Birong Li

SOS regulatory elements are essential for UPEC pathogenesis.

Epithelial cells are highly regarded as the first line of defense against microorganisms, but the mechanisms used to control bacterial diseases are poorly understood. A component of the DNA damage repair regulon, SulA, is essential for UPEC virulence in a mouse model for human urinary tract infection, suggesting that DNA damage is a key mediator in the primary control of pathogens within the epithelium. In this study, we examine the role of DNA damage repair regulators in the intracellular lifestyle of UPEC within superficial bladder epithelial cells. LexA and RecA coordinate various operons for repair of DNA damage due to exogenous and endogenous agents and are known regulators of sulA. UPEC strains defective in regulation of the SOS response mediated by RecA and LexA display attenuated virulence in immunocompetent mice within the first 6 h post infection. RecA and LexA regulation of the SOS regulon is dispensable in immunocompromised mice. These data suggest that epithelial cells produce sufficient levels of DNA damaging agents, such that the bacterial DNA damage repair response is essential, as a means to control invading bacteria. Since many pathogens interact with the epithelium before exposure to professional phagocytes, it is likely that adaptation to oxidative radicals during intracellular growth provides additional protection from killing by innate immune phagocytes.

Ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract

Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified Ribonuclease 6 (RNase 6) as the RNase A Superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are up-regulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14+ monocytes and murine bone marrow derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility.

Expression and antimicrobial function of beta-defensin 1 in the lower urinary tract.

Beta defensins (BDs) are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1) expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC) in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1 -/-) mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1 -/- and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract.

Aberrant community architecture and attenuated persistence of uropathogenic Escherichia coli in the absence of individual IHF subunits.

Uropathogenic Escherichia coli (UPEC) utilizes a complex community-based developmental pathway for growth within superficial epithelial cells of the bladder during cystitis. Extracellular DNA (eDNA) is a common matrix component of organized bacterial communities. Integration host factor (IHF) is a heterodimeric protein that binds to double-stranded DNA and produces a hairpin bend. IHF-dependent DNA architectural changes act both intrabacterially and extrabacterially to regulate gene expression and community stability, respectively. We demonstrate that both IHF subunits are required for efficient colonization of the bladder, but are dispensable for early colonization of the kidney. The community architecture of the intracellular bacterial communities (IBCs) is quantitatively different in the absence of either IhfA or IhfB in the murine model for human urinary tract infection (UTI). Restoration of Type 1 pili by ectopic production does not restore colonization in the absence of IhfA, but partially compensates in the absence of IhfB. Furthermore, we describe a binding site for IHF that is upstream of the operon that encodes for the P-pilus. Taken together, these data suggest that both IHF and its constituent subunits (independent of the heterodimer), are able to participate in multiple aspects of the UPEC pathogenic lifestyle, and may have utility as a target for treatment of bacterial cystitis.

The Interaction between Enterobacteriaceae and Calcium Oxalate Deposits.

Background The role of calcium oxalate crystals and deposits in UTI pathogenesis has not been established. The objectives of this study were to identify bacteria present in pediatric urolithiasis and, using in vitro and in vivo models, to determine the relevance of calcium oxalate deposits during experimental pyelonephritis. Methods Pediatric kidney stones and urine were collected and both cultured and sequenced for bacteria. Bacterial adhesion to calcium oxalate was compared. Murine kidney calcium oxalate deposits were induced by intraperitoneal glyoxalate injection and kidneys were transurethrally inoculated with uropathogenic Escherichia coli to induce pyelonephritis Results E. coli of the family Enterobacteriaceae was identified in patients by calcium oxalate stone culture. Additionally Enterobacteriaceae DNA was sequenced from multiple calcium oxalate kidney stones. E. coli selectively aggregated on and around calcium oxalate monohydrate crystals. Mice inoculated with glyoxalate and uropathogenic E. coli had higher bacterial burdens, increased kidney calcium oxalate deposits and an increased kidney innate immune response compared to mice with only calcium oxalate deposits or only pyelonephritis. Conclusions In a murine model, the presence of calcium oxalate deposits increases pyelonephritis risk, likely due to preferential aggregation of bacteria on and around calcium oxalate crystals. When both calcium oxalate deposits and uropathogenic bacteria were present, calcium oxalate deposit number increased along with renal gene transcription of inner stone core matrix proteins increased. Therefore renal calcium oxalate deposits may be a modifiable risk factor for infections of the kidney and urinary tract. Furthermore, bacteria may be present in calcium oxalate deposits and potentially contribute to calcium oxalate renal disease.

Comparison of the microbiological milieu of patients randomized to either hydrophilic or conventional PVC catheters for clean intermittent catheterization.

INTRODUCTION Control of bacteriuria is problematic in patients who perform clean intermittent catheterization for management of neurogenic bladder. This population is often burdened with multiple urinary tract infections (UTIs), placing them at increased risk of end-stage renal disease. Hydrophilic catheters are a potential way to improve smooth and clean insertion, reduce disruption of the urothelium, and reduce bacterial colonization. OBJECTIVE The goal of the study was to compare the type and virulence of microorganisms recovered from the urine of patients that use either a hydrophilic or conventional polyvinyl chloride (PVC) catheter. METHODS Fifty patients with an underlying diagnosis of myelomeningocele were recruited for a 12-month prospective, randomized, investigator-blinded study. Twenty-five patients were allocated to the hydrophilic catheter intervention, and 25 continued use of a PVC catheter. Cultures were performed on urine obtained by catheterization at enrollment, and 3, 6, and 12 months. Bacterial species were assigned a designation as either potentially pathogenic or non-pathogenic. Escherichia coli isolates were the most predominant and were serotyped to further stratify the pathogenicity of the strains. Lastly, patients were surveyed at enrollment, and at the two later time points evaluating their current catheter for satisfaction. RESULTS A total of 232 different bacterial isolates were obtained from the 182 collected urine cultures. In addition, seven species were recovered from the two UTI reported during the study period. Bacterial growth was not detected in 29 of the samples (16%). Although not statistically significant, collectively there was a 40% decrease in the average number of potentially pathogenic species recovered from those patients using hydrophilic catheters (0.81 per urine sample) compared with PVC catheter use (1.24 per urine sample). Since E. coli species can be either pathogenic or non-pathogenic, we examined 14 of the most commonly implicated serotypes associated with uropathogenic E. coli (UPEC). We identified the serotype of 57% of E. coli strains recovered. There was a trend for the recovery of fewer UPEC serotypes from the hydrophilic group (54% hydrophilic verses 64% PVC), further suggesting that the catheter type may influence the microbiological milieu. Although no significant differences were reported in patient satisfaction, almost half of the patients from the hydrophilic catheter cohort continue use of this type of catheter. CONCLUSIONS There was a trend for reduced recovery of potentially pathogenic bacteria with the use of hydrophilic catheters. The reduction in potentially pathogenic species will reduce antibiotic exposures and some patients may prefer the comfort hydrophilic catheters provide.

New paradigms of urinary tract infections: Implications for patient management

Urinary tract infections (UTIs) represent one of the most commonly acquired diseases among the general population as well as hospital in-patients, yet remain difficult to effectively and consistently treat. High rates of recurrence, anatomic abnormalities, and functional disturbances of the urinary tract all contribute to the difficulty in management of these infections. However, recent advances reveal important molecular and genetic factors that contribute to bacterial invasion and persistence in the urinary tract, particularly for the most common causative agent, uropathogenic Escherichia coli. Recent studies using animal models of experimental UTIs have recently provided mechanistic insight into the clinical observations that question the effectiveness of antibiotic therapy in treatment. Ultimately, continuing research will be necessary to identify the best targets for effective treatment of this costly and widespread infectious disease.

Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection

Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine 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 UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain.

Renal epithelial miR-205 expression correlates with disease severity in a mouse model of congenital obstructive nephropathy.

Background:Congenital obstructive nephropathy (CON) is a leading cause of pediatric chronic kidney disease (CKD). Despite optimal surgical and medical care, there is a high rate of CKD progression. Better understanding of molecular and cellular changes is needed to facilitate development of improved biomarkers and novel therapeutic approaches in CON.Methods:The megabladder (mgb) mouse is an animal model of CKD with impaired bladder emptying, hydronephrosis, and progressive renal injury. In this study, we characterize a particular microRNA, miR-205, whose expression changes with the degree of hydronephrosis in the mgb−/− kidney.Results:Expression of miR-205 is progressively increased in the adult mgb−/− mouse with worsening severity of hydronephrosis. miR-205 expression is correlated with altered expression of cytokeratins and uroplakins, which are markers of cellular differentiation in urothelium. We describe the spatial pattern of miR-205 expression, including increased expression in renal urothelium and novel miR-205 expression in medullary collecting duct epithelium in the congenitally obstructed kidney.Conclusion:miR-205 is increased with severity of CON and CKD in the mgb−/− mouse and may regulate urothelial differentiation.

Urine Stasis Predisposes to Urinary Tract Infection by an Opportunistic Uropathogen in the Megabladder (Mgb) Mouse.

Purpose Urinary stasis is a risk factor for recurrent urinary tract infection (UTI). Homozygous mutant Megabladder (Mgb-/-) mice exhibit incomplete bladder emptying as a consequence of congenital detrusor aplasia. We hypothesize that this predisposes Mgb-/- mice to spontaneous and experimental UTI. Methods Mgb-/-, Mgb+/-, and wild-type female mice underwent serial ultrasound and urine cultures at 4, 6, and 8 weeks to detect spontaneous UTI. Urine bacterial isolates were analyzed by Gram stain and speciated. Bladder stones were analyzed by x-ray diffractometry. Bladders and kidneys were subject to histologic analysis. The pathogenicity of coagulase-negative Staphylococcus (CONS) isolated from Mgb-/- urine was tested by transurethral administration to culture-negative Mgb-/- or wild-type animals. The contribution of urinary stasis to CONS susceptibility was evaluated by cutaneous vesicostomy in Mgb-/- mice. Results Mgb-/- mice develop spontaneous bacteriuria (42%) and struvite bladder stones (31%) by 8 weeks, findings absent in Mgb+/- and wild-type controls. CONS was cultured as a solitary isolate from Mgb-/- bladder stones. Bladders and kidneys from mice with struvite stones exhibit mucosal injury, inflammation, and fibrosis. These pathologic features of cystitis and pyelonephritis are replicated by transurethral inoculation of CONS in culture-negative Mgb-/- females, whereas wild-type animals are less susceptible to CONS colonization and organ injury. Cutaneous vesicostomy prior to CONS inoculation significantly reduces the quantity of CONS recovered from Mgb-/- urine, bladders, and kidneys. Conclusions CONS is an opportunistic uropathogen in the setting of urinary stasis, leading to enhanced UTI incidence and severity in Mgb-/- mice.