical medication experienced adverse effects of moderate or high degree. About half the patients being treated with laser or surgery felt their situation had improved afterwards. Nine-tenths of the individuals were satisfied with the information and care given, although their knowledge about glaucoma was rather incomplete. One-fifth missed information, mainly on causes, treatment and prognosis of the disease. The younger patients were more anxious and inquiring, reported more side effects and were less satisfied than the older patients. The women were in general more dissatisfied than the men. CONCLUSION: Giving a patient a diagnosis of glaucoma influences his quality of life negatively. Only half of our patients experienced any visual difficulties, whereas one-fourth reported adverse reactions due to the therapy. Most of the patients were very satisfied with the information and care given. Ophthalmologists in private practice are quite central in the management and care of the glaucoma patients in a medical setting like ours.

The impact of glaucoma on the quality of life of patients in Norway. II. Patient response correlated to objective data.

PURPOSE: To elucidate the relationship between the visual difficulties reported by patients treated for glaucoma and their objective functional damage, and to evaluate the reliability of the patient responses. METHODS: Questionnaires concerning quality of life filled in at home by 589 patients treated for chronic open angle glaucoma were correlated to corresponding questionnaires returned from their ophthalmologists. RESULTS: Few of our patients had a visual field damage judged to be of functional significance. There was a weak to moderate association between both visual field defects and decreased visual acuity and self-reported visual difficulties. A high proportion of the patients had normal binocular visual field and a stable disease, raising the suspicion that some of them were treated for ocular hypertension. The agreement between the responses from the patients and the ophthalmologists concerning the topical treatment was good, regarding treatment duration and other diseases of the patients the agreement was moderate. CONCLUSION: The association between subjective visual disability and presence of visual field defects was weak to moderate in our patients treated for glaucoma, and this association was further weakened by adjusting for visual acuity. Some patients might be treated unnecessarily, and a favourable prognosis might be given to most of them. The reliability of the patients in general was good.

Cpx signaling pathway monitors biogenesis and affects assembly and expression of P pili.

P pili are important virulence factors in uropathogenic Escherichia coli. The Cpx two-component signal transduction system controls a stress response and is activated by misfolded proteins in the periplasm. We have discovered new functions for the Cpx pathway, indicating that it may play a critical role in pathogenesis. P pili are assembled via the chaperone/usher pathway. Subunits that go 'OFF-pathway' during pilus biogenesis generate a signal. This signal is derived from the misfolding and aggregation of subunits that failed to come into contact with the chaperone in the periplasm. In response, Cpx not only controls the stress response, but also controls genes necessary for pilus biogenesis, and is involved in regulating the phase variation of pap expression and, potentially, the expression of a panoply of other virulence factors. This study demonstrates how the prototypic chaperone/usher pathway is intricately linked and dependent upon a signal transduction system.

Bacterial invasion augments epithelial cytokine responses to Escherichia coli through a lipopolysaccharide-dependent mechanism.

One mechanism of initiating innate host defenses against uropathogenic Escherichia coli (UPEC) is the production of cytokines by bladder epithelial cells; however, the means by which these cells recognize bacterial pathogens is poorly understood. Type 1 pili, expressed by the majority of UPEC, have been shown to have a critical role in inducing the expression of IL-6 in bladder epithelial cells after exposure to E. coli. In this study, we demonstrate that type 1 pili are not sufficient to activate IL-6 production by bladder epithelial cells. Instead, it was shown that bacterial invasion mediated by type 1 pili augments bladder epithelial responses to E. coli via an LPS-dependent mechanism, leading to the production of IL-6. RNA transcripts for the LPSR Toll-like receptor 4 (TLR4) was detected in cultured bladder epithelial cells. The in vivo role of TLR4 was assessed using C3H/HeJ mice, which express a dominant negative form of TLR4. After infection with UPEC, C3H/HeJ mice have large foci of intracellular bacteria that persist within the bladder epithelium in the absence of any notable inflammatory response. These results indicate that LPS is required for bacterial invasion to enhance host responses to E. coli within the bladder.

Role of intestinal surfactant-like particles as a potential reservoir of uropathogenic Escherichia coli.

The binding of uropathogenic Escherichia coli is mediated at the tips of pili by the PapG adhesin, which recognizes the Galalpha(1-4)Gal disaccharide on the uroepithelial surface. These receptors have been identified unequivocally in the human and murine urinary tracts but not in intestinal epithelium, yet uropathogenic E. coli strains are commonly found in normal colonic microflora. The gastrointestinal tract from duodenum to rectum elaborates a phospholipid-rich membrane particle with surfactant-like properties. In these studies, we report that purified murine particles contain a receptor recognized by the class I PapG adhesin because: (1) PapD-PapG complexes and class I pili bound to surfactant-like particles in a solid-phase assay, whereas binding was not detected in microvillous membranes derived from the same tissues, (2) purified PapD-PapG complex bound to a glycolipid receptor detectable in lipid extracts from the particles, and (3) soluble Galalpha(1-4)Gal inhibited the adhesin by 72% from binding to surfactant-like particles. The Galalpha(1-4)Gal receptor present in the intestinal surfactant-like particle which overlies the intestinal mucosa could provide one means to establish an intestinal habitat for uropathogenic E. coli.

Chaperone-assisted pilus assembly and bacterial attachment.

Bacterial pili assembled by the chaperone-usher pathway can mediate microbial attachment, an early step in the establishment of an infection, by binding specifically to sugars present in host tissues. Recent work has begun to reveal the structural basis both of chaperone function in the biogenesis of these pili and of bacterial attachment.

Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story.

Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.

Cell biology. Bacterial spelunkers.

Bacteria that are engulfed by phagocytic cells of the immune system are usually destroyed once inside the host cell but not always. Why is it that sometimes engulfed bacteria survive and thrive quite happily inside the host cell? As Mulvey and Hultgren explain in their Perspective, the answer may lie in small indentations in the host cell plasma membrane called caveolae that direct certain signal transduction pathways inside the host cell (Shin et al.). If bacteria adhere to regions of the host cell surface that is rich in caveolae, they are better able to survive once inside the cell.

Bad bugs and beleaguered bladders: interplay between uropathogenic Escherichia coli and innate host defenses.

Strains of uropathogenic Escherichia coli (UPEC) are the causative agents in the vast majority of all urinary tract infections. Upon entering the urinary tract, UPEC strains face a formidable array of host defenses, including the flow of urine and a panoply of antimicrobial factors. To gain an initial foothold within the bladder, most UPEC strains encode filamentous surface adhesive organelles called type 1 pili that can mediate bacterial attachment to, and invasion of, bladder epithelial cells. Invasion provides UPEC with a protective environment in which bacteria can either replicate or persist in a quiescent state. Infection with type 1-piliated E. coli can trigger a number of host responses, including cytokine production, inflammation, and the exfoliation of infected bladder epithelial cells. Despite numerous host defenses and even antibiotic treatments that can effectively sterilize the urine, recent studies demonstrate that uropathogens can persist within the bladder tissue. These bacteria may serve as a reservoir for recurrent infections, a common problem affecting millions each year.

Snapshots of usher-mediated protein secretion and ordered pilus assembly.

Type 1 pilus biogenesis was used as a paradigm to investigate ordered macromolecular assembly at the outer cell membrane. The ability of Gram-negative bacteria to secrete proteins across their outer membrane and to assemble adhesive macromolecular structures on their surface is a defining event in pathogenesis. We elucidated genetic, biochemical, and biophysical requirements for assembly of functional type 1 pili. We discovered that the minor pilus protein FimG plays a critical role in nucleating the formation of the adhesive tip fibrillum. Genetic methods were used to trap pilus subunits during their translocation through the outer membrane usher protein, providing data on the structural interactions that occur between subunit components during type 1 pilus formation. Electron microscopic and biochemical analyses of these stepwise assembly intermediates demonstrated that translocation of pilus subunits occurs linearly through the usher's central channel, with formation of the pilus helix occurring extracellularly. Specialized pilin subunits play unique roles both in this multimerization and in the final ultrastructure of the adhesive pilus.