Biology subjects

Zimmern, P. E.

Publications and source records attributed to Zimmern, P. E..

18 recordsLinked to original sources

bioRxiv2026

Gain of Function Cytolysin Variant Expressed by Clinically Isolated Urinary Enterococcus faecalis

Cytolysin is a toxin-bacteriocin commonly encoded by Enterococcus faecalis that can target erythrocytes, neutrophils, and macrophages as well as other Gram-positive bacteria. Cytolysin can lyse human, horse, rabbit, and mouse erythrocytes, but not sheep erythrocytes (SEs). It is hypothesized that SEs are resistant to cytolysin because they lack phosphatidylcholine (PC) in their membranes, while susceptible erythrocytes have PC-rich membranes, suggesting PC as a cytolysin binding target. Here, we isolated a urinary E. faecalis strain C33 that can efficiently lyse SEs and found that this activity was conserved among several E. faecalis clinical isolates. We demonstrated that although C33 cytolysin was necessary for SE lysis, it was not sufficient, suggesting a secondary factor enhances cytolysin activity against SEs. Through liposome assays we found that while PC was required for the activity of DS16 cytolysin, PC was dispensable for C33 cytolysin activity. To assess the biological significance of C33 cytolysin in the bladder microenvironment, we assessed C33 cytolysin activity against bladder epithelial cells and the urinary microbiota. We found that C33 cytolysin was more cytotoxic to human bladder epithelial cells than the canonical DS16 cytolysin. We also found greater inhibition of co-isolated urinary microbiota species Streptococcus parasanguinis, Enterococcus raffinosus, Staphylococcus capitis, and Corynebacterium coyleae by C33 cytolysin. Interestingly, E. faecalis C33 also inhibited the protective urogenital taxa Lactobacillus crispatus and Bifidobacterium longum. Together, these results reveal a new PC-independent cytolysin activity among clinical E. faecalis isolates that may support survival and competition in the urinary microenvironment.

microbiology↗

bioRxiv2026

Engineered living materials suppress uropathogenic E. coli growth and invasion of urothelial cells through sustained probiotic release

Recurrent urinary tract infection (rUTI) is a significant public health problem. The most common cause of rUTI is uropathogenic Escherichia coli (UPEC). Antimicrobial prophylactic and therapeutic regimens for rUTI disrupts the microbiome and leads to infection with antimicrobial resistant organisms. Therefore, there is an urgent need to develop microbiome-sparing alternative approaches for preventing rUTI. Asymptomatic bacteriuria (ABU) E. coli strain 83972 (ABEC) outcompetes UPEC in the urinary tract without causing UTI symptoms, but its limited persistence in the bladder restricts its efficacy. Here, we investigate a device made from an engineered living material (ELM) that releases ABEC in a sustained manner as an antibiotic-free platform against UTIs and rUTIs. Using physiologically relevant in vitro models incorporating human urothelial cells, human urine, and periodic urine exchange, we show that ABEC-releasing ELMs suppress UPEC proliferation and inhibit UPEC attachment to and invasion into urothelial cells, particularly when ABEC is maintained at equal or higher levels than UPEC. Because ELMs continuously release ABEC, they sustain competitive pressure even as planktonic bacteria are cleared during voiding, outperforming a single dose of free-floating ABEC. In a rUTI model, sustained ABEC release from ELMs reduces the proliferation and re-invasion of UPEC expelled from infected urothelial cells, while UPEC infiltration into fractured ELMs remains negligible. Finally, we design a first-generation ELM device that can be transurethrally delivered and retained within the mouse urinary bladder, achieving sustained ABEC release in vivo, with ABEC persisting in the bladder, kidneys, and urine for at least 4 days. In summary, we report the development of an ELM device that continuously releases ABEC to suppress UPEC proliferation and urothelial cell invasion in in vitro models of rUTI, and demonstrate sustained ABEC release in vivo in a mouse bladder.

bioengineering↗

bioRxiv2026

Quantitative Analysis of External Urethral Sphincter Stimulation Parameters for Modulating Urinary Output

ObjectiveNeurogenic lower urinary tract dysfunction (NLUTD) impairs bladder control and remains difficult to treat. We aim to define how electrical stimulation (ES) parameters of the external urethral sphincter (EUS) affect urinary leakage thresholds to guide neuromodulation strategies for NLUTD. MethodsWe performed direct EUS stimulation in anesthetized rats using charge-balanced biphasic pulses while systematically varying current amplitude (0.5-3.0 mA), frequency (20-100 Hz), and pulse duration (0.5-3 ms). Urine leakage thresholds were mapped across the multidimensional parameter space. ResultsStimulation parameters exhibited strong nonlinear interdependence in determining leakage onset. At a fixed pulse duration, higher current amplitudes required lower stimulation frequencies to evoke leakage. Increasing pulse duration substantially reduced both current and frequency thresholds. Age and sex caused modest shifts in absolute thresholds but did not alter the fundamental parameter-response relationships. ConclusionPulse duration, current amplitude, and frequency jointly govern urinary leakage thresholds, with pulse duration serving as the dominant modulator of stimulation efficiency. SignificanceThis work establishes a quantitative framework for charge-efficient stimulation parameter selection, enabling the design of energy-aware, precision neuromodulation protocols and implantable systems for NLUTD rehabilitation.

neuroscience↗

bioRxiv2026

PREFERENTIAL INVASION OF DIFFERENTIATED BLADDER CARCINOMA CELLS BY FLAGELLATED GROUP B2 ESCHERICHIA COLI

Phylogenetic group B2 Escherichia coli is associated with urinary tract infections and other pathologies, but the basis for this phylogenetic skew is not understood. One aspect of urinary tract infections is binding to and entering uroepithelial cells. To test whether a phylogenetic skew exists for cell invasion, we examined invasion of 10 E. coli strains from three phylogenetic groups into CRL2169 and HTB-9 cells, which are derived from grade 1 and grade 2 bladder carcinomas, respectively. The top four strains that invaded CRL2169 were from group B2: three of these strains had more flagella gene transcripts than the other seven strains. The seven strains that invaded HTB-9 were from different phylogenetic groups. For the model uropathogenic group B2 strain UTI89, which expresses pili over flagella, loss of flagella or pili impacted invasion into CRL2169 to similar extents, but loss of pili had a greater effect on invasion into HTB-9 and a murine infection model than loss of flagella. A hyperflagellated variant of a group A strain did not invade either cell line better than the parental strain. Reported transcript differences, which were confirmed experimentally, showed that CRL2169 was more differentiated. The endocytosis stimulator tanshinone enhanced invasion into HTB-9, but not into CRL2169, which suggests differences in endocytic pathways and is consistent with differences in differentiation states. If the initial or recurring event in urinary tract infection is invasion into differentiated urothelial cells, as opposed to tight junctions, then the role of flagella may have been underestimated.

molecular biology↗

bioRxiv2026

Strain level variation in Proteus mirabilis chondroitin sulfate degradation kinetics and regulation by urea

To establish infection, uropathogens must overcome several host defenses including the glycosaminoglycan (GAG) layer coating the apical surface of the bladder urothelium. GAGs are thought to protect against urinary tract infection (UTI) by serving as scaffolding sites for commensals, providing barrier function and preventing uropathogen adherence. However, the ability of uropathogens to degrade and utilize GAGs and the contribution of these activities toward UTI progression is largely unknown. We previously discovered that the uropathogen Proteus mirabilis, a common cause of catheter-associated UTI (CAUTI), degrades the GAG chondroitin sulfate (CS). In this study we sought to define the kinetics and regulation of CS degradation by diverse P. mirabilis strains clinically isolated from both recurrent UTI and CAUTI patients. We found variation in CS degradation kinetics between P. mirabilis strains and media types. However, CS degradation depended on conserved putative chondroitin sulfate ABC endo- and exolyases in all strains. Furthermore, we found that CS degradation in Pm123 was repressed by urea and that this repression was dependent on P. mirabilis urease activity. Complementation of the Pm123 endolyase into urea-insensitive HI4320 resulted in a urea-sensitive CS degradation phenotype suggesting functional differences between the Pm123 and HI4320 endolyases. Sequence alignment and structural modeling analysis identified two unique point mutations within the Pm123 endolyase that may contribute to urea sensitivity. Finally, unlike urea-insensitive P. mirabilis strains, Pm123 demonstrated attenuated swarming and loss of chondroitin endolyase activity had no effect on Pm123 virulence in a mouse CAUTI model. Our results suggest that the kinetics and regulation of CS degradation differ between P. mirabilis strains and in urea-sensitive strains, thus reduces the contribution of CS degradation to urovirulence during murine CAUTI. ImportanceThis work demonstrates that the ability to degrade a common component of bladder mucosal surfaces, chondroitin sulfate, is a phenotype that is shared by multiple strains of the common catheter-associated UTI (CAUTI) pathogen P. mirabilis. We find that this activity is dependent on encoded chondroitin ABC endo- and exolyases, first described in Proteus vulgaris. Additionally, we discovered that for P. mirabilis strain Pm123, degradation of CS is negatively regulated by the presence of urea, a major component of urine. The repression of CS degradation by urea is dependent on the activity of the P. mirabilis urease enzyme, which breaks down urea producing ammonia which raises pH. We found expression of the Pm123 CS endolyase was sufficient to confer a urea-sensitive CS-degradation phenotype and identified two unique mutations within the Pm123 enzyme that may contribute to urea sensitivity. Finally, we find that while CS-degradation plays a role in progression and severity of murine CAUTI model in urea-insensitive P. mirabilis, there was not significant difference in CAUTI outcomes between the urea-sensitive Pm123 wild-type and chondroitinase knockout strains. This study represents a major step forward in understanding the diversity of CS degradation activity and regulation among clinical strains of the critically important CAUTI pathogen P. mirabilis as well as its contribution to urovirulence.

microbiology↗

bioRxiv2025

Enterococcus faecalis Influences the Transcription and Metabolism of Pathogenic Escherichia coli when Grown in Co-Culture

UTI involves bacterial growth in the disparate environments of the bladder and within uroepithelial cells. The bladder is a low-nutrient environment that nonetheless supports rapid growth. Phylogenetic group B2 Escherichia coli (Ec) is frequently isolated from urinary tract infection (UTI) patients. Non-B2 strains have also been isolated and are often co-isolated with Enterococcus faecalis (Ef). We characterized the interaction between three co-isolated Ec-Ef pairs and other Ec-Ef combinations in a nutrient-rich medium which was intended to emulate the rapid growth condition of the bladder. In this medium, Ef had little effect on Ec growth but resulted in major transcriptome differences. Ef affected the non-B2 and B2 strains differently. For the non-B2 Ec strains, Ef induced transcript for genes whose products degrade ornithine via putrescine to succinate which is subsequently metabolized by the TCA cycle. For a control B2 Ec strain, Ef induced transcripts for growth rate-associated genes of macromolecular synthesis, and for similar metabolic enzymes, except for those that degrade putrescine to succinate. Unexpectedly, Ef induced transcripts for glyoxylate shunt enzymes in both non-B2 and B2 Ec strains. The bacterial disparate and constantly changing environments during UTI suggest the potential for different types of Ec-Ef interactions. Our results provide evidence for nutrient cross-feeding as one type of Ec-Ef interaction.

microbiology↗

bioRxiv2025

Daily and Weekly Fluctuations in the Microbiome of the Urinary Tract of Postmenopausal Women with No History of Urinary Tract Infections

At least half of all women will experience a urinary tract infection (UTI) in their lifetime. Age, sexual activity, hormonal status, and the urinary tract microbiome (urobiome) contribute to the probability of infection. While studies have assessed urobiome composition during and after infection, no study has tracked the daily and weekly fluctuations of healthy urobiomes to determine urobiome stability. In this study, we sampled the clean catch urine of 3 women with no history of UTIs over the span of two weeks sampling three times a day on two days each week to track urobiome changes, and whether the urobiome in individuals with no UTI history resembles that of individuals after a UTI. Two participants were dominantly colonized by Lactobacillus while the third had a more diverse set of taxa albeit at a lower total amount of bacteria. We previously reported that the urine from only one of the less diverse urobiomes supported the growth of recently isolated uropathogenic Escherichia coli or Enterococcus faecalis, while the other two participants urines did not support growth. We conclude that 1) urobiomes exist in healthy post-menopausal women 2) these urobiomes have a stable and robust community structure and 3) a combination of colonization resistance, including nutrient limitation, and urobiome structure contributes to resistance to UTIs. ImportancePresented here is the first analysis of urinary tract microbiomes (urobiome) of post-menopausal women with no history of urinary tract infections collected at multiple times. These data contribute to the growing understanding of urobiomes of post-menopausal women during a healthy state. Minimal fluctuations were observed on an individual basis, confirming that the time of day or day of the week has no impact on the findings in longitudinal urine sample studies.

genetics↗

bioRxiv2024

Urinary biochemical ecology reveals microbiome-metabolite interactions and metabolic markers of recurrent urinary tract infection

Recurrent urinary tract infections (rUTIs) are a major clinical challenge and their increasing prevalence underscores the need to define host-microbiome interactions underlying susceptibility. How the urinary microbiota engages with the biochemical environment of the urogenital tract is yet to be fully defined. Here, we leverage paired metagenomic and quantitative metabolomic data to establish a microbe-metabolite association network of the female urinary microbiome and define metabolic signatures of rUTI. We observe unique metabolic networks of uropathogens and uroprotective species, highlighting potential metabolite-driven ecological shifts influencing rUTI susceptibility. We find distinct metabolites are associated with urinary microbiome diversity and identify a lipid signature of active rUTI that accurately distinguishes cases from controls. Finally, we identify deoxycholic acid as a prognostic indicator for UTI recurrence. Together these findings provide insight into microbiome-metabolite interactions within the female urinary tract and highlight new biomarkers for the development of new diagnostic tools to improve patient outcomes.

microbiology↗

bioRxiv2024

Controlled release of microorganisms from engineered living materials

Probiotics offer therapeutic benefits by modulating the local microbiome, the host immune response, and the proliferation of pathogens. Probiotics have the potential to treat complex diseases, but their persistence or colonization is required at the target site for effective treatment. Although probiotic persistence can be achieved by repeated delivery, no biomaterial that releases clinically relevant doses of metabolically active probiotics in a sustained manner has been previously described. Here, we encapsulate stiff probiotic microorganisms within relatively less stiff hydrogels and show a generic mechanism where these microorganisms proliferate and induce hydrogel fracture, resulting in microbial release. Importantly, this fracture-based mechanism leads to microorganism release with zero-order release kinetics. Using this mechanism, small ([~]1 L) engineered living materials (ELMs) release >108 colony-forming-units (CFUs) of E. coli in 2 h. This release is sustained for at least 10 days. Cell release can be varied by more than three orders of magnitude by varying initial cell loading and modulating the mechanical properties of encapsulating matrix. As the governing mechanism of microbial release is entirely mechanical, we demonstrate controlled release of model Gram-negative, Gram-positive, and fungal probiotics from multiple hydrogel matrices. SIGNIFICANCEProbiotics offer therapeutic benefits and have the potential to treat complex diseases, but their persistence at the target site is often required for effective treatment. Although probiotic persistence can be achieved by repeated delivery, no biomaterial that releases metabolically active probiotics in a sustained manner has been developed yet. This work demonstrates a generic mechanism where stiff probiotics encapsulated within relatively less stiff hydrogels proliferate and induce hydrogel fracture. This allows a zero-order release of probiotics which can be easily controlled by adjusting the properties of the encapsulating matrices. This generic mechanism is applicable for a wide range of probiotics with different synthetic matrices and has the potential to be used in the treatment of a broad range of diseases.

bioengineering↗

bioRxiv2024

Control of pili synthesis and putrescine homeostasis in Escherichia coli

Polyamines are biologically ubiquitous cations that bind to nucleic acids, ribosomes, and phospholipids and, thereby, modulate numerous processes, including surface motility in Escherichia coli. We characterized the metabolic pathways that contribute to polyamine-dependent control of surface motility in the commonly used strain W3110 and the transcriptome of a mutant lacking a putrescine synthetic pathway that was required for surface motility. Genetic analysis showed that surface motility required type 1 pili, the simultaneous presence of two independent putrescine anabolic pathways, and modulation by putrescine transport and catabolism. An immunological assay for FimA--the major pili subunit, reverse transcription quantitative PCR of fimA, and transmission electron microscopy confirmed that pili synthesis required putrescine. Comparative RNAseq analysis of a wild type and {Delta}speB mutant which exhibits impaired pili synthesis showed that the latter had fewer transcripts for pili structural genes and for fimB which codes for the phase variation recombinase that orients the fim operon promoter in the ON phase, although loss of speB did not affect the promoter orientation. Results from the RNAseq analysis also suggested (a) changes in transcripts for several transcription factor genes that affect fim operon expression, (b) compensatory mechanisms for low putrescine which implies a putrescine homeostatic network, and (c) decreased transcripts of genes for oxidative energy metabolism and iron transport which a previous genetic analysis suggests may be sufficient to account for the pili defect in putrescine synthesis mutants. We conclude that pili synthesis requires putrescine and putrescine concentration is controlled by a complex homeostatic network that includes the genes of oxidative energy metabolism.

microbiology↗

bioRxiv2023

Functional and genetic adaptations contributing to Enterococcus faecalis persistence in the female urinary tract

Enterococcus faecalis is the leading Gram-positive bacterial species implicated in urinary tract infection (UTI). An opportunistic pathogen, E. faecalis is a commensal of the human gastrointestinal tract (GIT) and its presence in the GIT is a predisposing factor for UTI. The mechanisms by which E. faecalis colonizes and survives in the urinary tract (UT) are poorly understood, especially in uncomplicated or recurrent UTI. The UT is distinct from the GIT and is characterized by a sparse nutrient landscape and unique environmental stressors. In this study, we isolated and sequenced a collection of 37 clinical E. faecalis strains from the urine of primarily postmenopausal women. We generated 33 closed genome assemblies and four highly contiguous draft assemblies and conducted a comparative genomics to identify genetic features enriched in urinary E. faecalis with respect to E. faecalis isolated from the human GIT and blood. Phylogenetic analysis revealed high diversity among urinary strains and a closer relatedness between urine and gut isolates than blood isolates. Plasmid replicon (rep) typing further underscored possible UT-GIT interconnection identifying nine shared rep types between urine and gut E. faecalis. Both genotypic and phenotypic analysis of antimicrobial resistance among urinary E. faecalis revealed infrequent resistance to front-line UTI antibiotics nitrofurantoin and fluoroquinolones and no vancomycin resistance. Finally, we identified 19 candidate genes enriched among urinary strains that may play a role in adaptation to the UT. These genes are involved in the core processes of sugar transport, cobalamin import, glucose metabolism, and post-transcriptional regulation of gene expression. IMPORTANCEUrinary tract infection (UTI) is a global health issue that imposes substantial burden on healthcare systems. Women are disproportionately affected by UTI with >60% of women experiencing at least one UTI in their lifetime. UTIs can recur, particularly in postmenopausal women, leading to diminished quality of life and potentially life-threatening complications. Understanding how pathogens colonize and survive in the urinary tract is necessary to identify new therapeutic targets that are urgently needed due to rising rates of antimicrobial resistance. How Enterococcus faecalis, a bacterium commonly associated with UTI, adapts to the urinary tract remains understudied. Here, we generated a collection of high-quality closed genome assemblies of clinical urinary E. faecalis isolated from the urine of postmenopausal women that we used alongside detailed clinical metadata to perform a robust comparative genomic investigation of genetic factors that may mediate urinary E. faecalis adaptation to the female urinary tract.

microbiology↗

bioRxiv2023

Nitrogen-limitation independent control of glnA (glutamine synthetase) expression in Escherichia coli by urea, several amino acids, and post-transcriptional regulation.

The expression of glnA (ammonia-assimilating glutamine synthetase) is high for uropathogenic E. coli grown in urine. Because glnA is part of an operon that codes for regulators of the nitrogen-regulated (Ntr) response, high glnA expression has been interpreted to suggest nitrogen limitation, which is unexpected because of the high urinary ammonia concentration and the extremely rapid bacterial growth. We present evidence that glnA expression does not result from nitrogen limitation. First, in the presence of ammonia, urea induced expression of glnA from the cAMP receptor protein (Crp)- dependent glnAp1 promoter, which circumvents control from the nitrogen-regulated glnAp2 promoter. This urea effect on glnA expression has not been previously described. Second, the most abundant amino acids in urine inhibited GS activity, based on reversal of the inhibition by glutamate and glutamine, and increased glnA expression. The relevance of these inhibitory amino acids in natural environments has not been previously demonstrated. Third, neither urea nor the inhibitory amino acids induced other Ntr genes, i.e., high glnA expression can be independent of other Ntr genes. Finally, the urea-dependent induction did not result in GlnA synthesis because of a previously undescribed translational control. We conclude that glnA expression in urea-containing environments does not imply growth rate-limiting nitrogen restriction and is consistent with rapid growth of uropathogenic E. coli. SignificanceUrinary tract infections (UTIs), often caused by E. coli, frequently become resistant to antibiotic treatment. Expressed metabolic genes during an infection could guide development of urgently-needed alternate or adjunct therapies. glnA (glutamine synthetase) is expressed during growth in urine, which implies growth-restricting nitrogen limitation. We show that glnA expression results from urinary amino acids that inhibit GlnA activity and urea, but not from nitrogen limitation. Urinary components will vary greatly between individuals which suggests corresponding variations in glnA expression. GlnA may be a metabolic vulnerability during UTIs, which may depend on a variable urinary composition. glnA expression may be important in a complex host-pathogen interaction, but may not be a good therapeutic target.

physiology↗

bioRxiv2023

Urinary Glycosaminoglycans are Associated with Recurrent UTI and Urobiome Ecology in Postmenopausal Women

Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides composed of repeating disaccharide units of uronic acid and amino sugars. The luminal surface of the bladder epithelium is coated with a GAG layer. These urothelial GAGs are thought to provide a protective barrier and serve as a potential interaction site with the urinary microbiome (urobiome). Previous studies have profiled urinary GAG composition in mixed cohorts, but the urinary GAG composition in postmenopausal women remains undefined. To investigate the relationship between GAGs and recurrent UTI (rUTI), we profiled urinary GAGs in a controlled cohort of postmenopausal women. We found that chondroitin sulfate (CS) is the major urinary GAG in postmenopausal women and that urinary CS was elevated in women with active rUTI. We also associated urinary GAGs with urobiome composition and identified bacterial species that significantly associated with urinary GAG concentration. Corynebacterium amycolatum, Porphyromonas somerae, and Staphylococcus pasteuri were positively associated with heparin sulfate or hyaluronic acid and bacterial species associated with vaginal dysbiosis were negatively correlated to urinary CS. Altogether, this work defines changes in urinary GAG composition associated with rUTI and identifies new associations between urinary GAGs and the urobiome that may play a role in rUTI pathobiology.

biochemistry↗

bioRxiv2022

Bacterial growth-promoting properties of pooled urine and individual urines from post-menopausal women with or without a urinary tract infection can vary substantially

Urinary tract infections (UTIs), mostly caused by uropathogenic E. coli (UPEC), are common bacterial infections that affect a majority of women and often recur. Genomic and transcriptomic analyses have not identified a common set of virulence genes which has suggested conserved virulence functions instead of virulence genes, multiple virulence mechanisms, and complex host-pathogen interactions. One aspect of the host-pathogen interaction is rapid UPEC growth in urine in vivo. When bacteria are grown in pooled urine, an averaged urine is assumed to diminish individual variation. We grew a non-pathogenic and pathogenic E. coli strains in urine from individuals who never had a UTI, had a UTI history but no current infection, and had a UTI history with a current infection. Bacterial growth showed large variations in individual urines and pooled urine supported significantly more growth than predicted for never and history groups but not the current group. UPEC strains, but not the non-pathogenic strain, were resistant to urinary inhibitory factors e.g., antimicrobial peptides based on an indirect inoculation-density effect assay. Total nutrient content tended to be higher in current group urine than never and history group urine. We propose that pooling optimizes a nutrient mixture in never and history group urines, which are often studied, whereas urine from current group individuals appear to have a more optimal nutrient mixture. We conclude that pooled urine is not "an average urine", and that the best comparisons of results between labs using pooled urine would also include results with a standardized synthetic urine.

microbiology↗

bioRxiv2022

Conserved FimK truncation coincides with increased expression of type 3 fimbriae and cultured bladder epithelial cell association in Klebsiella quasipneumoniae

Klebsiella spp. commonly cause both uncomplicated urinary tract infection (UTI) and recurrent UTI (rUTI). Klebsiella quasipneumoniae, a relatively newly defined species of Klebsiella, has been shown to be metabolically distinct from Klebsiella pneumoniae, but its urovirulence mechanisms have not been defined. K. pneumoniae uses both type 1 and type 3 fimbriae to attach to host epithelial cells. The type 1 fimbrial operon is well-conserved between Escherichia coli and K. pneumoniae with the exception of fimK, which is unique to Klebsiella spp. FimK contains an N-terminal DNA binding domain and a C-terminal phosphodiesterase (PDE) domain that has been hypothesized to cross-regulate type 3 fimbriae expression via modulation of cellular levels of cyclic di-GMP. Here, we find that a conserved premature stop codon in K. quasipneumoniae fimK results in truncation of the C-terminal PDE domain and that the bladder epithelial cell association and invasion of uropathogenic K. quasipneumoniae strain KqPF9 is dependent on type 3 but not type 1 fimbriae. Further, we show that basal expression of both type 1 and type 3 fimbrial operons as well as bladder epithelial cell association is elevated in KqPF9 relative to uropathogenic K. pneumoniae TOP52 and that complementation of KqPF9{Delta}fimK with the TOP52 fimK allele reduced type 3 fimbrial expression and bladder epithelial cell attachment. Taken together these data suggest that the C-terminal PDE of FimK can modulate type 3 fimbrial expression in K. pneumoniae and its absence in K. quasipneumoniae may lead to a loss of type 3 fimbrial cross-regulation. ImportanceK. quasipneumoniae is often indicated as the cause of many opportunistic infections including urinary tract infection (UTI), which affects >50% of women worldwide. However, virulence mechanisms of K. quasipneumoniae remain uninvestigated. Prior to this work, K. quasipneumoniae and K. pneumoniae had only been distinguished phenotypically by metabolic differences. This work contributes to the understanding of K. quasipneumoniae virulence phenotypes by evaluating the contribution of type 1 and type 3 fimbriae, which are critical colonization factors encoded by all Klebsiella spp., to K. quasipneumoniae bladder epithelial cell attachment. We identify clear phenotypic differences in both bladder epithelial cell attachment between uropathogenic K. pneumoniae and K. quasipneumoniae. Importantly, we find that a structural difference in the fimbrial regulatory gene fimK may contribute to differential co-regulation of type 1 and type 3 fimbriae between the two Klebsiella species.

microbiology↗

bioRxiv2021

Recurrent urinary tract infection and estrogen shape the taxonomic ecology and functional potential of the postmenopausal urobiome

Community-acquired urinary tract infection (UTI) is among the most common bacterial infections observed in humans. Postmenopausal women are a rapidly growing and underserved demographic group who are severely affected by recurrent UTI (rUTI) with a >50% recurrence rate. In this population, rUTI can persist for years, reducing quality of life and imposing a significant healthcare burden. rUTI is most often treated by antibiotics, but development of antibiotic resistance and allergy limit therapeutic options. The female urinary microbiome (urobiome) has been identified as a key component of the urogenital environment. However, compositional and functional changes in the urobiome underlying rUTI susceptibility in postmenopausal women are not well understood. Here, we used a controlled, cross-sectional cohort of postmenopausal women, to interrogate changes in urobiome structure and function linked to rUTI susceptibility by whole genome metagenomic sequencing (WGMS), advanced urine culture, estrogen metabolite profiling, and antibiotic sensitivity testing. Overall, we detected 276 bacterial, archaeal, and fungal species representing 106 genera. We find a putative commensal population consisting of species known to protect against bacterial vaginosis, such as Lactobacillus crispatus, within the urobiome of postmenopausal women who do not experience UTI. Integration of clinical metadata detected an almost exclusive enrichment of lactobacilli, including L. crispatus and L. vaginalis, in women taking estrogen hormone therapy (EHT). Integrating quantitative metabolite profiling of urinary estrogens with WGMS, we observed robust correlations between urobiome taxa, such as Bifidobacterium breve and L. crispatus, and urinary estrogen conjugate concentrations in women with no history of UTI that were absent in women with rUTI history. We further used functional metagenomic profiling and patient-derived isolate phenotyping to identify microbial metabolic pathways, antimicrobial resistance genes (ARGs), and clinically relevant antimicrobial resistance phenotypes enriched between disease-states. Our data indicate that distinct urobiome metabolic and ARG signatures are associated with current rUTI status and history. Importantly, we observed that rUTI history leaves an imprint of enriched ARGs even in women not currently experiencing UTI. Taken together, our data suggests that rUTI history and estrogen strongly shape the functional and taxonomic composition of the urobiome in postmenopausal women.

microbiology↗

bioRxiv2020

The Nutrient and Energy Pathway Requirements for Surface Motility of Nonpathogenic and Uropathogenic Escherichia coli

Uropathogenic E. coli (UPEC) is the causative pathogen for most uncomplicated urinary tract infections. Flagellar-mediated motility is essential for virulence and colonization for ascending urinary tract infections. The appendage requirement for surface motility depends on the strain: nonpathogenic E. coli (NPEC) lab strains use pili, NPEC hypermotile derivatives use flagella, and UPEC strains use flagella. E. coli flagella-dependent surface motility had been previously shown to require glucose and amino acids. We examined the nutritional and pathway requirements of the NPEC strain W3110 for pili-dependent surface motility, which have not been previously examined. We then compared these requirements to those for two strains with flagella-dependent surface motility: a variant of W3110, W3110-J1, in which the synthesis of the activator of flagella synthesis has been upregulated and the UPEC strain UTI89. The glucose requirement for W3110 was higher than that for either W3110-J1 or UTI89. The pathways required for motility were also different. W3110, but not UTI89, required the Embden-Meyerhof-Parnas pathway via PfkA; conversely, UTI89, but not W3110, required the Entner-Doudoroff pathway, acetogenesis, and the TCA cycle. Glucose did not control flagella synthesis for W3110-J1 and UTI89. The differing requirements for surface motility are likely to reflect major metabolic differences between strains. The metabolic requirements for UTI89 motility suggest a specific adaptation to the urinary tract environment. IMPORTANCEUrinary tract infections affect over 50% of women and E. coli is the most common uropathogen. Virulence requires both pili and flagella, and both appendages can contribute to surface motility. Previous studies of E. coli surface motility did not consider the appendage requirement and the ability to switch the surface appendage. The nutrient and pathway requirements for surface motility of a non-pathogenic E. coli strain with either pili- or flagella-dependent surface motility and the uropathogen UTI89 were examined. Pili-dependent surface motility required glycolysis, while flagella-dependent motility required the TCA cycle and oxidative phosphorylation and was less dependent on glycolysis. The distinctive nutrient and pathway requirements for UTI89 motility probably result from metabolic adaptations to the urinary tract.

microbiology↗

bioRxiv2019

FIMBRIAE AND FLAGELLA MEDIATED SURFACE MOTILITY AND THE EFFECT OF GLUCOSE ON NONPATHOGENIC AND UROPATHOGENIC ESCHERICHIA COLI

We characterized the surface motility of nonpathogenic and pathogenic E. coli strains with respect to the appendage requirement, flagella versus fimbriae, and the glucose requirement. Nonpathogenic lab strains exhibited either slow or fast surface movement. The slow strains required type 1 fimbriae for movement, while the fast strains required flagella and had an insertion in the flhDC promoter region. Surface movement of three uropathogenic E. coli (UPEC) strains was fast and required flagella, but these strains did not have an insertion in the flhDC promoter region. We assessed swimming motility as an indicator of flagella synthesis and found that glucose inhibited swimming of the slow nonpathogenic strains but not of the fast nonpathogenic or pathogenic strains. Fimbriae-based surface motility requires glucose, which inhibits cyclic-AMP (cAMP) and flagella synthesis; therefore, we examined whether surface motility required cAMP. The surface motility of a slow, fimbriae-dominant, nonpathogenic strain did not require cAMP, which was expected because fimbriae synthesis does not require cAMP. In contrast, the surface motility of a faster, flagella-dominant, UPEC strain required cAMP, which was unexpected because swarming was unaffected by the presence of glucose. Electron microscopy verified the presence or absence of fimbriae or flagella. In summary, surface motilities of the nonpathogenic and uropathogenic E. coli strains of this study differed in the appendage used and the effects of glucose on flagella synthesis. IMPORTANCEUropathogenic Escherichia coli strains cause 80-90% of community-acquired urinary tract infections, and recurrent urinary tract infections, which can last for years, and often become antibiotic resistant. Urinary tract infections can be associated with intra-vesical lesions extending from localized trigonitis/cystitis to widely distributed pancystitis: motility may be a factor that distinguishes between these infection patterns. Nonpathogenic and uropathogenic E. coli were shown to exhibit fimbriae- and flagella-dependent surface motility, respectively, and the difference was attributed to altered control of flagella synthesis by glucose. Uropathogenic E. coli strains grow more rapidly in urine than nonpathogenic strains, which implies differences in metabolism. Understanding the basis for glucose-insensitive control of flagella-dependent motility could provide insight into uropathogenic E. coli metabolism and virulence.

microbiology↗