A novel chaperone-effector-immunity system identified in uropathogenic Escherichia coli UMN026.

Background: Urinary tract infections (UTIs) are very common worldwide. According to their symptomatology, these infections are classified as pyelonephritis, cystitis, or asymptomatic bacteriuria (AB). Approximately 75-95% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which is an extraintestinal bacterium that possesses virulence factors for bacterial adherence and invasion in the urinary tract. In addition, UPEC possesses type 6 secretion systems (T6SS) as virulence mechanisms that can participate in bacterial competition and in bacterial pathogenicity. UPEC UMN026 carries three genes, namely, ECUMN_0231, ECUMN_0232, and ECUMN_0233, which encode three uncharacterized proteins related to the T6SS that are conserved in strains from phylogroups B2 and D and have been proposed as biomarkers of UTIs. Aim: To analyze the frequency of the ECUMN_0231, ECUMN_0232, ECUMN_0233, and vgrG genes in UTI isolates, as well as their expression in Luria Bertani (LB) medium and urine; to determine whether these genes are related to UTI symptoms or bacterial competence and to identify functional domains on the putative proteins. Methods: The frequency of the ECUMN and vgrG genes in 99 clinical isolates from UPEC was determined by endpoint PCR. The relationship between gene presence and UTI symptomatology was determined using the chi2 test, with p < 0.05 considered to indicate statistical significance. The expression of the three ECUMN genes and vgrG was analyzed by RT-PCR. The antibacterial activity of strain UMN026 was determined by bacterial competence assays. The identification of functional domains and the docking were performed using bioinformatic tools. Results: The ECUMN genes are conserved in 33.3% of clinical isolates from patients with symptomatic and asymptomatic UTIs and have no relationship with UTI symptomatology. Of the ECUMN+ isolates, only five (15.15%, 5/33) had the three ECUMN and vgrG genes. These genes were expressed in LB broth and urine in UPEC UMN026 but not in all the clinical isolates. Strain UMN026 had antibacterial activity against UPEC clinical isolate 4014 (ECUMN-) and E. faecalis but not against isolate 4012 (ECUMN+). Bioinformatics analysis suggested that the ECUMN genes encode a chaperone/effector/immunity system. Conclusions: The ECUMN genes are conserved in clinical isolates from symptomatic and asymptomatic patients and are not related to UTI symptoms. However, these genes encode a putative chaperone/effector/immunity system that seems to be involved in the antibacterial activity of strain UMN026.

Multidrug-resistant Escherichia coli causing canine pyometra and urinary tract infections are genetically related but distinct from those causing prostatic abscesses.

Despite extensive characterisation of uropathogenic Escherichia coli (UPEC) causing urinary tract infections (UTIs), the genetic background of non-urinary extraintestinal pathogenic E. coli (ExPEC) in companion animals remains inadequately understood. In this study, we characterised virulence traits of 104 E. coli isolated from canine pyometra (n = 61) and prostatic abscesses (PAs) (n = 38), and bloodstream infections (BSIs) in dogs (n = 2), and cats (n = 3). A stronger association with UPEC of pyometra strains in comparison to PA strains was revealed. Notably, 44 isolates exhibited resistance to third-generation cephalosporins and/or fluoroquinolones, 15 were extended-spectrum ß-lactamase-producers. Twelve multidrug-resistant (MDR) strains, isolated from pyometra (n = 4), PAs (n = 5), and BSIs (n = 3), along with 7 previously characterised UPEC strains from dogs and cats, were sequenced. Genomic characteristics revealed that MDR E. coli associated with UTIs, pyometra, and BSIs belonged to international high-risk E. coli clones, including sequence type (ST) 38, ST131, ST617, ST648, and ST1193. However, PA strains belonged to distinct lineages, including ST12, ST44, ST457, ST744, and ST13037. The coreSNPs, cgMLST, and pan-genome illustrated intra-clonal variations within the same ST from different sources. The high-risk ST131 and ST1193 (phylogroup B2) contained high numbers of ExPEC virulence genes on pathogenicity islands, predominating in pyometra and UTI. Hybrid MDR/virulence IncF multi-replicon plasmids, containing aerobactin genes, were commonly found in non-B2 phylogroups from all sources. These findings offer genomic insights into non-urinary ExPEC, highlighting its potential for invasive infections in pets beyond UTIs, particularly with regards to high-risk global clones.

Escherichia coli isolated from pyometra and cystitis in the same animal exhibit a wide phenotypic similarity.

AIMS: Pyometra and cystitis caused by Escherichia coli are common diseases identified in canine or feline females. The origin of pyometra infection remains uncertain, and effective prevention strategies for this disease are still unknown. This study aimed to provide a phenotypic characterization, including antimicrobial resistance and virulence profiles, of endometrial pathogenic (EnPEC) and uropathogenic (UPEC) E. coli strains isolated simultaneously from the same animal. METHODS AND RESULTS: Sixteen E. coli strains, from eight different animals, were analyzed in this study. The antimicrobial susceptibility profile of EnPEC and UPEC strains was determined using the disc diffusion method, which showed a similar susceptibility profile among strains (EnPEC and UPEC) from the same animal. The virulence profile of the strains was assessed through biofilm formation, as well as serum resistance abilities. EnPEC and UPEC strains from the same animal exhibited slight variations in their virulence and antimicrobial resistance capabilities. Overall, most of the strain pairs showed a high similarity in their ability to establish biofilms and survive in serum complement activity. CONCLUSIONS: Overall, strains of E. coli isolated from both pyometra and cystitis in the same animal, despite presenting distinct clinical diseases, exhibit a wide phenotypic similarity, suggesting a common origin for the strains.

Peptidoglycan endopeptidase MepM of uropathogenic Escherichia coli contributes to competitive fitness during urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) are common bacterial infections, primarily caused by uropathogenic Escherichia coli (UPEC), leading to significant health issues and economic burden. Although antibiotics have been effective in treating UPEC infections, the rise of antibiotic-resistant strains hinders their efficacy. Hence, identifying novel bacterial targets for new antimicrobial approaches is crucial. Bacterial factors required for maintaining the full virulence of UPEC are the potential target. MepM, an endopeptidase in E. coli, is involved in the biogenesis of peptidoglycan, a major structure of bacterial envelope. Given that the bacterial envelope confronts the hostile host environment during infections, MepM's function could be crucial for UPEC's virulence. This study aims to explore the role of MepM in UPEC pathogenesis. RESULTS: MepM deficiency significantly impacted UPEC's survival in urine and within macrophages. Moreover, the deficiency hindered the bacillary-to-filamentous shape switch which is known for aiding UPEC in evading phagocytosis during infections. Additionally, UPEC motility was downregulated due to MepM deficiency. As a result, the mepM mutant displayed notably reduced fitness in causing UTIs in the mouse model compared to wild-type UPEC. CONCLUSIONS: This study provides the first evidence of the vital role of peptidoglycan endopeptidase MepM in UPEC's full virulence for causing UTIs. MepM's contribution to UPEC pathogenesis may stem from its critical role in maintaining the ability to resist urine- and immune cell-mediated killing, facilitating the morphological switch, and sustaining motility. Thus, MepM is a promising candidate target for novel antimicrobial strategies.

Nucleoside-diphosphate kinase of uropathogenic Escherichia coli inhibits caspase-1-dependent pyroptosis facilitating urinary tract infection.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection (UTI). UPEC invades bladder epithelial cells (BECs) via fusiform vesicles, escapes into the cytosol, and establishes biofilm-like intracellular bacterial communities (IBCs). Nucleoside-diphosphate kinase (NDK) is secreted by pathogenic bacteria to enhance virulence. However, whether NDK is involved in UPEC pathogenesis remains unclear. Here, we find that the lack of ndk impairs the colonization of UPEC CFT073 in mouse bladders and kidneys owing to the impaired ability of UPEC to form IBCs. Furthermore, we demonstrate that NDK inhibits caspase-1-dependent pyroptosis by consuming extracellular ATP, preventing superficial BEC exfoliation, and promoting IBC formation. UPEC utilizes the reactive oxygen species (ROS) sensor OxyR to indirectly activate the regulator integration host factor, which then directly activates ndk expression in response to intracellular ROS. Here, we reveal a signaling transduction pathway that UPEC employs to inhibit superficial BEC exfoliation, thus facilitating acute UTI.

Insulin receptor signaling engages bladder urothelial defenses that limit urinary tract infection.

Urinary tract infections (UTIs) commonly afflict people with diabetes. To better understand the mechanisms that predispose diabetics to UTIs, we employ diabetic mouse models and altered insulin signaling to show that insulin receptor (IR) shapes UTI defenses. Our findings are validated in human biosamples. We report that diabetic mice have suppressed IR expression and are more susceptible to UTIs caused by uropathogenic Escherichia coli (UPEC). Systemic IR inhibition increases UPEC susceptibility, while IR activation reduces UTIs. Localized IR deletion in bladder urothelium promotes UTI by increasing barrier permeability and suppressing antimicrobial peptides. Mechanistically, IR deletion reduces nuclear factor κB (NF-κB)-dependent programming that co-regulates urothelial tight junction integrity and antimicrobial peptides. Exfoliated urothelial cells or urine samples from diabetic youths show suppressed expression of IR, barrier genes, and antimicrobial peptides. These observations demonstrate that urothelial insulin signaling has a role in UTI prevention and link IR to urothelial barrier maintenance and antimicrobial peptide expression.

Protective role for C3aR in experimental chronic pyelonephritis.

Emerging evidence suggest that C3aR plays important roles in homeostasis, host defense and disease. Although it is known that C3aR is protective in several models of acute bacterial infections, the role for C3aR in chronic infection is largely unknown. Here we show that C3aR is protective in experimental chronic pyelonephritis. Global C3aR deficient (C3ar-/- ) mice had higher renal bacterial load, more pronounced renal histological lesions, increased renal apoptotic cell accumulation, tissue inflammation and extracellular matrix deposition following renal infection with uropathogenic E. coli (UPEC) strain IH11128, compared to WT control mice. Myeloid C3aR deficient (Lyz2-C3ar-/- ) mice exhibited a similar disease phenotype to global C3ar-/- mice. Pharmacological treatment with a C3aR agonist reduced disease severity in experimental chronic pyelonephritis. Furthermore, macrophages of C3ar-/- mice exhibited impaired ability to phagocytose UPEC. Our data clearly demonstrate a protective role for C3aR against experimental chronic pyelonephritis, macrophage C3aR plays a major role in the protection, and C3aR is necessary for phagocytosis of UPEC by macrophages. Our observation that C3aR agonist curtailed the pathology suggests a therapeutic potential for activation of C3aR in chronic infection.

Role of aggregate-forming pilus (AFP) in adherence and colonization of both intestinal and urinary tracts.

Hybrid-pathogenic Escherichia coli represent an important group of strains associated with intestinal and extraintestinal infections. Recently, we described strain UPEC-46, a uropathogenic/enteroaggregative E. coli (UPEC/EAEC) strain presenting the aggregative adherence (AA) pattern on bladder and colorectal epithelial cells mediated by aggregate-forming pili (AFP). However, the role of AFP and other uninvestigated putative fimbriae operons in UPEC-46 pathogenesis remains unclear. Thus, this study evaluated the involvement of AFP and other adhesins in uropathogenicity and intestinal colonization using different in vitro and in vivo models. The strain UPEC-46 was able to adhere and invade intestinal and urinary cell lines. A library of transposon mutants also identified the involvement of type I fimbriae (TIF) in the adherence to HeLa cells, in addition to colorectal and bladder cell lines. The streptomycin-treated mouse in vivo model also showed an increased number of bacterial counts in the colon in the presence of AFP and TIF. In the mouse model of ascending urinary tract infection (UTI), AFP was more associated with kidney colonization, while TIF appears to mediate bladder colonization. Results observed in in vivo experiments were also confirmed by electron microscopy (EM) analyses. In summary, the in vitro and in vivo analyses show a synergistic role of AFP and TIF in the adherence and colonization of intestinal and urinary epithelia. Therefore, we propose that hybrid E. coli strains carrying AFP and TIF could potentially cause intestinal and urinary tract infections in the same patient.

Dendrobium officinale polysaccharides attenuate uropathogenic Escherichia coli (UPEC)-induced pyroptosis in macrophage cells.

Urinary tract infections (UTI) are recognized as one of the most common infectious diseases worldwide, and uropathogenic Escherichia coli (UPEC) is the main causative agent of UTI. Dendrobium officinale polysaccharides (DOPs), the main effective ingredient in Dendrobium officinale, have been reported to possess an anti-inflammatory role. Whether DOPs can attenuate the inflammatory injury (pyroptosis) induced by UPEC remains unknown. The present study aimed to assess the protective effect and potential mechanism of DOPs in UPEC-induced pyroptosis. Cell viability of THP-1 differentiated macrophage cells with DOPs was determined using MTT assay. Pyroptosis by UPEC in macrophage cells with or not DOPs pre-treatment was evaluated with flow cytometry analysis, lactate dehydrogenase (LDH) assay, and proinflammatory cytokines secretion. Expression level of key proteins in the NLRP3/Caspase-1/GSDMD pyroptotic pathway was analyzed with western blot. Furthermore the effect of DOPs on ROS activation was investigated. Results indicated that DOPs attenuated UPEC-induced cell damage in macrophage cells, inhibited the activation of NLRP3 mediated inflammasome, subsequently decreased induction and activation of caspase-1/GSDMD, and reduced the secretion of pro-inflammatory cytokine (IL-1ß et al.). Moreover, pretreatment with DOPs significantly reduces ROS production, an important/putative pyroptosis stimulus signal. These results suggested that DOPs successfully mitigate UPEC-promoted pyroptosis in macrophage cells. The protective effects of DOPs are associated with the inhibition of the NLRP3/Caspase-1/GSDMD pathway and ROS signal activation.

Studies on Virulence and Extended-Spectrum ß-Lactamase-Producing Uropathogenic Escherichia coli Isolates and Therapeutic Effect of Fosfomycin in Acute Pyelonephritis Mice.

The understanding about virulence factors (VFs) and the drug resistance of uropathogenic Escherichia coli (UPEC) helps us understand the pathogenesis of urinary tract infections (UTIs) and make better decisions for clinical treatment. This study examined the correlation between the extended-spectrum ß-lactamases (ESBLs) phenotype and VFs in UPEC strains. In addition, we validated the therapeutic potential of fosfomycin in acute pyelonephritis mice. From May 2017 to November 2018, 22 nonduplicate E coli. strains were isolated from UTI patients. PCR was utilized to detect the distribution of virulence genes. We also analyzed the ESBL phenotype in E coli. We further evaluated the therapeutic effect of intravenous fosfomycin treatment in the acute pyelonephritis (APN) model. All 22 UPEC strains expressed the type 1 fimbriae (FimH) gene and more than 50% (12/22) of strains produced ESBLs. The detection rates of the iron acquisition-associated genes ChuT and IutA were 77.3% (n = 17) and 50% (n = 11) and those of P fimbria papA and papC genes were 45% (n = 10) and 50% (n = 11), respectively. Though the VFs were closely related with pathologenicity, the relationship between VFs and ESBLs still needs further investigation. Furthermore, intravenous fosfomycin 800 mg/kg significantly reduced the bacterial load and the inflammatory infiltration in the bladder and kidney, maintaining the structural integrity of the kidney. Intravenous fosfomycin administration can be used for the treatment of acute pyelonephritis caused by highly pathogenic and drug-resistant UPEC strains.

Isolation and Characterization of Novel Lytic Phages Infecting Multidrug-Resistant Escherichia coli.

Urinary tract infections (UTIs) are the second most frequent bacterial infections worldwide, with Escherichia coli being the main causative agent. The increase of antibiotic-resistance determinants among isolates from clinical samples, including UTIs, makes the development of novel therapeutic strategies a necessity. In this context, the use of bacteriophages as a therapeutic alternative has been proposed, due to their ability to efficiently kill bacteria. In this work, we isolated and characterized three novel bacteriophages, microbes laboratory phage 1 (MLP1), MLP2, and MLP3, belonging to the Chaseviridae, Myoviridae, and Podoviridae families, respectively. These phages efficiently infect and kill laboratory reference strains and multidrug-resistant clinical E. coli isolates from patients with diagnosed UTIs. Interestingly, these phages are also able to infect intestinal pathogenic Escherichia coli strains, such as enteroaggregative E. coli and diffusely adherent E. coli. Our data show that the MLP phages recognize different regions of the lipopolysaccharide (LPS) molecule, an important virulence factor in bacteria that is also highly variable among different E. coli strains. Altogether, our results suggest that these phages may represent an interesting alternative for the treatment of antibiotic-resistant E. coli. IMPORTANCE Urinary tract infections affect approximately 150 million people annually. The current antibiotic resistance crisis demands the development of novel therapeutic alternatives. Our results show that three novel phages, MLP1, MLP2, and MLP3 are able to infect both laboratory and multidrug-resistant clinical isolates of Escherichia coli. Since these phages (i) efficiently kill antibiotic-resistant clinical isolates of uropathogenic Escherichia coli (UPEC), (ii) recognize different portions of the LPS molecule, and (iii) are able to efficiently infect intestinal pathogenic Escherichia coli hosts, we believe that these novel phages are good candidates to be used as a therapeutic alternative to treat antibiotic-resistant E. coli strains generating urinary tract and/or intestinal infections.

The Promoter of the Immune-Modulating Gene TIR-Containing Protein C of the Uropathogenic Escherichia coli Strain CFT073 Reacts to the Pathogen's Environment.

The TIR-containing protein C (TcpC) of the uropathogenic Escherichia coli strain CFT073 modulates innate immunity by interfering with the Toll-like receptor and NALP3 inflammasome signaling cascade. During a urinary tract infection the pathogen encounters epithelial and innate immune cells and replicates by several orders of magnitude. We therefore analyzed whether these cell types and also the density of the pathogen would induce the recently defined promoter of the CFT073 tcpC gene to, in time, dampen innate immune responses. Using reporter constructs we found that the uroepithelial cell line T24/83 and the monocytic cell line THP-1 induced the tcpC promoter. Differentiation of monocytic THP-1 cells to macrophages increased their potential to switch on the promoter. Cell-associated CFT073 displayed the highest promoter activity. Since potassium represents the most abundant intracellular ion and is secreted to induce the NLRP3 inflammasome, we tested its ability to activate the tcpC promoter. Potassium induced the promoter with high efficiency. Sodium, which is enriched in the renal cortex generating an antibacterial hypersalinity, also induced the tcpC promoter. Finally, the bacterial density modulated the tcpC promoter activity. In the search for promoter-regulating proteins, we found that the DNA-binding protein H-NS dampens the promoter activity. Taken together, different cell types and salts, present in the kidney, are able to induce the tcpC promoter and might explain the mechanism of TcpC induction during a kidney infection with uropathogenic E. coli strains.

Differential Afa/Dr Fimbriae Expression in the Multidrug-Resistant Escherichia coli ST131 Clone.

Many antibiotic resistant uropathogenic Escherichia coli (UPEC) strains belong to clones defined by their multilocus sequence type (ST), with ST131 being the most dominant. Although we have a good understanding of resistance development to fluoroquinolones and third-generation cephalosporins by ST131, our understanding of the virulence repertoire that has contributed to its global dissemination is limited. Here we show that the genes encoding Afa/Dr fimbriae, a group of adhesins strongly associated with UPEC that cause gestational pyelonephritis and recurrent cystitis, are found in approximately one third of all ST131 strains. Sequence comparison of the AfaE adhesin protein revealed a unique allelic variant carried by 82.9% of afa-positive ST131 strains. We identify the afa regulatory region as a hotspot for the integration of insertion sequence (IS) elements, all but one of which alter afa transcription. Close investigation demonstrated that the integration of an IS1 element in the afa regulatory region leads to increased expression of Afa/Dr fimbriae, promoting enhanced adhesion to kidney epithelial cells and suggesting a mechanism for altered virulence. Finally, we provide evidence for a more widespread impact of IS1 on ST131 genome evolution, suggesting that IS dynamics contribute to strain level microevolution that impacts ST131 fitness. IMPORTANCE E. coli ST131 is the most common antibiotic resistant UPEC clone associated with human urinary tract and bloodstream infections. Understanding the features of ST131 that have driven its global dissemination remains a critical priority if we are to counter its increasing antibiotic resistance. Here, we utilized a large collection of ST131 isolates to investigate the prevalence, regulation, and function of Afa/Dr fimbriae, a well-characterized UPEC colonization and virulence factor. We show that the afa genes are found frequently in ST131 and demonstrate how the integration of IS elements in the afa regulatory region modulates Afa expression, presenting an example of altered virulence capacity. We also exploit a curated set of ST131 genomes to map the integration of the antibiotic resistance-associated IS1 element in the ST131 pangenome, providing evidence for its widespread impact on ST131 genome evolution.

Bladder infection with uropathogenic Escherichia coli increases the excitability of afferent neurons.

Urinary tract infections (UTIs) cause bladder hyperactivity and pelvic pain, but the underlying causes of these symptoms remain unknown. We investigated whether afferent sensitization contributes to the bladder overactivity and pain observed in mice suffering from experimentally induced bacterial cystitis. Inoculation of mouse bladders with the uropathogenic Escherichia coli strain UTI89 caused pelvic allodynia, increased voiding frequency, and prompted an acute inflammatory process marked by leukocytic infiltration and edema of the mucosa. Compared with controls, isolated bladder sensory neurons from UTI-treated mice exhibited a depolarized resting membrane potential, lower action potential threshold and rheobase, and increased firing in response to suprathreshold stimulation. To determine whether bacterial virulence factors can contribute to the sensitization of bladder afferents, neurons isolated from naïve mice were incubated with supernatants collected from bacterial cultures with or depleted of lipopolysaccharide (LPS). Supernatants containing LPS prompted the sensitization of bladder sensory neurons with both tetrodotoxin (TTX)-resistant and TTX-sensitive action potentials. However, bladder sensory neurons with TTX-sensitive action potentials were not affected by bacterial supernatants depleted of LPS. Unexpectedly, ultrapure LPS increased the excitability only of bladder sensory neurons with TTX-resistant action potentials, but the supplementation of supernatants depleted of LPS with ultrapure LPS resulted in the sensitization of both population of bladder sensory neurons. In summary, the results of our study indicate that multiple virulence factors released from UTI89 act on bladder sensory neurons to prompt their sensitization. These sensitized bladder sensory neurons mediate, at least in part, the bladder hyperactivity and pelvic pain seen in mice inoculated with UTI89.NEW & NOTEWORTHY Urinary tract infection (UTI) produced by uropathogenic Escherichia coli (UPEC) promotes sensitization of bladder afferent sensory neurons with tetrodotoxin-resistant and tetrodotoxin-sensitive action potentials. Lipopolysaccharide and other virulence factors produced by UPEC contribute to the sensitization of bladder afferents in UTI. In conclusion, sensitized afferents contribute to the voiding symptoms and pelvic pain present in mice bladder inoculated with UPEC.

Differences in recipient ability of uropathogenic Escherichia coli strains in relation with their pathogenic potential.

Conjugation is recognized as a mechanism driving dissemination of antibacterial resistances and virulence factors among bacteria. In the presented work conjugative transfer frequency into clinical uropathogenic Escherichia coli strains (UPEC) isolated from patients with symptomatic urinary tract infections was investigated. From 93 obtained UPEC strains only 29 were suitable for conjugation experiments with the plasmid pOX38, a well-known F-plasmid derivative. The study was focused on comparison of conjugation frequencies in plankton and biofilm, including comparison of conjugation frequencies in high and low biofilm biomass with their virulence potential. It was shown that the conjugation frequency depended on the biofilm biomass and was significantly higher in thin (OD580 < 0.3) than in thick biofilm (OD580 ≥ 0.3). Nonmetric multidimensional scaling analysis revealed that higher conjugation frequencies in plankton and biofilm were directly positively correlated with the sum of virulence-associated genes of the recipient strain and presence of multidrug antibiotic resistances. On the other hand, the sum of insensitivities to different bacteriocins was negatively correlated with an increase in the conjugative transfer level. Our results obtained hence indicate that the evolution of potentially more pathogenic strains via conjugation is depended on the strains' ability to be a "good" recipient in the conjugative transfer, possibly due to the ability to form thinner biofilms.

Human Renal Fibroblasts, but Not Renal Epithelial Cells, Induce IL-1ß Release during a Uropathogenic Escherichia coli Infection In Vitro.

Understanding how uropathogenic Escherichia coli (UPEC) modulates the immune response in the kidney is essential to prevent UPEC from reaching the bloodstream and causing urosepsis. The purpose of this study was to elucidate if renal fibroblasts can release IL-1ß during a UPEC infection and to investigate the mechanism behind the IL-1ß release. We found that the UPEC strain CFT073 induced an increased IL-1ß and LDH release from renal fibroblasts, but not from renal epithelial cells. The UPEC-induced IL-1ß release was found to be NLRP3, caspase-1, caspase-4, ERK 1/2, cathepsin B and serine protease dependent in renal fibroblasts. We also found that the UPEC virulence factor α-hemolysin was necessary for IL-1ß release. Conditioned medium from caspase-1, caspase-4 and NLRP3-deficient renal fibroblasts mediated an increased reactive oxygen species production from neutrophils, but reduced UPEC phagocytosis. Taken together, our study demonstrates that renal fibroblasts, but not renal epithelial cells, release IL-1ß during a UPEC infection. This suggest that renal fibroblasts are vital immunoreactive cells and not only structural cells that produce and regulate the extracellular matrix.

Osteopontin Deficiency Ameliorates Prostatic Fibrosis and Inflammation.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

Differences of virulence factors, and antimicrobial susceptibility according to phylogenetic group in uropathogenic Escherichia coli strains isolated from Korean patients.

BACKGROUND: Escherichia coli is among the most common uropathogens. Increased antibiotic resistance in Gram negative bacilli is global concern. Alternative therapeutic options including vaccines against uropathogenic E. coli (UPEC) have been developed. In this study, we compared the genotypic characteristics and antimicrobial susceptibility of UPEC according to phylogenetic groups. METHODS: We retrospectively reviewed the medical records of pyelonephritis patients with UPEC between February 2015 and June 2018. The study was conducted at a medical center in Korea. We compared the clinical and genotypic characteristics of UPEC according to phylogenetic groups. The phylogenetic groups and 29 virulence factors were identified using multiplex polymerase chain reaction. RESULTS: Phylogenetic group analysis revealed that most uropathogenic E. coli belonged to groups B2 and D: B2 (276, 77.7%), D (62, 17.5%), B1 (12, 3.4%), and A (5, 1.4%). Among the virulence factors, fyuA, fimH, traT, iutA, papG allele II, and papC were the most frequently observed. Phylogenetic group B2 was more closely related to virulence factors, including fimH, sfa/focED, focG, hlyA, cnf1, fyuA, and PAI, than group D. Groups B2 and D showed similar clinical presentations and complications. Group B2 had mostly healthcare-associated infections and antimicrobial resistance. Group D mostly had community-acquired infections. The K1 serotype was prevalent in group B2, and K5 was the most prevalent in group D. CONCLUSIONS: Phylogenetic group B2 had more proportions and types of virulence factors than group D. Group B2 showed a high presentation of virulence factors related to adhesions and toxins. An increased presentation of antimicrobial resistance and healthcare-associated infections was also noted. Considering the genetic characteristics of UPEC, alternative therapeutic options targeting frequent virulence factors might be considered in addition to antibiotics.

A previously uncharacterized two-component signaling system in uropathogenic Escherichia coli coordinates protection against host-derived oxidative stress with activation of hemolysin-mediated host cell pyroptosis.

Uropathogenic Escherichia coli (UPEC) deploy an array of virulence factors to successfully establish urinary tract infections. Hemolysin is a pore-forming toxin, and its expression correlates with the severity of UPEC infection. Two-component signaling systems (TCSs) are a major mechanism by which bacteria sense environmental cues and respond by initiating adaptive responses. Here, we began this study by characterizing a novel TCS (C3564/C3565, herein renamed orhK/orhR for oxidative resistance and hemolysis kinase/regulator) that is encoded on a UPEC pathogenicity island, using bioinformatic and biochemical approaches. A prevalence analysis indicates that orhK/orhR is highly associated with the UPEC pathotype, and it rarely occurs in other E. coli pathotypes tested. We then demonstrated that OrhK/OrhR directly activates the expression of a putative methionine sulfoxide reductase system (C3566/C3567) and hemolysin (HlyA) in response to host-derived hydrogen peroxide (H2O2) exposure. OrhK/OrhR increases UPEC resistance to H2O2 in vitro and survival in macrophages in cell culture via C3566/C3567. Additionally, OrhK/OrhR mediates hemolysin-induced renal epithelial cell and macrophage death via a pyroptosis pathway. Reducing intracellular H2O2 production by a chemical inhibitor impaired OrhK/OrhR-mediated activation of c3566-c3567 and hlyA. We also uncovered that UPEC links the two key virulence traits by cotranscribing the c3566-c3567 and hlyCABD operons. Taken together, our data suggest a paradigm in which a signal transduction system coordinates both bacterial pathogen defensive and offensive traits in the presence of host-derived signals; and this exquisite mechanism likely contributes to hemolysin-induced severe pathological outcomes.

Escherichia coli type-1 fimbriae are critical to overcome initial bottlenecks of infection upon low-dose inoculation in a porcine model of cystitis.

Most uropathogenic Escherichia coli (UPEC) express type-1 fimbriae (T1F), a key virulence factor for urinary tract infection (UTI) in mice. Evidence that conclusively associates this pilus with uropathogenesis in humans has, however, been difficult to obtain. We used an experimental porcine model of cystitis to assess the role of T1F in larger mammals more closely related to humans. Thirty-one pigs were infected with UPEC strain UTI89 or its T1F deficient mutant, UTI89ΔfimH, at inoculum titres of 102 to 108 colony forming units per millilitre. Urine and blood samples were collected and analysed 7 and 14 days post-inoculation, and whole bladders were removed at day 14 and analysed for uroepithelium-associated UPEC. All animals were consistently infected and reached high urine titres independent of inoculum titre. UTI89ΔfimH successfully colonized the bladders of 1/6 pigs compared to 6/6 for the wild-type strain. Intracellular UPEC were detectable in low numbers in whole bladder explants. In conclusion, low doses of UPEC are able to establish robust infections in pigs, similar to what is presumed in humans. T1F are critical for UPEC to surpass initial bottlenecks during infection but may be dispensable once infection is established. While supporting the conclusions from mice studies regarding a general importance of T1F in successfully infecting the host, the porcine UTI models' natural high, more human-like, susceptibility to infection, allowed us to demonstrate a pivotal role of T1F in initial establishment of infection upon a realistic low-inoculum introduction of UPEC in the bladder.