Proteus mirabilis UreR coordinates cellular functions required for urease activity.

A hallmark of Proteus mirabilis infection of the urinary tract is the formation of stones. The ability to induce urinary stone formation requires urease, a nickel metalloenzyme that hydrolyzes urea. This reaction produces ammonia as a byproduct, which can serve as a nitrogen source and weak base that raises the local pH. The resulting alkalinity induces the precipitation of ions to form stones. Transcriptional regulator UreR activates expression of urease genes in a urea-dependent manner. Thus, urease genes are highly expressed in the urinary tract where urea is abundant. Production of mature urease also requires the import of nickel into the cytoplasm and its incorporation into the urease apoenzyme. Urease accessory proteins primarily acquire nickel from one of two nickel transporters and facilitate incorporation of nickel to form mature urease. In this study, we performed a comprehensive RNA-seq to define the P. mirabilis urea-induced transcriptome as well as the UreR regulon. We identified UreR as the first defined regulator of nickel transport in P. mirabilis. We also offer evidence for the direct regulation of the Ynt nickel transporter by UreR. Using bioinformatics, we identified UreR-regulated urease loci in 15 Morganellaceae family species across three genera. Additionally, we located two mobilized UreR-regulated urease loci that also encode the ynt transporter, implying that UreR regulation of nickel transport is a conserved regulatory relationship. Our study demonstrates that UreR specifically regulates genes required to produce mature urease, an essential virulence factor for P. mirabilis uropathogenesis. IMPORTANCE: Catheter-associated urinary tract infections (CAUTIs) account for over 40% of acute nosocomial infections in the USA and generate $340 million in healthcare costs annually. A major causative agent of CAUTIs is Proteus mirabilis, an understudied Gram-negative pathogen noted for its ability to form urinary stones via the activity of urease. Urease mutants cannot induce stones and are attenuated in a murine UTI model, indicating this enzyme is essential to P. mirabilis pathogenesis. Transcriptional regulation of urease genes by UreR is well established; here, we expand the UreR regulon to include regulation of nickel import, a function required to produce mature urease. Furthermore, we reflect on the role of urea catalysis in P. mirabilis metabolism and provide evidence for its importance.

Data highlighting phenotypic diversity of urine-associated Escherichia coli isolates.

This article provides a reusable dataset describing detailed phenotypic and associated clinical parameters in n=303 clinical isolates of urinary Escherichia coli collected at Vanderbilt University Medical Center. De-identified clinical data collected with each isolate are detailed here and correlated to biofilm abundance and metabolomics data. Biofilm-abundance data were collected for each isolate under different in vitro conditions along with datasets quantifying biofilm abundance of each isolate under different conditions. Metabolomics data were collected from a subset of bacterial strains isolated from uncomplicated cases of cystitis or cases with no apparent symptoms accompanying colonization. For more insight, please see "Defining a Molecular Signature for Uropathogenic versus Urocolonizing Escherichia coli: The Status of the Field and New Clinical Opportunities" [1].

Defining a Molecular Signature for Uropathogenic versus Urocolonizing Escherichia coli: The Status of the Field and New Clinical Opportunities.

Urinary tract infections (UTIs) represent a major burden across the population, although key facets of their pathophysiology and host interaction remain unclear. Escherichia coli epitomizes these obstacles: this gram-negative bacterial species is the most prevalent agent of UTIs worldwide and can also colonize the urogenital tract in a phenomenon known as asymptomatic bacteriuria (ASB). Unfortunately, at the level of the individual E. coli strains, the relationship between UTI and ASB is poorly defined, confounding our understanding of microbial pathogenesis and strategies for clinical management. Unlike diarrheagenic pathotypes of E. coli, the definition of uropathogenic E. coli (UPEC) remains phenomenologic, without conserved phenotypes and known genetic determinants that rigorously distinguish UTI- and ASB-associated strains. This article provides a cross-disciplinary review of the current issues from interrelated mechanistic and diagnostic perspectives and describes new opportunities by which clinical resources can be leveraged to overcome molecular challenges. Specifically, we present our work harnessing a large collection of patient-derived isolates to identify features that do (and do not) distinguish UTI- from ASB-associated E. coli strains. Analyses of biofilm formation, previously reported to be higher in ASB strains, revealed extensive phenotypic heterogeneity that did not correlate with symptomatology. However, metabolomic experiments revealed distinct signatures between ASB and cystitis isolates, including in the purine pathway (previously shown to be critical for intracellular survival during acute infection). Together, these studies demonstrate how large-scale, wild-type approaches can help dissect the physiology of colonization versus infection, suggesting that the molecular definition of UPEC may rest at the level of global bacterial metabolism.

The Proton Pump Inhibitor Omeprazole Does Not Promote Clostridioides difficile Colonization in a Murine Model.

Proton pump inhibitor (PPI) use has been associated with microbiota alterations and susceptibility to Clostridioides difficile infections (CDIs) in humans. We assessed how PPI treatment alters the fecal microbiota and whether treatment promotes CDIs in a mouse model. Mice receiving a PPI treatment were gavaged with 40 mg of omeprazole per kg of body weight during a 7-day pretreatment phase, the day of C. difficile challenge, and the following 9 days. We found that mice treated with omeprazole were not colonized by C. difficile When omeprazole treatment was combined with a single clindamycin treatment, one cage of mice remained resistant to C. difficile colonization, while the other cage was colonized. Treating mice with only clindamycin followed by challenge resulted in C. difficile colonization. 16S rRNA gene sequencing analysis revealed that omeprazole had minimal impact on the structure of the murine microbiota throughout the 16 days of omeprazole exposure. These results suggest that omeprazole treatment alone is not sufficient to disrupt microbiota resistance to C. difficile infection in mice that are normally resistant in the absence of antibiotic treatment.IMPORTANCE Antibiotics are the primary risk factor for Clostridioides difficile infections (CDIs), but other factors may also increase a person's risk. In epidemiological studies, proton pump inhibitor (PPI) use has been associated with CDI incidence and recurrence. PPIs have also been associated with alterations in the human intestinal microbiota in observational and interventional studies. We evaluated the effects of the PPI omeprazole on the structure of the murine intestinal microbiota and its ability to disrupt colonization resistance to C. difficile We found omeprazole treatment had minimal impact on the murine fecal microbiota and did not promote C. difficile colonization. Further studies are needed to determine whether other factors contribute to the association between PPIs and CDIs seen in humans or whether aspects of murine physiology may limit its utility to test these types of hypotheses.

Respiratory Heterogeneity Shapes Biofilm Formation and Host Colonization in Uropathogenic Escherichia coli.

Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization to the biofilm community such that biofilm residents can benefit from the production of common goods while being protected from exogenous insults. Spatial organization is driven by the presence of chemical gradients, such as oxygen. Here we show that two quinol oxidases found in Escherichia coli and other bacteria organize along the biofilm oxygen gradient and that this spatially coordinated expression controls architectural integrity. Cytochrome bd, a high-affinity quinol oxidase required for aerobic respiration under hypoxic conditions, is the most abundantly expressed respiratory complex in the biofilm community. Depletion of the cytochrome bd-expressing subpopulation compromises biofilm complexity by reducing the abundance of secreted extracellular matrix as well as increasing cellular sensitivity to exogenous stresses. Interrogation of the distribution of quinol oxidases in the planktonic state revealed that ∼15% of the population expresses cytochrome bd at atmospheric oxygen concentration, and this population dominates during acute urinary tract infection. These data point toward a bet-hedging mechanism in which heterogeneous expression of respiratory complexes ensures respiratory plasticity of E. coli across diverse host niches.IMPORTANCE Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization in the biofilm community. Here we demonstrate that oxygen gradients in uropathogenic Escherichia coli (UPEC) biofilms lead to spatially distinct expression programs for quinol oxidases-components of the terminal electron transport chain. Our studies reveal that the cytochrome bd-expressing subpopulation is critical for biofilm development and matrix production. In addition, we show that quinol oxidases are heterogeneously expressed in planktonic populations and that this respiratory heterogeneity provides a fitness advantage during infection. These studies define the contributions of quinol oxidases to biofilm physiology and suggest the presence of respiratory bet-hedging behavior in UPEC.

Biofilm Formation by Uropathogenic Escherichia coli Is Favored under Oxygen Conditions That Mimic the Bladder Environment.

One of the most common urologic problems afflicting millions of people worldwide is urinary tract infection (UTI). The severity of UTIs ranges from asymptomatic bacteriuria to acute cystitis, and in severe cases, pyelonephritis and urosepsis. The primary cause of UTIs is uropathogenic Escherichia coli (UPEC), for which current antibiotic therapies often fail. UPEC forms multicellular communities known as biofilms on urinary catheters, as well as on and within bladder epithelial cells. Biofilm formation protects UPEC from environmental conditions, antimicrobial therapy, and the host immune system. Previous studies have investigated UPEC biofilm formation in aerobic conditions (21% oxygen); however, urine oxygen tension is reduced (4-6%), and urine contains molecules that can be used by UPEC as alternative terminal electron acceptors (ATEAs) for respiration. This study was designed to determine whether these different terminal electron acceptors utilized by E. coli influence biofilm formation. A panel of 50 urine-associated E. coli isolates was tested for the ability to form biofilm under anaerobic conditions and in the presence of ATEAs. Biofilm production was reduced under all tested sub-atmospheric levels of oxygen, with the notable exception of 4% oxygen, the reported concentration of oxygen within the bladder.