Evasion of toll-like receptor recognition by Escherichia coli is mediated via population level regulation of flagellin production.

Uropathogenic Escherichia coli is a major cause of urinary tract infections. Analysis of the innate immune response in immortalized urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defenses. A panel of 48 clinical uro-associated E. coli isolates recovered from either cystitis, pyelonephritis asymptomatic bacteriuria (ABU) or UTI-associated bacteraemia infections were characterized for motility and their ability to induce an innate response in urothelial cells stably transfected with a NF-κB luciferase reporter. Thirty-two isolates (67%) were identified as motile with strains recovered from cystitis patients exhibiting an uneven motility distribution pattern; seven of the cystitis isolates were associated with a > 5-fold increase in NF-κB signaling. To explore whether the NF-κB signaling response reflected antigenic variation, flagellin was purified from 14 different isolates. Purified flagellin filaments generated comparable NF-κB signaling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. Investigations also argued that neither TLR4 dependent recognition of bacterial lipopolysaccharide nor growth fitness of the isolates played key roles in leading to the variable host response. To determine the roles, if any, of flagellar abundance in inducing these variable responses, flagellar hook numbers of a range of cystitis and ABU isolates were quantified. Images suggested that up to 60% of the isolate population exhibited flagella with the numbers averaging between 1 and 2 flagella per bacterial cell. These data suggest that selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility, but exploit population heterogeneity, which together function to prevent host TLR5 recognition and bacterial killing.

Multidrug-resistant Uro-associated Escherichia coli Populations and Recurrent Urinary Tract Infections in Patients Performing Clean Intermittent Self-catheterisation.

BACKGROUND: The AnTIC trial linked continuous low-dose antibiotic prophylaxis treatments to a lower incidence of symptomatic urinary tract infections (UTIs) among individuals performing clean intermittent self-catheterisation (CISC). OBJECTIVE: To explore potential mechanisms underlying the protective effects of low-dose antibiotic prophylaxis treatments, blood and urine samples and uro-associated Escherichia coli isolates from AnTIC participants were analysed. DESIGN SETTING AND PARTICIPANTS: Blood samples (n = 204) were analysed for TLR gene polymorphisms associated with UTI susceptibility and multiple urine samples (n = 558) were analysed for host urogenital responses. E.coli sequence data for 45 temporal isolates recovered from the urine samples of 16 trial participants in the prophylaxis (n = 9) and no-prophylaxis (n = 7) study arms, and characterised by multidrug resistance (MDR), were used to classify individual strains. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: TLR polymorphism data were analysed using Poisson regression. Concentrations of urine host defence markers were analysed using linear mixed-effects models, which accounted for repeated urine samples. RESULTS AND LIMITATIONS: Urine samples from CISC users, irrespective of antibiotic treatment regimens, were associated with robust urothelial innate responses. No links were identified between TLR genotype and CISC user susceptibility to recurrent UTIs. Microbiological study data were limited to the predominant MDR E. coli population; participants prescribed low-dose prophylactic antibiotics were predominantly colonised by a single uro-associated E. coli strain, while participants given acute antibiotic treatments were each colonised by more than one E. coli strain. CONCLUSIONS: Antibiotic treatments did not impact urogenital responses to infection in CISC users. Host genetics in terms of TLR polymorphisms played no role in determining CISC user susceptibility to or protection from recurrent UTIs. Prophylactic antibiotic treatments associated with MDR E. coli were associated with colonisation by stable uro-associated E. coli genotypes. PATIENT SUMMARY: Our findings show that the natural urogenital defences of clean intermittent self-catheterisation (CISC) users were not impacted by antibiotic treatments. For some CISC users, prophylaxis with low-dose antibiotics selected for a stable, predominantly, Esherichia coli rich uromicrobiota.

Topical Estrogen Treatment Augments the Vaginal Response to Escherichia coli Flagellin.

The female climacteric or menopausal process characterised by reduced estrogen, associates with an increased risk of recurrent urinary tract infections (rUTIs) linked to uropathogenic Escherichia coli (UPEC). Clinically, topical vaginal estrogen treatment has a prophylactic effect against such infections. The aim of this study was to investigate, in vitro, the effects of a topical estrogen treatment on vaginal epithelial responses following challenge with E.coli flagellin mimicking an UPEC challenge. Immortalised vaginal epithelial cells (VK2 E6/E7), modelling the vaginal epithelium were treated with either 4 nM 17ß-estradiol (E) for seven days, 50 ng/ml E.coli flagellin (F) for 12 h, or 4 nM 17ß-estradiol plus 50 ng/ml flagellin (E + F(12 h)). RNA was analysed by microarray gene profiling using the Illumina HumanHT-12 v 4 Expression Beadchip. Following E + F treatments expression of genes encoding host defence molecules including DEFß4A, DEFB103A, LCN2 as well as those associated with keratinisation eg CNFN and SPRR family genes were significantly enhanced (P < 0.05) compared to either E or F treatments alone. Mutation of estrogen responsive elements (EREs) identified in the DEFß4 gene promoter abolished the augmented gene expression suggesting estrogen functioned directly through a regulatory mechanism involving ESR1/2. Ingenuity pathway analyses also suggested the pro-inflammatory cytokine IL-17A to regulate the vaginal host defences during infection. Pre-treating VK2 E6/E7 cells with estrogen (4 nM) and challenging with 1L-17A & F (12 h) significantly enhanced DEFß4, DEF103A and S100A7 expression (P < 0.05). Origins of vaginal IL-17 in vivo remain unclear, but patient biopsies support γδ T cells located within the vaginal epithelium. These data suggest that the vaginal antimicrobial response induced by flagellin activation of Toll-like Receptor 5 cell signalling is augmented following topical estrogen application.

Elevated urine IL-10 concentrations associate with Escherichia coli persistence in older patients susceptible to recurrent urinary tract infections.

BACKGROUND: Age is a significant risk factor for recurrent urinary tract (rUTI) infections, but the clinical picture is often confused in older patients who also present with asymptomatic bacteriuria (ASB). Yet, how bacteriuria establishes in such patients and the factors underpinning and/or driving symptomatic UTI episodes are still not understood. To explore this further a pilot study was completed in which 30 male and female community based older patients (mean age 75y) presenting clinically with ASB / rUTIs and 15 control volunteers (72y) were recruited and monitored for up to 6 months. During this period symptomatic UTI episodes were recorded and urines collected for urinary cytokine and uropathogenic Escherichia coli (UPEC) analyses. RESULTS: Eighty-six per cent of patients carried E. coli (102 ≥ 105 CFU/ml urine) at some point throughout the study and molecular typing identified 26 different E. coli strains in total. Analyses of urine samples for ten different cytokines identified substantial patient variability. However, when examined longitudinally the pro-inflammatory markers, IL-1 and IL-8, and the anti-inflammatory markers, IL-5 and IL-10, were significantly different in the patient urines compared to those of the controls (P < 0.0001). Furthermore, analysing the cytokine data of the rUTI susceptible cohort in relation to E. coli carriage, showed the mean IL-10 concentration to be significantly elevated (P = 0.04), in patients displaying E. coli numbers ≥105 CFU/ml. CONCLUSIONS: These pilot study data suggest that bacteriuria, characteristic of older rUTI patients, is associated with an immune homeostasis in the urinary tract involving the synthesis and activities of the pro and anti-inflammatory cytokines IL-1, IL-5, IL-8 and IL-10. Data also suggests a role for IL-10 in regulating bacterial persistence.

Bladder-Drained Pancreas Transplantation: Urothelial Innate Defenses and Urinary Track Infection Susceptibility.

BACKGROUND: Pancreas transplantation restores insulin secretion in type 1 diabetes mellitus. The graft also produces exocrine secretions that can be drained enterically (enteric drainage [ED]) or via the bladder (bladder drainage [BD]). We suggest that in BD transplants, such secretions destroy bladder innate immunity, specifically host defense peptides/proteins (HDPs), which increases patient susceptibility to recurrent urinary tract infections (rUTIs). MATERIALS AND METHODS: BD and ED patient records were reviewed retrospectively for UTIs. Urine samples from ED and BD transplant recipients were analyzed for pH, the HDPs ß-defensin 2 (HBD2) and lipocalin-2, and amylase concentrations. In vitro, bacterial growth curves and antimicrobial assays were used to evaluate the effects of pH, HBD2, and HBD2 + pancreatic digestive enzymes (pancreatin) on uropathogenic Escherichia coli (UPEC) survival and growth. RESULTS: Urinalysis revealed a significant difference in pH between the BD and ED cohorts (7.2 ± 0.8 versus 6.7 ± 0.8; P = 0.012). Urinary HDPs were measured and BD, but not ED, lipocalin-2 concentrations were significantly decreased compared with those of diabetics awaiting transplant (P < 0.05). In vitro, an alkaline environment, pH 8.0, concomitant with the urine of the patient who underwent BD transplantation, significantly reduced UPEC growth (P < 0.05); addition of pancreatin to the growth medium was associated with a significant increase (P < 0.001) in growth rate. Antimicrobial data suggested significant UPEC killing in the presence of HBD2 (P < 0.01), but not in the presence of HBD2 + pancreatin (>12,500 amylase units). CONCLUSIONS: These in vivo and in vitro data suggest that BD pancreatic exocrine secretions inactivate the bladder innate defenses, which facilitate UPEC growth and underpins the increased susceptibility of patients who underwent BD pancreas transplantation to rUTIs.

Gene expression of AvBD6-10 in broiler chickens is independent of AvBD6, 9, and 10 peptide potency.

The Avian ß-defensin (AvBD) gene cluster contains fourteen genes; within this, two groups (AvBD6/7 and AvBD8 -10) encode charged peptides of >+5 (AvBD6/7), indicative of potent microbial killing activities, and ≤+4 (AvBD8-10), suggestive of reduced antimicrobial activities. Chicken broiler gut tissues are constantly exposed to microbes in the form of commensal bacteria. This study examined whether tissue expression patterns of AvBD6-10 reflected microbial exposure and the encoded peptides a functional antimicrobial hierarchy. Gut AvBD6-10 gene expression was observed in hatch to day 21 birds, although the AvBD8-10 profiles were eclipsed by those detected in the liver and kidney tissues. In vitro challenges of chicken CHCC-OU2 cells using the gut commensal Lactobacillus johnsonii (104 CFU) did not significantly affect AvBD8-10 gene expression patterns, although upregulation (P < 0.05) of IL-Iß gene expression was observed. Similarly, in response to Bacteriodes doreii, IL-Iß and IL-6 gene upregulation were detected (P < 0.05), but AvBD10 gene expression remained unaffected. These data suggested that AvBD8-10 gene expression was not induced by commensal gut bacteria. Bacterial time-kill assays employing recombinant (r)AvBD6, 9 and 10 peptides (0.5µM - 12µM), indicated an antimicrobial hierarchy, linked to charge, of AvBD6 > AvBD9 > AvBD10 against Escherichia coli, but AvBD10 > AvBD9 > AvBD6 using Enterococcus faecalis. rAvBD10, selected due to its reduced cationic charge was, using CHCC-OU2 cells, investigated for cell proliferation and wound healing properties, but none were observed. These data suggest that in healthy broiler chicken tissues AvBD6/7 and AvBD8-10 gene expression profiles are independent of the in vitro antimicrobial hierarchies of the encoded AvBD6, 9 and 10 peptides.

High molecular weight hyaluronic acid: a two-pronged protectant against infection of the urogenital tract?

OBJECTIVES: Recurrent urinary tract infections are associated with uropathogenic Escherichia coli (UPEC) ascending and infecting the urinary tract. Antibiotics provide only symptomatic relief, not prevent recurrence. Clinical evidence suggests that intravesical glycosaminoglycan therapy, such as hyaluronic acid (HA), helps reduce UTI recurrence. This has been investigated here using in vitro systems modelling the urogenital tract tissues. METHODS: RT4 bladder cells were preconditioned with high molecular weight HA (> 1500 kDa) at 2 mg mL-1 and challenged with UPEC to analyse barrier protection and bacterial adherence. Untreated and HA-preconditioned VK2 E6/E7 vaginal cells were challenged with E. coli flagellin (50 ng mL-1) to mimic bacterial challenge, and media analysed for lipocalin-2, human ß-defensin 2 and interleukin-8 by ELISA. Experiments were repeated after siRNA knockdown of Toll-like receptors 2, 4 and 5, and CD44 to investigate signalling. RESULTS: Microscopic analyses showed reduced bacterial adherence and urothelial disruption with HA, suggesting that HA functions as a barrier protecting the epithelium from bacterial infection. Cells treated with HA and flagellin simultaneously produced more of the host antimicrobial peptide LCN2 and pro-inflammatory IL-8 (P < 0.05) compared to the no HA/flagellin challenges. Increased gene expression of DEFB4 (P < 0.05), but not the hBD2 peptide, was observed in the HA/flagellin-challenged cells. CONCLUSION: These data suggest that exogenous HA has potential to protect the urogenital epithelia from UPEC infection via a two-pronged approach that involves the physical enhancement of the epithelial barrier and augmentation of its innate immune response.

Targeting Deficiencies in the TLR5 Mediated Vaginal Response to Treat Female Recurrent Urinary Tract Infection.

The identification of the host defence peptides as target effectors in the innate defence of the uro-genital tract creates new translational possibilities for immunomodulatory therapies, specifically vaginal therapies to treat women suffering from rUTI, particularly those carrying the TLR5_C1174T SNP. Urinary tract infections (UTIs) are a microbial disease reported worldwide. Women are particularly susceptible with many suffering debilitating recurrent (r) infections. Treatment is by antibiotics, but such therapy is linked to antibiotic resistance and re-infection. This study explored the innate protective mechanisms of the urogenital tract with the aim of boosting such defences therapeutically. Modelling UTIs in vitro, human vaginal and bladder epithelial cells were challenged with uropathogenic Escherichia coli (CFT073) and microbial PAMPs including flagellin, LPS and peptidoglycan. Flagellin functioning via the TLR5/NFκB pathway was identified as the key UPEC virulence factor causing a significant increase (P < 0.05) in the production of the host-defence peptide (HDP), BD2. BD2-depleted urine samples from bladder infected mice supported increased UPEC growth, strengthening the significance of the HDPs in protecting the urogenital tissues from infection. Clinically, vaginal-douche BD2 concentrations were reduced (p < 0.05) in women suffering rUTIs, compared to age-matched healthy controls with concentrations further decreased (p < 0.05) in a TLR5392Stop SNP rUTI subgroup. Topical vaginal estrogen treatment increased (p < 0.001) BD2 concentrations in all women, including those carrying the SNP. These data identify therapeutic and antibiotic sparing roles for vaginal immunomodulatory agents that specifically target HDP induction, facilitate bacterial killing and disrupt the UPEC infection cycle.

AvBD1 nucleotide polymorphisms, peptide antimicrobial activities and microbial colonisation of the broiler chicken gut.

BACKGROUND: The importance of poultry as a global source of protein underpins the chicken genome and associated SNP data as key tools in selecting and breeding healthy robust birds with improved disease resistance. SNPs affecting host peptides involved in the innate defences tend to be rare, but three non-synonymous SNPs in the avian ß-defensin (AvBD1) gene encoding the variant peptides NYH, SSY and NYY were identified that segregated specifically to three lines of commercial broiler chickens Line X (LX), Line Y(LY) and Line Z. The impacts of such amino acid changes on peptide antimicrobial properties were analysed in vitro and described in relation to the caecal microbiota and gut health of LX and LY birds. RESULTS: Time-kill and radial immune diffusion assays indicated all three peptides to have antimicrobial properties against gram negative and positive bacteria with a hierarchy of NYH > SSY > NYY. Calcein leakage assays supported AvBD1 NYH as the most potent membrane permeabilising agent although no significant differences in secondary structure were identified to explain this. However, distinct claw regions, identified by 3D modelling and proposed to play a key role in microbial membrane attachment, and permeation, were more distinct in the NYH model. In vivo AvBD1 synthesis was detected in the bird gut epithelia. Analyses of the caecal gut microbiota of young day 4 birds suggested trends in Lactobacilli sp. colonisation at days 4 (9% LX vs × 30% LY) and 28 (20% LX vs 12% LY) respectively, but these were not statistically significant (P > 0.05). CONCLUSION: Amino acid changes altering the killing capacity of the AvBD1 peptide were associated with two different bird lines, but such changes did not impact significantly on caecal gut microbiota.

Towards Profiles of Resistance Development and Toxicity for the Small Cationic Hexapeptide RWRWRW-NH2.

RWRWRW-NH2 (MP196) is an amphipathic hexapeptide that targets the bacterial cytoplasmic membrane and inhibits cellular respiration and cell wall synthesis. In previous studies it showed promising activity against Gram-positive bacteria and no significant cytotoxicity or hemolysis. MP196 is therefore used as lead structure for developing more potent antibiotic derivatives. Here we present a more comprehensive study on the parent peptide MP196 with regard to clinically relevant parameters. We found that MP196 acts rapidly bactericidal killing 97% of initial CFU within 10 min at two times MIC. We were unable to detect resistance in standard 24 and 48 h resistance frequency assays. However, MP196 was effective against some but not all MRSA and VISA strains. Serum binding of MP196 was intermediate and we confirmed its low toxicity against mammalian cell lines. MP196 did neither induce NFκB activation nor cause an increase in IL8 levels at 250 µg/mL, and no IgE-dependent activation of basophil granulocytes was detected at 500 µg/mL. Yet, MP196 demonstrated acute toxicity in mice upon injection into the blood stream. Phase contrast microscopy of mouse blood treated with MP196 revealed a shrinking of erythrocytes at 250 µg/mL and severe morphological changes and lysis of erythrocytes at 500 µg/mL. These data suggest that MP196 derivatization directed at further lowering hemolysis could be instrumental in overcoming acute toxicity. The assessment of hemolysis is a critical step in the evaluation of the clinical potential of promising antimicrobial peptides and should be accompanied by microscopy-based morphological analysis of blood cells.

Effects of rearing environment on the gut antimicrobial responses of two broiler chicken lines.

To reduce the risk of enteric disease in poultry, knowledge of how bird gut innate defences mature with age while also responding to different rearing environments is necessary. In this study the gut innate responses of two phylogenetically distinct lines of poultry raised from hatch to 35days, in conditions mimicing high hygiene (HH) and low hygiene (LH) rearing environments, were compared. Analyses focussed on the proximal gut antimicrobial activities and the duodenal and caecal AvBD1, 4 and 10 defensin profiles. Variability in microbial killing was observed between individual birds in each of the two lines at all ages, but samples from day 0 birds (hatch) of both lines exhibited marked killing properties, Line X: 19±11% (SEM) and Line Y: 8.5±12% (SEM). By day 7 a relaxation in killing was observed with bacterial survival increased from 3 (Line Y (LY)) to 11 (Line X (LX)) fold in birds reared in the HH environment. A less marked response was observed in the LH environment and delayed until day 14. At day 35 the gut antimicrobial properties of the two lines were comparable. The AvBD 1, 4 and 10 data relating to the duodenal and caecal tissues of day 0, 7 and 35 birds LX and LY birds revealed gene expression trends specific to each line and to the different rearing environments although the data were confounded by inter-individual variability. In summary elevated AvBD1 duodenal expression was detected in day 0 and day 7 LX, but not LY birds, maintained in LH environments; Line X and Y duodenal AvBD4 profiles were detected in day 7 birds reared in both environments although duodenal AvBD10 expression was less sensitive to bird age and rearing background. Caecal AvBD1 expression was particularly evident in newly hatched birds. These data suggest that proximal gut antimicrobial activity is related to the bird rearing environments although the roles of the AvBDs in such activities require further investigation.

An investigation of mitochondrial haplogroups in autism.

Family and twin studies provide strong evidence of a major genetic influence in autism, but the underlying gene defects have yet to be characterized. The mothers of boys with autism share autistic traits, raising the possibility of a maternally inherited factor. Mitochondrial DNA (mtDNA) is almost exclusively inherited down the maternal line. We therefore explored the possibility that a particular mtDNA lineage contributes to the risk of developing autism. The mtDNA haplogroup was determined in 162 autism probands, and compared to two sets of population controls. Results show no compelling evidence of an association of any mitochondrial haplogroup in autism.

Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background.

Leber hereditary optic neuropathy (LHON) is due primarily to one of three common point mutations of mitochondrial DNA (mtDNA), but the incomplete penetrance implicates additional genetic or environmental factors in the pathophysiology of the disorder. Both the 11778G-->A and 14484T-->C LHON mutations are preferentially found on a specific mtDNA genetic background, but 3460G-->A is not. However, there is no clear evidence that any background influences clinical penetrance in any of these mutations. By studying 3,613 subjects from 159 LHON-affected pedigrees, we show that the risk of visual failure is greater when the 11778G-->A or 14484T-->C mutations are present in specific subgroups of haplogroup J (J2 for 11778G-->A and J1 for 14484T-->C) and when the 3460G-->A mutation is present in haplogroup K. By contrast, the risk of visual failure is significantly less when 11778G-->A occurs in haplogroup H. Substitutions on MTCYB provide an explanation for these findings, which demonstrate that common genetic variants have a marked effect on the expression of an ostensibly monogenic mtDNA disorder.