Project description:Antimicrobial peptides (AMPs) serve key proposed roles in defending the urinary tract against invading uropathogens, but individual AMPs bearing greatest responsibility for these functions remain largely unknown. We identified RegIIIγ as the most transcriptionally upregulated AMP in bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with cystitis and pyelonephritis exhibit increased urine levels of the orthologous HIP/PAP protein. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. RegIIIγ knockout and control urinary tracts contain comparable bacterial burden following experimental inoculation of UPEC as well as Gram-positive uropathogens. Thus, while RegIIIγ and HIP/PAP expression occurs in human and murine UTI, their specific functions in the urinary tract remain uncertain.
Project description:The NLRP3 inflammasome, estrogen and antimicrobial peptides have all been emphasised to have a vital role in the protection of the bladder urothelium. However, the interdependence between these protective factors during a bladder infection is currently unknown. Our aim was to investigate the role of NLRP3 in regulation of antimicrobial peptides and estrogen signaling in bladder epithelial cells during a UPEC infection. Human bladder epithelial cells and CRISPR/Cas9 generated NLRP3-deficient cells were stimulated with the UPEC strain CFT073 and estradiol. The gene and protein expression were evaluated with microarray, qRT-PCR, western blot and ELISA. Microarray results showed that the expression of most antimicrobial peptides was reduced in CFT073-infected NLRP3-deficient cells compared to Cas9 control cells. Conditioned medium from NLRP3-deficient cells also lost the ability to suppress CFT073 growth. Moreover, NLRP3-deficient cells had lower basal release of Beta-defensin-1, Beta-defensin-2 and RNase7. The ability of estradiol to induce an increased expression of antimicrobial peptides was also abrogated in NLRP3-deficient cells. The decreased antimicrobial peptide expression might be linked to the observed reduced expression and activity of estradiol receptor beta in NLRP3-deficient cells. This study suggests that NLRP3 may regulate the release and expression of antimicrobial peptides and affect estrogen signaling in bladder epithelial cells.
Project description:Uropathogenic Escherichia coli (UPEC) are the most common cause of urinary tract infection (UTI). UPEC normally reside in the intestine, and during establishment of UTI, it undergoes metabolic adaptations, first to urine and then upon tissue invasion to the bladder cell interior. In order to understand these adaptations, we used quantitative proteomic profiling to characterize protein expression of UPEC strain UTI89 growing in human urine and when inside J82 bladder cells. In order to facilitate detection of UPEC proteins over the excess amount of eukaryotic proteins in bladder cells, we developed a method where proteins from UTI89 grown in MOPS and urine was spiked-in to enhance detection of bacterial proteins. More than 2000 E. coli proteins were detected. During growth in urine, proteins associated with iron acquisition and several amino acid uptake and biosynthesis systems, in particular arginine metabolism, were significantly upregulated. During growth in J82 cells, proteins related to iron uptake and arginine metabolisms were upregulated together with proteins involved in sulphur compound turnover. Results suggested that UPEC experience a richer environment in bladder cells compared to urine. There was no direct correlation between upregulated proteins and proteins reported to be essential for infections, showing that upregulation during growth does not signify that the proteins are essential for growth under a condition.
Project description:Urinary tract infections (UTI) are common and recurrent. Both host genetics and UTI history impact susceptibility to recurrent UTI (rUTI) in women and in animal models. To identify shared patterns of host response that correlate with susceptibility, we investigated bladder inflammatory and transcriptional kinetics in acute and rUTI models. We found that TNFɑ signaling kinetics differed with mouse strain and infection history. Mice resistant to severe UTI/rUTI displayed a robust TNFɑ-dependent inflammation during the first 6 hours of acute cystitis, which waned by 24 hours; mice that are susceptible varied in their early responses but were prone to severe inflammation at 24 hours post-infection. Depletion of TNFɑ in an rUTI model revealed that early TNFɑ signaling promoted colonization resistance via exfoliation of infected bladder cells, but prolonged TNFɑ signaling exacerbated inflammation, thereby worsening infection. Host genetics and disease history impacts susceptibility by regulating the kinetics of a common TNFɑ pathway.
Project description:Urinary bladder wound healing is today pooorly chracterized. MicroRNAs are small non-coding RNA molecules with regulatory functions. In this study we aimed at identifying microRNAs expressed during bladder wound healing. We performed Affymetrix microRNA profiling of the rodent urinary bladder during healing of a surgically created wound.
Project description:Vesicoureteral reflux (VUR) is a common pediatric condition that predisposes children to renal damage after urinary tract infection (UTI). We profiled the urinary proteome of VUR patients with recurrent UTI and renal scarring to identify potential biomarkers characterizing this condition. Urine was obtained from 22 age-matched controls and 22 patients with low grade VUR (1-3 out of 5), renal scarring, and history of recurrent UTI. Proteins extracted from these samples were analyzed by mass spectrometry for protein identification and quantitation for comparison.
Project description:Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, with ~400 million cases across the globe each year. Uropathogenic E. coli (UPEC) is the major cause of UTI and increasingly associated with antibiotic resistance. This scenario has been worsened by the emergence and spread of pandemic UPEC sequence type 131 (ST131), a multidrug-resistant clone associated with extraordinarily high rates of infection. Here, we employed transposon-directed insertion site sequencing in combination with metabolomic profiling to identify genes and biochemical pathways required for growth and survival of the UPEC ST131 reference strain EC958 in human urine (HU). We identified 24 UPEC genes required for growth in HU, which mapped to diverse pathways involving small peptide, amino acid and nucleotide metabolism, the stringent response pathway, and lipopolysaccharide (LPS) biosynthesis. We also discovered a role for UPEC resistance to fluoride during growth in HU, most likely associated with fluoridation of drinking water. Complementary NMR-based metabolomics identified changes in a range of HU metabolites following UPEC growth, the most pronounced being L-lactate, which was utilized as a carbon source via the L-lactate dehydrogenase LldD. Using a mouse UTI model with mixed competitive infection experiments, we demonstrated a role for nucleotide metabolism and the stringent response in UPEC colonization of the mouse bladder. Together, our application of multiple omics technologies combined with different infection-relevant settings has uncovered new factors required for UPEC growth in HU, thus enhancing our understanding of this pivotal step in the UPEC infection pathway.