Identification and recombinant expression of a cutinase from Papiliotrema laurentii that hydrolyzes natural and synthetic polyesters.

Given the multitude of extracellular enzymes at their disposal, many of which are designed to degrade nature's polymers (lignin, cutin, cellulose, etc.), fungi are adept at targeting synthetic polyesters with similar chemical composition. Microbial-influenced deterioration of xenobiotic polymeric surfaces is an area of interest for material scientists as these are important for the conservation of the underlying structural materials. Here, we describe the isolation and characterization of the Papiliotrema laurentii 5307AH (P. laurentii) cutinase, Plcut1. P. laurentii is basidiomycete yeast with the ability to disperse Impranil-DLN (Impranil), a colloidal polyester polyurethane, in agar plates. To test whether the fungal factor involved in this clearing was a secreted enzyme, we screened the ability of P. laurentii culture supernatants to disperse Impranil. Using size exclusion chromatography (SEC), we isolated fractions that contained Impranil-clearing activity. These fractions harbored a single ~22 kD band, which was excised and subjected to peptide sequencing. Homology searches using the peptide sequences identified, revealed that the protein Papla1 543643 (Plcut1) displays similarities to serine esterase and cutinase family of proteins. Biochemical assays using recombinant Plcut1 confirmed that this enzyme has the capability to hydrolyze Impranil, soluble esterase substrates, and apple cutin. Finally, we confirmed the presence of the Plcut1 in culture supernatants using a custom antibody that specifically recognizes this protein. The work shown here supports a major role for the Plcut1 in the fungal degradation of natural polyesters and xenobiotic polymer surfaces.IMPORTANCEFungi play a vital role in the execution of a broad range of biological processes that drive ecosystem function through production of a diverse arsenal of enzymes. However, the universal reactivity of these enzymes is a current problem for the built environment and the undesired degradation of polymeric materials in protective coatings. Here, we report the identification and characterization of a hydrolase from Papiliotrema laurentii 5307AH, an aircraft-derived fungal isolate found colonizing a biodeteriorated polymer-coated surface. We show that P. laurentii secretes a cutinase capable of hydrolyzing soluble esters as well as ester-based compounds forming solid surface coatings. These findings indicate that this fungus plays a significant role in biodeterioration through the production of a cutinase adept at degrading ester-based polymers, some of which form the backbone of protective surface coatings. The work shown here provides insights into the mechanisms employed by fungi to degrade xenobiotic polymers.

Differential Regulation of Escherichia coli fim Genes following Binding to Mannose Receptors.

Regulation of the uropathogenic Escherichia coli (UPEC) fimB and fimE genes was examined following type 1 pili binding to mannose-coated Sepharose beads. Within 25 min after mannose attachment, fimE expression dropped eightfold, whereas fimB transcription increased about two- to fourfold. Because both fim genes encode site-specific recombinases that affect the position of the fimS element containing the fimA promoter, the positioning of fimS was also examined. The fimS element changed to slightly more Phase-OFF in bacteria mixed with plain beads, whereas UPEC cells interacting with mannose-coated beads had significantly less Phase-OFF orientation of fimS under pH 7 conditions. On the other hand, Phase-OFF oriented fimS increased fourfold when UPEC cells were mixed with plain beads in a pH 5.5 environment. Positioning of fimS was also affected by fimH mutations, demonstrating that the FimH ligand binding to its receptor facilitates the changes. Moreover, enzyme immunoassays showed that UPEC cells had greater type 1 pili expression when mixed with mannose-coated beads versus plain beads. These results indicate that, after type 1 pilus binding to tethered mannose receptors, the physiology of the E. coli cells changes to maintain the expression of type 1 pili even when awash in an acidic environment.

The Yersiniabactin-Associated ATP Binding Cassette Proteins YbtP and YbtQ Enhance Escherichia coli Fitness during High-Titer Cystitis.

The Yersinia high-pathogenicity island (HPI) is common to multiple virulence strategies used by Escherichia coli strains associated with urinary tract infection (UTI). Among the genes in this island are ybtP and ybtQ, encoding distinctive ATP binding cassette (ABC) proteins associated with iron(III)-yersiniabactin import in Yersinia pestis In this study, we compared the impact of ybtPQ on a model E. coli cystitis strain during in vitro culture and experimental murine infections. A ybtPQ-null mutant exhibited no growth defect under standard culture conditions, consistent with nonessentiality in this background. A growth defect phenotype was observed and genetically complemented in vitro during iron(III)-yersiniabactin-dependent growth. Following inoculation into the bladders of C3H/HEN and C3H/HeOuJ mice, this strain exhibited a profound, 10(6)-fold competitive infection defect in the subgroup of mice that progressed to high-titer bladder infections. These results identify a virulence role for YbtPQ in the highly inflammatory microenvironment characteristic of high-titer cystitis. The profound competitive defect may relate to the apparent selection of Yersinia HPI-positive E. coli in uncomplicated clinical UTIs.

Metal selectivity by the virulence-associated yersiniabactin metallophore system.

Uropathogenic Escherichia coli secrete siderophores during human infections. Although siderophores are classically defined by their ability to bind iron(III) ions, the virulence-associated siderophore yersiniabactin was recently found to bind divalent copper ions during urinary tract infections. Here we use a mass spectrometric approach to determine the extent of non-iron(III) metal interactions by yersiniabactin and its TonB-dependent outer membrane importer FyuA. In addition to copper, iron and gallium ions, yersiniabactin was also observed to form stable nickel, cobalt, and chromium ion complexes. In E. coli, copper(II) and all other non-iron(III) yersiniabactin complexes were imported by FyuA in a TonB-dependent manner. Among metal-yersiniabactin complexes, copper(II) yersiniabactin is predicted to be structurally distinctive and was the only complex not to competitively inhibit iron(III) yersiniabactin import. These results are consistent with yersiniabactin as part of a metallophore system able to prioritize iron(III) complex uptake in high copper environments.

Human Urinary Composition Controls Antibacterial Activity of Siderocalin.

During Escherichia coli urinary tract infections, cells in the human urinary tract release the antimicrobial protein siderocalin (SCN; also known as lipocalin 2, neutrophil gelatinase-associated lipocalin/NGAL, or 24p3). SCN can interfere with E. coli iron acquisition by sequestering ferric iron complexes with enterobactin, the conserved E. coli siderophore. Here, we find that human urinary constituents can reverse this relationship, instead making enterobactin critical for overcoming SCN-mediated growth restriction. Urinary control of SCN activity exhibits wide ranging individual differences. We used these differences to identify elevated urinary pH and aryl metabolites as key biochemical host factors controlling urinary SCN activity. These aryl metabolites are well known products of intestinal microbial metabolism. Together, these results identify an innate antibacterial immune interaction that is critically dependent upon individualistic chemical features of human urine.

Metabolomic analysis of siderophore cheater mutants reveals metabolic costs of expression in uropathogenic Escherichia coli.

Bacterial siderophores are a group of chemically diverse, virulence-associated secondary metabolites whose expression exerts metabolic costs. A combined bacterial genetic and metabolomic approach revealed differential metabolomic impacts associated with biosynthesis of different siderophore structural families. Despite myriad genetic differences, the metabolome of a cheater mutant lacking a single set of siderophore biosynthetic genes more closely approximate that of a non-pathogenic K12 strain than its isogenic, uropathogen parent strain. Siderophore types associated with greater metabolomic perturbations are less common among human isolates, suggesting that metabolic costs influence success in a human population. Although different siderophores share a common iron acquisition function, our analysis shows how a metabolomic approach can distinguish their relative metabolic impacts in E. coli.

Cupric yersiniabactin is a virulence-associated superoxide dismutase mimic.

Many Gram-negative bacteria interact with extracellular metal ions by expressing one or more siderophore types. Among these, the virulence-associated siderophore yersiniabactin (Ybt) is an avid copper chelator, forming stable cupric (Cu(II)-Ybt) complexes that are detectable in infected patients. Here we show that Ybt-expressing E. coli are protected from intracellular killing within copper-replete phagocytic cells. This survival advantage is highly dependent upon the phagocyte respiratory burst, during which superoxide is generated by the NADPH oxidase complex. Chemical fractionation links this phenotype to a previously unappreciated superoxide dismutase (SOD)-like activity of Cu(II)-Ybt. Unlike previously described synthetic copper-salicylate (Cu(II)-SA) SOD mimics, the salicylate-based natural product Cu(II)-Ybt retains catalytic activity at physiologically plausible protein concentrations. These results reveal a new virulence-associated adaptation based upon spontaneous assembly of a non-protein catalyst.

The siderophore yersiniabactin binds copper to protect pathogens during infection.

Bacterial pathogens secrete chemically diverse iron chelators called siderophores, which may exert additional distinctive functions in vivo. Among these, uropathogenic Escherichia coli often coexpress the virulence-associated siderophore yersiniabactin (Ybt) with catecholate siderophores. Here we used a new MS screening approach to reveal that Ybt is also a physiologically favorable Cu(II) ligand. Direct MS detection of the resulting Cu(II)-Ybt complex in mice and humans with E. coli urinary tract infections demonstrates copper binding to be a physiologically relevant in vivo interaction during infection. Ybt expression corresponded to higher copper resistance among human urinary tract isolates, suggesting a protective role for this interaction. Chemical and genetic characterization showed that Ybt helps bacteria resist copper toxicity by sequestering host-derived Cu(II) and preventing its catechol-mediated reduction to Cu(I). Together, these studies reveal a new virulence-associated function for Ybt that is distinct from iron binding.

Development of an integrated metabolomic profiling approach for infectious diseases research.

Metabolomic profiling offers direct insights into the chemical environment and metabolic pathway activities at sites of human disease. During infection, this environment may receive important contributions from both host and pathogen. Here we apply an untargeted metabolomics approach to identify compounds associated with an E. coli urinary tract infection population. Correlative and structural data from minimally processed samples were obtained using an optimized LC-MS platform capable of resolving ~2300 molecular features. Principal component analysis readily distinguished patient groups and multiple supervised chemometric analyses resolved robust metabolomic shifts between groups. These analyses revealed nine compounds whose provisional structures suggest candidate infection-associated endocrine, catabolic, and lipid pathways. Several of these metabolite signatures may derive from microbial processing of host metabolites. Overall, this study highlights the ability of metabolomic approaches to directly identify compounds encountered by, and produced from, bacterial pathogens within human hosts.

Early severe inflammatory responses to uropathogenic E. coli predispose to chronic and recurrent urinary tract infection.

Chronic infections are an increasing problem due to the aging population and the increase in antibiotic resistant organisms. Therefore, understanding the host-pathogen interactions that result in chronic infection is of great importance. Here, we investigate the molecular basis of chronic bacterial cystitis. We establish that introduction of uropathogenic E. coli (UPEC) into the bladders of C3H mice results in two distinct disease outcomes: resolution of acute infection or development of chronic cystitis lasting months. The incidence of chronic cystitis is both host strain and infectious dose-dependent. Further, development of chronic cystitis is preceded by biomarkers of local and systemic acute inflammation at 24 hours post-infection, including severe pyuria and bladder inflammation with mucosal injury, and a distinct serum cytokine signature consisting of elevated IL-5, IL-6, G-CSF, and the IL-8 analog KC. Mice deficient in TLR4 signaling or lymphocytes lack these innate responses and are resistant, to varying degrees, to developing chronic cystitis. Treatment of C3H mice with the glucocorticoid anti-inflammatory drug dexamethasone prior to UPEC infection also suppresses the development of chronic cystitis. Finally, individuals with a history of chronic cystitis, lasting at least 14 days, are significantly more susceptible to redeveloping severe, chronic cystitis upon bacterial challenge. Thus, we have discovered that the development of chronic cystitis in C3H mice by UPEC is facilitated by severe acute inflammatory responses early in infection, which subsequently are predisposing to recurrent cystitis, an insidious problem in women. Overall, these results have significant implications for our understanding of how early host-pathogen interactions at the mucosal surface determines the fate of disease.

Enterococcal biofilm formation and virulence in an optimized murine model of foreign body-associated urinary tract infections.

Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial UTIs and pose significant clinical challenges. Enterococcal species are among the predominant causative agents of CAUTIs. However, very little is known about the pathophysiology of Enterococcus-mediated UTIs. We optimized a murine model of foreign body-associated UTI in order to mimic conditions of indwelling catheters in patients. In this model, the presence of a foreign body elicits major histological changes and induces the expression of several proinflammatory cytokines in the bladder. In addition, in contrast to naïve mice, infection of catheter-implanted mice with Enterococcus faecalis induced the specific expression of interleukin 1ß (IL-1ß) and macrophage inflammatory protein 1α (MIP-1α) in the bladder. These responses resulted in a favorable niche for the development of persistent E. faecalis infections in the murine bladders and kidneys. Furthermore, biofilm formation on the catheter implant in vivo correlated with persistent infections. However, the enterococcal autolytic factors GelE and Atn (also known as AtlA), which are important in biofilm formation in vitro, are dispensable in vivo. In contrast, the housekeeping sortase A (SrtA) is critical for biofilm formation and virulence in CAUTIs. Overall, this murine model represents a significant advance in the understanding of CAUTIs and underscores the importance of urinary catheterization during E. faecalis uropathogenesis. This model is also a valuable tool for the identification of virulence determinants that can serve as potential antimicrobial targets for the treatment of enterococcal infections.

Virulence plasmid harbored by uropathogenic Escherichia coli functions in acute stages of pathogenesis.

Urinary tract infections (UTIs), the majority of which are caused by uropathogenic Escherichia coli (UPEC), afflict nearly 60% of women within their lifetimes. Studies in mice and humans have revealed that UPEC strains undergo a complex pathogenesis cycle that involves both the formation of intracellular bacterial communities (IBC) and the colonization of extracellular niches. Despite the commonality of the UPEC pathogenesis cycle, no specific urovirulence genetic profile has been determined; this is likely due to the fluid nature of the UPEC genome as the result of horizontal gene transfer and numerous genes of unknown function. UTI89 has a large extrachromosomal element termed pUTI89 with many characteristics of UPEC pathogenicity islands and that likely arose due to horizontal gene transfer. The pUTI89 plasmid has characteristics of both F plasmids and other known virulence plasmids. We sought to determine whether pUTI89 is important for virulence. Both in vitro and in vivo assays were used to examine the function of pUTI89 using plasmid-cured UTI89. No differences were observed between UTI89 and plasmid-cured UTI89 based on growth, type 1 pilus expression, or biofilm formation. However, in a mouse model of UTI, a significant decrease in bacterial invasion, CFU and IBC formation of the pUTI89-cured strain was observed at early time points postinfection compared to the wild type. Through directed deletions of specific operons on pUTI89, the cjr operon was partially implicated in this observed defect. Our findings implicate pUTI89 in the early aspects of infection.

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.

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.

Contribution of autolysin and Sortase a during Enterococcus faecalis DNA-dependent biofilm development.

Biofilm production is a major attribute of Enterococcus faecalis clinical isolates. Although some factors, such as sortases, autolysin, and extracellular DNA (eDNA), have been associated with E. faecalis biofilm production, the mechanisms underlying the contributions of these factors to this process have not been completely elucidated yet. In this study we define important roles for the major E. faecalis autolysin (Atn), eDNA, and sortase A (SrtA) during the developmental stages of biofilm formation under static and hydrodynamic conditions. Deletion of srtA affects the attachment stage and results in a deficiency in biofilm production. Atn-deficient mutants are delayed in biofilm development due to defects in primary adherence and DNA release, which we show to be particularly important during the accumulative phase for maturation and architectural stability of biofilms. Confocal laser scanning and freeze-dry electron microscopy of biofilms grown under hydrodynamic conditions revealed that E. faecalis produces a DNase I-sensitive fibrous network, which is important for biofilm stability and is absent in atn-deficient mutant biofilms. This study establishes the stage-specific requirements for SrtA and Atn and demonstrates a role for Atn in the pathway leading to DNA release during biofilm development in E. faecalis.

Dammarane-type triterpene glycosides from Oncoba manii active against methicillin-resistant Staphylococcus aureus.

Drug-resistant bacteria are becoming more prevalent both in the community and in hospitals. In a search for new antibiotic leads, we used a high-throughput natural products chemistry approach to isolate one new (1) and two known (2, 3) dammarane-type triterpenes with mass-limited material from the African plant Oncoba manii. The new compound was determined by spectroscopic methods to be 1beta,2alpha,3beta,20(R)-tetrahydroxydammar-24-ene 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranoside. Compounds 1 and 2 inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA).

Suppression of bladder epithelial cytokine responses by uropathogenic Escherichia coli.

Urinary tract infections are most commonly caused by uropathogenic strains of Escherichia coli (UPEC), which invade superficial bladder epithelial cells via a type 1 pilus-dependent mechanism. Inside these epithelial cells, UPEC organisms multiply to high numbers to form intracellular bacterial communities, allowing them to avoid immune detection. Bladder epithelial cells produce interleukin-6 (IL-6) and IL-8 in response to laboratory strains of E. coli in vitro. We investigated the ability of UPEC to alter epithelial cytokine signaling by examining the in vitro responses of bladder epithelial cell lines to the cystitis strains UTI89 and NU14. The cystitis strains induced significantly less IL-6 than did the laboratory E. coli strain MG1655 from 5637 and T24 bladder epithelial cells. The cystitis strains also suppressed epithelial cytokine responses to exogenous lipopolysaccharide (LPS) and to laboratory E. coli. We found that insertional mutations in the rfa and rfb operons and in the surA gene all abolished the ability of UTI89 to suppress cytokine induction. The rfa and rfb operons encode LPS biosynthetic genes, while surA encodes a periplasmic cis-trans prolyl isomerase important in the biogenesis of outer membrane proteins. We conclude that, in this in vitro model system, cystitis strains of UPEC have genes encoding factors that suppress proinflammatory cytokine production by bladder epithelial cells.

Toll-like receptor 4 on stromal and hematopoietic cells mediates innate resistance to uropathogenic Escherichia coli.

Innate host defenses at mucosal surfaces are critical in the early stages of many bacterial infections. In addition to cells of the traditional innate immune system, epithelial cells can also produce inflammatory mediators during an infection. However, the role of the epithelium in innate host defense in vivo is unclear. Recent studies have shown that lipopolysaccharide (LPS) recognition is critical for bladder epithelial cells to recognize and respond to Escherichia coli. Moreover, the LPS-nonresponsive mouse strain C3HHeJ, which has a mutation in the primary LPS receptor, Toll-like receptor 4 (TLR4), is extremely susceptible to infection with uropathogenic strains of E. coli. In this study, a bone marrow transplant approach was used to investigate the specific contributions of the bladder epithelium (and other stromal cells) in the TLR4-mediated innate immune response to the invading E. coli pathogen. Mice expressing the mutant TLR4 in the epithelialstromal compartment were not able to mount a protective inflammatory response to control the early infection even when their hematopoietic cells expressed wild-type TLR4. However, the presence of TLR4(+) epithelialstromal cells was not sufficient to activate an acute inflammatory response unless the hematopoietic cells were also TLR4(+). These results demonstrated that bladder epithelial cells play a critical role in TLR4-mediated innate immunity in vivo during a mucosal bacterial infection.