Dihydrothiazolo ring-fused 2-pyridone antimicrobial compounds treat Streptococcus pyogenes skin and soft tissue infection.

We have developed GmPcides from a peptidomimetic dihydrothiazolo ring-fused 2-pyridone scaffold that has antimicrobial activities against a broad spectrum of Gram-positive pathogens. Here, we examine the treatment efficacy of GmPcides using skin and soft tissue infection (SSTI) and biofilm formation models by Streptococcus pyogenes. Screening our compound library for minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations identified GmPcide PS757 as highly active against S. pyogenes. Treatment of S. pyogenes biofilm with PS757 revealed robust efficacy against all phases of biofilm formation by preventing initial biofilm development, ceasing biofilm maturation and eradicating mature biofilm. In a murine model of S. pyogenes SSTI, subcutaneous delivery of PS757 resulted in reduced levels of tissue damage, decreased bacterial burdens, and accelerated rates of wound healing, which were associated with down-regulation of key virulence factors, including M protein and the SpeB cysteine protease. These data demonstrate that GmPcides show considerable promise for treating S. pyogenes infections.

Dihydrothiazolo ring-fused 2-pyridone antimicrobial compounds treat Streptococcus pyogenes skin and soft tissue infection.

We have developed GmPcides from a peptidomimetic dihydrothiazolo ring-fused 2-pyridone scaffold that have antimicrobial activities against a broad-spectrum of Gram-positive pathogens. Here we examine the treatment efficacy of GmPcides using skin and soft tissue infection (SSTI) and biofilm formation models by Streptococcus pyogenes. Screening our compound library for minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations identified GmPcide PS757 as highly active against S. pyogenes. Treatment of S. pyogenes biofilm with PS757 revealed robust efficacy against all phases of biofilm formation by preventing initial biofilm development, ceasing biofilm maturation and eradicating mature biofilm. In a murine model of S. pyogenes SSTI, subcutaneous delivery of PS757 resulted in reduced levels of tissue damage, decreased bacterial burdens and accelerated rates of wound-healing, which were associated with down-regulation of key virulence factors, including M protein and the SpeB cysteine protease. These data demonstrate that GmPcides show considerable promise for treating S. pyogenes infections.

Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice.

Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic Escherichia coli (UPEC) causes most of these primary infections and leads to 25% becoming recurrent or chronic. To repel invading pathogens, the urinary tract mounts a vigorous innate immune response that includes the secretion of antimicrobial peptides (AMPs), rapid recruitment of phagocytes, and exfoliation of superficial umbrella cells. Here, we investigate secretory leukocyte protease inhibitor (SLPI), an AMP with antiprotease, antimicrobial, and immunomodulatory functions, known to play protective roles at other mucosal sites, but not well characterized in UTIs. Using a preclinical model of UPEC-caused UTI, we show that urine SLPI increases in infected mice and that SLPI is localized to bladder epithelial cells. UPEC-infected SLPI-deficient (Slpi-/-) mice suffer from higher urine bacterial burdens, prolonged bladder inflammation, and elevated urine neutrophil elastase (NE) levels compared to wild-type (Slpi+/+) controls. Combined with bulk bladder RNA sequencing, our data indicate that Slpi-/- mice have a dysregulated immune and tissue repair response following UTI. We also measure SLPI in urine samples from a small group of female subjects 18-49 years old and find that SLPI tends to be higher in the presence of a uropathogen, except in patients with a history of recent or recurrent UTI, suggesting a dysregulation of SLPI expression in these women. Taken together, our findings show SLPI promotes clearance of UPEC in mice and provides preliminary evidence that SLPI is likewise regulated in response to uropathogen exposure in women.IMPORTANCEAnnually, millions of people suffer from urinary tract infections (UTIs) and more than $3 billion are spent on work absences and treatment of these patients. While the early response to UTI is known to be important in combating urinary pathogens, knowledge of host factors that help curb infection is still limited. Here, we use a preclinical model of UTI to study secretory leukocyte protease inhibitor (SLPI), an antimicrobial protein, to determine how it protects the bladder against infection. We find that SLPI is increased during UTI, accelerates the clearance of bacteriuria, and upregulates genes and pathways needed to fight an infection while preventing prolonged bladder inflammation. In a small clinical study, we show SLPI is readily detectable in human urine and is associated with the presence of a uropathogen in patients without a previous history of UTI, suggesting SLPI may play an important role in protecting from bacterial cystitis.

A prospective randomized clinical trial to assess antibiotic pocket irrigation on tissue expander breast reconstruction.

Bacterial infection is the most common complication following staged post-mastectomy breast reconstruction initiated with a tissue expander (TE). To limit bacterial infection, antibiotic irrigation of the surgical site is commonly performed despite little high-quality data to support this practice. We performed a prospective randomized control trial to compare the impact of saline irrigation alone to a triple antibiotic irrigation regimen (1 g cefazolin, 80 mg gentamicin, and 50,000 units of bacitracin in 500 mL of saline) for breast implant surgery. The microbiome in breasts with cancer (n = 16) was compared to those without (n = 16), as all patients (n = 16) had unilateral cancers but bilateral mastectomies (n = 32). Biologic and prosthetic specimens procured both at the time of mastectomy and during TE removal months later were analyzed for longitudinal comparison. Outcomes included clinical infection, bacterial abundance, and relative microbiome composition. No patient in either group suffered a reconstructive failure or developed an infection. Triple antibiotic irrigation administered at the time of immediate TE reconstruction did not reduce bacterial abundance or impact microbial diversity relative to saline irrigation at the time of planned exchange. Implanted prosthetic material adopted the microbial composition of the surrounding host tissue. In cancer-naïve breasts, relative to saline, antibiotic irrigation increased bacterial abundance on periprosthetic capsules (P = 0.03) and acellular dermal matrices (P = 0.04) and altered the microbiota on both. These data show that, relative to saline only, the use of triple antibiotic irrigation in TE breast reconstruction does impact the bacterial abundance and diversity of certain biomaterials from cancer-naïve breasts. IMPORTANCE The lifetime risk of breast cancer is ~13% in women and is treated with a mastectomy in ~50% of cases. The majority are reconstructed, usually starting with a tissue expander to help restore the volume for a subsequent permanent breast implant or the women's own tissues. The biopsychosocial benefits of breast reconstruction, though, can be tempered by a high complication rate of at least 7% but over 30% in some women. Bacterial infection is the most common complication, and can lead to treatment delays, patient physical and emotional distress and escalating health care cost. To limit this risk, plastic surgeons have tried a variety of strategies to limit bacterial infection including irrigating the pocket created after removing the breast implant with antibiotic solutions, but good-quality data are scarce. Herein, we study the value of antibiotics in pocket irrigation using a robust randomized clinical trial design and molecular microbiology approaches.

Uropathogenic Escherichia coli infection-induced epithelial trained immunity impacts urinary tract disease outcome.

Previous urinary tract infections (UTIs) can predispose one to future infections; however, the underlying mechanisms affecting recurrence are poorly understood. We previously found that UTIs in mice cause differential bladder epithelial (urothelial) remodelling, depending on disease outcome, that impacts susceptibility to recurrent UTI. Here we compared urothelial stem cell (USC) lines isolated from mice with a history of either resolved or chronic uropathogenic Escherichia coli (UPEC) infection, elucidating evidence of molecular imprinting that involved epigenetic changes, including differences in chromatin accessibility, DNA methylation and histone modification. Epigenetic marks in USCs from chronically infected mice enhanced caspase-1-mediated cell death upon UPEC infection, promoting bacterial clearance. Increased Ptgs2os2 expression also occurred, potentially contributing to sustained cyclooxygenase-2 expression, bladder inflammation and mucosal wounding-responses associated with severe recurrent cystitis. Thus, UPEC infection acts as an epi-mutagen reprogramming the urothelial epigenome, leading to urothelial-intrinsic remodelling and training of the innate response to subsequent infection.

Establishment and Characterization of Bacterial Infection of Breast Implants in a Murine Model.

BACKGROUND: Staphylococcus epidermidis and Pseudomonas aeruginosa are the most common causes of Gram-positive and Gram-negative breast implant-associated infection. Little is known about how these bacteria infect breast implants as a function of implant surface characteristics and timing of infection. OBJECTIVES: The aim of this work was to establish a mouse model for studying the impact of various conditions on breast implant infection. METHODS: Ninety-one mice were implanted with 273 breast implant shells and infected with S. epidermidis or P. aeruginosa. Smooth, microtextured, and macrotextured breast implant shells were implanted in each mouse. Bacterial inoculation occurred during implantation or 1 day later. Implants were retrieved 1 or 7 days later. Explanted breast implant shells were sonicated, cultured, and colony-forming units determined or analyzed with scanning electron microscopy. RESULTS: P. aeruginosa could be detected on all device surfaces at 1- and 7- days post infection (dpi), when mice were implanted and infected concurrently or when they were infected 1- day after implantation. However, P. aeruginosa infection was more robust on implant shells retrieved at 7 dpi and particularly on the macrotextured devices that were infected 1 day post implantation. S. epidermidis was mostly cleared from implants when mice were infected and implanted concurrently. Other the other hand, S. epidermidis could be detected on all device surfaces at 1 dpi and 2 days post implantation. However, S. epidermdis infection was suppressed by 7 dpi and 8 days post implantation. CONCLUSIONS: S. epidermidis required higher inoculating doses to cause infection and was cleared within 7 days. P. aeruginosa infected at lower inoculating doses, with robust biofilms noted 7 days later.

Deposition of Host Matrix Proteins on Breast Implant Surfaces Facilitates Staphylococcus Epidermidis Biofilm Formation: In Vitro Analysis.

BACKGROUND: Staphylococcus epidermidis is a primary cause of breast implant-associated infection. S epidermidis possesses several virulence factors that enable it to bind both abiotic surfaces and host factors to form a biofilm. In addition S epidermidis colocalizes with matrix proteins coating explanted human breast implants. OBJECTIVES: The authors sought to identify matrix proteins that S epidermidis may exploit to infect various breast implant surfaces in vitro. METHODS: A combination of in vitro assays was used to characterize S epidermidis strains isolated from human breast implants to gain a better understanding of how these bacteria colonize breast implant surfaces. These included determining the (1) minimum inhibitory and bactericidal concentrations for irrigation solutions commonly used to prevent breast implant contamination; (2) expression and carriage of polysaccharide intercellular adhesin and serine-aspartate repeat proteins, which bind fibrinogen (SdrG) and collagen (SdrF), respectively; and (3) biofilm formation on varying implant surface characteristics, in different growth media, and supplemented with fibrinogen and Types I and III collagen. Scanning electron microscopy and immunofluorescence staining analyses were performed to corroborate findings from these assays. RESULTS: Textured breast implant surfaces support greater bacterial biofilm formation at baseline, and the addition of collagen significantly increases biomass on all surfaces tested. We found that S epidermidis isolated from breast implants all encoded SdrF. Consistent with this finding, these strains had a clear affinity for Type I collagen, forming dense, highly structured biofilms in its presence. CONCLUSIONS: The authors found that S epidermidis may utilize SdrF to interact with Type I collagen to form biofilm on breast implant surfaces.

Mucosal infection rewires TNFɑ signaling dynamics to skew susceptibility to recurrence.

A mucosal infectious disease episode can render the host either more or less susceptible to recurrent infection, but the specific mechanisms that tip the balance remain unclear. We investigated this question in a mouse model of recurrent urinary tract infection and found that a prior bladder infection resulted in an earlier onset of tumor necrosis factor-alpha (TNFɑ)-mediated bladder inflammation upon subsequent bacterial challenge, relative to age-matched naive mice. However, the duration of TNFɑ signaling activation differed according to whether the first infection was chronic (Sensitized) or self-limiting (Resolved). TNFɑ depletion studies revealed that transient early-phase TNFɑ signaling in Resolved mice promoted clearance of bladder-colonizing bacteria via rapid recruitment of neutrophils and subsequent exfoliation of infected bladder cells. In contrast, sustained TNFɑ signaling in Sensitized mice prolonged damaging inflammation, worsening infection. This work reveals how TNFɑ signaling dynamics can be rewired by a prior infection to shape diverse susceptibilities to future mucosal infections.

Insights into the Microbiome of Breast Implants and Periprosthetic Tissue in Breast Implant-Associated Anaplastic Large Cell Lymphoma.

Though rare, breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), a CD30+ T-cell lymphoma associated with textured breast implants, has adversely impacted our perception of the safety of breast implants. Its etiology unknown, one hypothesis suggests an initiating inflammatory stimulus, possibly infectious, triggers BIA-ALCL. We analyzed microbiota of breast, skin, implant and capsule in BIA-ALCL patients (n = 7), and controls via culturing methods, 16S rRNA microbiome sequencing, and immunohistochemistry. Alpha and beta diversity metrics and relative abundance of Gram-negative bacteria were calculated, and phylogenetic trees constructed. Staphylococcus spp., the most commonly cultured microbes, were identified in both the BIA-ALCL and contralateral control breast. The diversity of bacterial microbiota did not differ significantly between BIA-ALCL and controls for any material analyzed. Further, there were no significant differences in the relative abundance of Gram-negative bacteria between BIA-ALCL and control specimens. Heat maps suggested substantial diversity in the composition of the bacterial microbiota of the skin, breast, implant and capsule between patients with no clear trend to distinguish BIA-ALCL from controls. While we identified no consistent differences between patients with BIA-ALCL-affected and contralateral control breasts, this study provides insights into the composition of the breast microbiota in this population.

Fimbriae reprogram host gene expression - Divergent effects of P and type 1 fimbriae.

Pathogens rely on a complex virulence gene repertoire to successfully attack their hosts. We were therefore surprised to find that a single fimbrial gene reconstitution can return the virulence-attenuated commensal strain Escherichia coli 83972 to virulence, defined by a disease phenotype in human hosts. E. coli 83972pap stably reprogrammed host gene expression, by activating an acute pyelonephritis-associated, IRF7-dependent gene network. The PapG protein was internalized by human kidney cells and served as a transcriptional agonist of IRF-7, IFN-ß and MYC, suggesting direct involvement of the fimbrial adhesin in this process. IRF-7 was further identified as a potent upstream regulator (-log (p-value) = 61), consistent with the effects in inoculated patients. In contrast, E. coli 83972fim transiently attenuated overall gene expression in human hosts, enhancing the effects of E. coli 83972. The inhibition of RNA processing and ribosomal assembly indicated a homeostatic rather than a pathogenic end-point. In parallel, the expression of specific ion channels and neuropeptide gene networks was transiently enhanced, in a FimH-dependent manner. The studies were performed to establish protective asymptomatic bacteriuria in human hosts and the reconstituted E. coli 83972 variants were developed to improve bacterial fitness for the human urinary tract. Unexpectedly, P fimbriae were able to drive a disease response, suggesting that like oncogene addiction in cancer, pathogens may be addicted to single super-virulence factors.

The Detection of Bacteria and Matrix Proteins on Clinically Benign and Pathologic Implants.

BACKGROUND: Bacterial contamination of breast implants causes infection, can lead to capsular contracture, and is implicated in breast implant-associated anaplastic large cell lymphoma. Bacteria, however, also colonize clinically benign breast implants and little is known about the biologic signals that trigger the switch from a benign to pathologic state. METHODS: Explanted smooth as well as Biocell and Siltex textured breast implants associated with clinically normal and pathologic conditions were analyzed in this observational study. Immunofluorescence and bacterial culture techniques were performed. To avoid sampling bias, implant surfaces >25 sq cm were analyzed. RESULTS: Bacteria were detected on 9 of 22 clinically normal explanted devices or periprosthetic capsules, including 40% of Biocell tissue expanders and 75% of Biocell textured implants. Staphylococcus epidermidis was identified in 67% of the bacteria-positive capsular contractures. Fibrinogen was present on 17 of 18, and collagen on 13 of 18 analyzed breast implants. S. epidermidis co-localized with collagen, while group B streptococci and Klebsiella pneumoniae co-localized with fibrinogen. CONCLUSIONS: Bacteria are often detectable on clinically benign breast implants when a multimodal approach is applied to a substantial proportion of the device surface to avoid sampling bias. The impact of bacteria on breast implant pathology should be studied in the presence of an adequate negative control group to account for clinically benign bacteria. Disruption of the interaction of bacteria with matrix proteins coating the surface of breast implants may represent a nonantibiotic strategy for the prevention of breast implant bacterial contamination.

Host restriction of Escherichia coli recurrent urinary tract infection occurs in a bacterial strain-specific manner.

Urinary tract infections (UTI) are extremely common and can be highly recurrent, with 1-2% of women suffering from six or more recurrent episodes per year. The high incidence of recurrent UTI, including recurrent infections caused by the same bacterial strain that caused the first infection, suggests that at least some women do not mount a protective adaptive immune response to UTI. Here we observed in a mouse model of cystitis (bladder infection) that infection with two different clinical uropathogenic Escherichia coli (UPEC) isolates, UTI89 or CFT073, resulted in different kinetics of bacterial clearance and different susceptibility to same-strain recurrent infection. UTI89 and CFT073 both caused infections that persisted for at least two weeks in similar proportions of mice, but whereas UTI89 infections could persist indefinitely, CFT073 infections began to clear two weeks after inoculation and were uniformly cleared within eight weeks. Mice with a history of CFT073 cystitis lasting four weeks were protected against recurrent CFT073 infection after antibiotic therapy, but were not protected against challenge with UTI89. In contrast, mice with a history of UTI89 cystitis lasting four weeks were highly susceptible to challenge infection with either strain after antibiotic treatment. We found that depletion of CD4+ and CD8+ T cell subsets impaired the ability of the host to clear CFT073 infections and rendered mice with a history of CFT073 cystitis lasting four weeks susceptible to recurrent CFT073 cystitis upon challenge. Our findings demonstrate the complex interplay between the broad genetic diversity of UPEC and the host innate and adaptive immune responses during UTI. A better understanding of these host-pathogen interactions is urgently needed for effective drug and vaccine development in the era of increasing antibiotic resistance.

Hydrogen Sulfide Sensing through Reactive Sulfur Species (RSS) and Nitroxyl (HNO) in Enterococcus faecalis.

Recent studies of hydrogen sulfide (H2S) signaling implicate low molecular weight (LMW) thiol persulfides and other reactive sulfur species (RSS) as signaling effectors. Here, we show that a CstR protein from the human pathogen Enterococcus faecalis ( E. faecalis), previously identified in Staphylococcus aureus ( S. aureus), is an RSS-sensing repressor that transcriptionally regulates a cst-like operon in response to both exogenous sulfide stress and Angeli's salt, a precursor of nitroxyl (HNO). E. faecalis CstR reacts with coenzyme A persulfide (CoASSH) to form interprotomer disulfide and trisulfide bridges between C32 and C61', which negatively regulate DNA binding to a consensus CstR DNA operator. A Δ cstR strain exhibits deficiency in catheter colonization in a catheter-associated urinary tract infection (CAUTI) mouse model, suggesting sulfide regulation and homeostasis is critical for pathogenicity. Cellular polysulfide metabolite profiling of sodium sulfide-stressed E. faecalis confirms an increase in both inorganic polysulfides and LMW thiols and persulfides sensed by CstR. The cst-like operon encodes two authentic thiosulfate sulfurtransferases and an enzyme we characterize here as an NADH and FAD-dependent coenzyme A (CoA) persulfide reductase (CoAPR) that harbors an N-terminal CoA disulfide reductase (CDR) domain and a C-terminal rhodanese homology domain (RHD). Both cysteines in the CDR (C42) and RHD (C508) domains are required for CoAPR activity and complementation of a sulfide-induced growth phenotype of a S. aureus strain lacking cstB, encoding a nonheme FeII persulfide dioxygenase. We propose that S. aureus CstB and E. faecalis CoAPR employ orthogonal chemistries to lower CoASSH that accumulates under conditions of cellular sulfide toxicity and signaling.

Host and bacterial proteases influence biofilm formation and virulence in a murine model of enterococcal catheter-associated urinary tract infection.

Enterococcus faecalis is a leading causative agent of catheter-associated urinary tract infection (CAUTI), the most common hospital-acquired infection. Its ability to grow and form catheter biofilm is dependent upon host fibrinogen (Fg). Examined here are how bacterial and host proteases interact with Fg and contribute to virulence. Analysis of mutants affecting the two major secreted proteases of E. faecalis OG1RF (GelE, SprE) revealed that while the loss of either had no effect on virulence in a murine CAUTI model or for formation of Fg-dependent biofilm in urine, the loss of both resulted in CAUTI attenuation and defective biofilm formation. GelE-, but not SprE- mutants, lost the ability to degrade Fg in medium, while paradoxically, both could degrade Fg in urine. The finding that SprE was activated independently of GelE in urine by a host trypsin-like protease resolved this paradox. Treatment of catheter-implanted mice with inhibitors of both host-derived and bacterial-derived proteases dramatically reduced catheter-induced inflammation, significantly inhibited dissemination from bladder to kidney and revealed an essential role for a host cysteine protease in promoting pathogenesis. These data show that both bacterial and host proteases contribute to CAUTI, that host proteases promote dissemination and suggest new strategies for therapeutic intervention.

Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

Highly conserved type 1 pili promote enterotoxigenic E. coli pathogen-host interactions.

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens.

Bacterial virulence phenotypes of Escherichia coli and host susceptibility determine risk for urinary tract infections.

Urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC) strains. In contrast to many enteric E. coli pathogroups, no genetic signature has been identified for UPEC strains. We conducted a high-resolution comparative genomic study using E. coli isolates collected from the urine of women suffering from frequent recurrent UTIs. These isolates were genetically diverse and varied in their urovirulence, that is, their ability to infect the bladder in a mouse model of cystitis. We found no set of genes, including previously defined putative urovirulence factors (PUFs), that were predictive of urovirulence. In addition, in some patients, the E. coli strain causing a recurrent UTI had fewer PUFs than the supplanted strain. In competitive experimental infections in mice, the supplanting strain was more efficient at colonizing the mouse bladder than the supplanted strain. Despite the lack of a clear genomic signature for urovirulence, comparative transcriptomic and phenotypic analyses revealed that the expression of key conserved functions during culture, such as motility and metabolism, could be used to predict subsequent colonization of the mouse bladder. Together, our findings suggest that UTI risk and outcome may be determined by complex interactions between host susceptibility and the urovirulence potential of diverse bacterial strains.

The Catabolite Repressor Protein-Cyclic AMP Complex Regulates csgD and Biofilm Formation in Uropathogenic Escherichia coli.

The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The catabolite repressor protein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaA and Δcrp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD IMPORTANCE The catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874-5893, 2004, https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibits E. coli biofilm formation, and ΔcyaA and Δcrp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406-3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms through csgD Additionally, we propose that cAMP may work as a signaling compound for uropathogenic E. coli (UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation.

Inflammation-Induced Adhesin-Receptor Interaction Provides a Fitness Advantage to Uropathogenic E. coli during Chronic Infection.

Uropathogenic E. coli (UPEC) is the dominant cause of urinary tract infections, clinically described as cystitis. UPEC express CUP pili, which are extracellular fibers tipped with adhesins that bind mucosal surfaces of the urinary tract. Here we identify the role of the F9/Yde/Fml pilus for UPEC persistence in the inflamed urothelium. The Fml adhesin FmlH binds galactose ß1-3 N-acetylgalactosamine found in core-1 and -2 O-glycans. Deletion of fmlH had no effect on UPEC virulence in an acute mouse model of cystitis. However, FmlH provided a fitness advantage during chronic cystitis, which is manifested as persistent bacteriuria, high bladder bacterial burdens, and chronic inflammation. In situ binding confirmed that FmlH bound avidly to the inflamed, but not the naive bladder. In accordance with its pathogenic profile, vaccination with FmlH significantly protected mice from chronic cystitis. Thus, UPEC employ separate CUP pili to adapt to the rapidly changing niche during bladder infection.

Human Urine Decreases Function and Expression of Type 1 Pili in Uropathogenic Escherichia coli.

UNLABELLED: Uropathogenic Escherichia coli (UPEC) is the primary cause of community-acquired urinary tract infections (UTIs). UPEC bind the bladder using type 1 pili, encoded by the fim operon in nearly all E. coli. Assembled type 1 pili terminate in the FimH adhesin, which specifically binds to mannosylated glycoproteins on the bladder epithelium. Expression of type 1 pili is regulated in part by phase-variable inversion of the genomic element containing the fimS promoter, resulting in phase ON (expressing) and OFF (nonexpressing) orientations. Type 1 pili are essential for virulence in murine models of UTI; however, studies of urine samples from human UTI patients demonstrate variable expression of type 1 pili. We provide insight into this paradox by showing that human urine specifically inhibits both expression and function of type 1 pili. Growth in urine induces the fimS phase OFF orientation, preventing fim expression. Urine also contains inhibitors of FimH function, and this inhibition leads to a further bias in fimS orientation toward the phase OFF state. The dual effect of urine on fimS regulation and FimH binding presents a potential barrier to type 1 pilus-mediated colonization and invasion of the bladder epithelium. However, FimH-mediated attachment to human bladder cells during growth in urine reverses these effects such that fim expression remains ON and/or turns ON. Interestingly, FimH inhibitors called mannosides also induce the fimS phase OFF orientation. Thus, the transduction of FimH protein attachment or inhibition into epigenetic regulation of type 1 pilus expression has important implications for the development of therapeutics targeting FimH function. IMPORTANCE: Urinary tract infections (UTIs) are extremely common infections, frequently caused by uropathogenic Escherichia coli (UPEC), that are treated with antibiotics but often recur. Therefore, UTI treatment both is complicated by and contributes to bacterial antibiotic resistance. Thus, it is important to understand UTI pathogenesis to devise novel strategies and targets for prevention and treatment. Based on evidence from disease epidemiology and mouse models of infection, UPEC relies heavily on type 1 pili to attach to and invade the bladder epithelium during initial stages of UTI. Here, we demonstrate that the negative effect of planktonic growth in human urine on both the function and expression of type 1 pili is overcome by attachment to bladder epithelial cells, representing a strategy to subvert this alternative innate defense mechanism. Furthermore, this dually inhibitory action of urine is a mechanism shared with recently developed anti-type 1 pilus molecules, highlighting the idea that further development of antivirulence strategies targeting pili may be particularly effective for UPEC.