Biology subjects

Hunt, B. C.

Publications and source records attributed to Hunt, B. C..

8 recordsLinked to original sources

bioRxiv2026

L Fucose Dependent Biofilm Formation by Escherichia coli Enhances Polymicrobial Interactions and Antibiotic Tolerance on Urinary Catheters

Urinary tract infections are common healthcare associated infections, a large subset of which are caused by indwelling catheters. Long term catheterization causes persistent, asymptomatic, polymicrobial colonization despite catheters changes and antibiotic usage. In these polymicrobial populations, P. mirabilis, E. faecalis, and E. coli were found as the most common co-colonizing species. We investigated how interactions between P. mirabilis, E. coli, and E. faecalis contribute to biofilm formation and colonization of urinary catheters. Our results show that the interaction between these three species leads to enhanced biofilm biomass driven by an increase in total protein content of the biofilm. Biofilm enhancement required all three species and was also media-dependent, especially for dual-species combinations. Importantly, triple species biofilms also demonstrate biofilm enhancement when established under flow conditions in a biofilm reactor model using silicone urinary catheters. Additionally, triple species biofilm enhancement occurred in co-colonizing isolates from catheterized patients and was found to be specific to interactions between these three species. Triple species biofilms also demonstrated a species-dependent resistance to two commonly used antibiotics, ciprofloxacin and nitrofurantoin. By examining priority effects, E. coli was found to be the main facilitator of biofilm enhancement in a flow model. Finally, proteomics revealed that an L-fucose utilization pathway in E. coli was a key contributor to triple species biofilm enhancement. Overall, our results demonstrate the significant impact of polymicrobial interactions on biofilm formation in the catheterized environment and highlight ways in which complex microbial interplay and priority effects can shape the establishment of persistent colonization.

microbiology↗

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↗

bioRxiv2026

Chondroitin sulfate degradation bolsters Proteus mirabilis growth and colonization of the catheterized urinary tract

Glycosaminoglycans (GAGs) are negatively charged polysaccharides composed of repeating disaccharide units and are essential components of the extracellular matrix throughout numerous tissues. The bladder urothelium has a thick protective GAG layer that primarily consists of chondroitin sulfate (CS), heparan sulfate (HS), and hyaluronic acid (HA), and urinary tract pathogens must either degrade or otherwise circumvent this layer to infect the urothelium. In this study, we investigated GAG degradation by Proteus mirabilis, a common and persistent colonizer of the catheterized urinary tract. Almost all P. mirabilis urinary tract isolates harbor a putative chondroitin endolyase (PMI2127), exolyase (PMI2128), and sulfatase (PMI2124). By generating mutant and complemented strains of these genes, we determined that P. mirabilis strain HI4320 degrades multiple forms of CS under numerous culture conditions, including during growth in human urine, and can use CS degradation products as a carbon source. Sulfatase and endolyase activity were required for efficient degradation of all CS types, while the exolyase only contributed to using CS-B or CS-C as carbon source. Interestingly, only endolyase activity contributed to colonization in a murine model of CAUTI, although the colonization defect was even more pronounced when the endolyase and exolyase were both disrupted. The colonization defect was specific to the CAUTI model, likely due to the impact of catheterization on the GAG landscape of the bladder. Limiting CS degradation by P. mirabilis may therefore represent a strategy for reducing risk of ascending infection in catheterized patients. ImportanceGlycosaminoglycans (GAGs) are a family of negatively charged heteropolysaccharides that are ubiquitously expressed throughout the body, forming a significant component of the extracellular matrix and a luminal GAG layer in the bladder. This GAG layer functions as a physical barrier for the bladder surface, protecting it from bacterial infection. Disruption of this barrier through physical forces, such as catheter insertion, or enzymatic degradation by bacteria may contribute to infection outcomes. In this study, we defined the contribution of three putative chondroitin sulfate degrading enzymes (PMI2124, PMI2127, PMI2128) to the pathogenesis of a common pathogen in the catharized urinary tract, Proteus mirabilis. We found that P. mirabilis can utilize chondroitin sulfate as a carbon source, and that chondroitin sulfate degradation contributes to infection in a model of catheterized urinary tract infection. This work contributes to a growing understanding of how uropathogens subvert host defenses and acquire nutrients within the bladder.

microbiology↗

bioRxiv2025

Contribution of Three tRNA Modification Enzymes to Proteus mirabilis Fitness and Catheter-Associated Urinary Tract Infection

RNA modifications play essential roles in cellular physiology by modulating RNA structure, stability, and translation efficiency. Among these, dihydrouridine is a conserved tRNA modification synthesized by the flavin-dependent enzymes DusA, DusB, and DusC, which introduces flexibility at defined uridine positions in the D-loop. While structural and biochemical studies in Escherichia coli have clarified Dus enzymes substrate specificities, their functional relevance in other bacterial pathogens remains uncharacterized. Here, we present the first functional characterization of the contribution of Dus enzymes to an important urinary tract pathogen, Proteus mirabilis. Insertional mutants were generated in dusA, dusB, and dusC in P. mirabilis strain HI4320 for evaluation of virulence-relevant phenotypes. All mutants grew similarly to wild-type under multiple culture conditions, although loss of dusB caused a fitness defect during growth in human urine. Disruption of dusB also resulted in increased biofilm biomass, impaired swimming motility, enhanced outer membrane permeability, and increased susceptibility to detergents and certain antibiotics, while disruption of dusA or dusC only impacted biofilm formation and susceptibility to detergents. In a murine model of catheter-associated urinary tract infection, dusB was also critical for fitness in all organs during co-challenge against wild-type as well as overall colonization and ascending infection during independent challenge. In contrast, dusC did not contribute to fitness and dusA showed a modest, compartment-specific contribution to fitness, as the dusA::Kan mutant was outcompeted in the bladder. Together, these findings identify DusB as a key regulator of motility, membrane integrity, and host fitness in P. mirabilis, linking tRNA modification to virulence.

microbiology↗

bioRxiv2024

Function and contribution of two putative Enterococcus faecalis glycosaminoglycan degrading enzymes to bacteremia and catheter-associated urinary tract infection

Enterococcus faecalis is a common cause of healthcare acquired bloodstream infections and catheter associated urinary tract infections (CAUTI) in both adults and children. Treatment of E. faecalis infection is frequently complicated by multi-drug resistance. Based on protein homology, E. faecalis encodes two putative hyaluronidases, EF3023 (HylA) and EF0818 (HylB). In other Gram-positive pathogens, hyaluronidases have been shown to contribute to tissue damage and immune evasion, but function in E. faecalis has yet to be explored. Here, we show that both hylA and hylB contribute to E. faecalis pathogenesis. In a CAUTI model, {Delta}hylA exhibited defects in bladder colonization and dissemination to the bloodstream, and {Delta}hylB exhibited a defect in kidney colonization. Furthermore, a {Delta}hylA{Delta}hylB double mutant exhibited a severe colonization defect in a model of bacteremia while the single mutants colonized to a similar level as the wild-type strain, suggesting potential functional redundancy within the bloodstream. We next examined enzymatic activity, and demonstrate that HylB is capable of digesting both HA and CS in vitro while HylA exhibits only a very modest activity against heparin. Importantly, HA degradation by HylB provided a modest increase in cell density during stationary phase and also contributed to dampening of LPS-mediated NF-B{kappa} activation. Overall, these data demonstrate that glycosaminoglycan degradation is important for E. faecalis pathogenesis in the urinary tract and during bloodstream infection.

microbiology↗

bioRxiv2023

Metabolic interplay between Proteus mirabilis and Enterococcus faecalis facilitates polymicrobial biofilm formation and invasive disease

Polymicrobial biofilms play an important role in the development and pathogenesis of CAUTI. Proteus mirabilis and Enterococcus faecalis are common CAUTI pathogens that persistently co-colonize the catheterized urinary tract and form biofilms with increased biomass and antibiotic resistance. In this study, we uncover the metabolic interplay that drives biofilm enhancement and examine the contribution to CAUTI severity. Through compositional and proteomic biofilm analyses, we determined that the increase in biofilm biomass stems from an increase in the protein fraction of the polymicrobial biofilm matrix. We further observed an enrichment in proteins associated with ornithine and arginine metabolism in polymicrobial biofilms compared to single-species biofilms. We show that L-ornithine secretion by E. faecalis promotes arginine biosynthesis in P. mirabilis, and that disruption of this metabolic interplay abrogates the biofilm enhancement we see in vitro and leads to significant decreases in infection severity and dissemination in a murine CAUTI model.

microbiology↗

bioRxiv2021

Nontypeable Haemophilus influenzae redox recycling of protein thiols promotes resistance to oxidative killing and bacterial survival in biofilms in a smoke related infection model

Smoke exposure is a risk factor for community acquired pneumonia, which is typically caused by host adapted opportunists like nontypeable Haemophilus influenzae (NTHi). Genomic analyses of NTHi revealed homologs of enzymes involved in thiol metabolism, which can have key roles in oxidant resistance. Using a clinical NTHi isolate (NTHi 7P49H1), we generated isogenic mutant bacterial strains in which homologs of glutathione reductase (NTHI 0251), thiol peroxidase (NTHI 0361), thiol peroxidase (NTHI 0907), thioredoxin reductase (NTHI 1327) and glutaredoxin/peroxiredoxin (NTHI 0705) were inactivated. Bacterial protein analyses revealed significant increases in protein oxidation after oxidative stress for all the mutant strains. Similarly, each of these mutants were less resistant to oxidative killing compared with the parental strain; these phenotypes were reversed by genetic complementation. Quantitative confocal analysis of biofilms showed reducted biofilm thickness and density, and significant sensitization of bacteria within the biofilm structure to oxidative killing for thiol mutant strains. Smoke-exposed mice infected with NTHi 7P49H1 showed significantly increased lung bacterial load, as compared to control mice. Immunofluorescent staining of lung tissues showed NTHi communities on the lung mucosa, interspersed with host neutrophil extracellular traps; these bacteria had surface moieties associated with the Hi biofilm matrix, and transcript profiles consistent with NTHi biofilms. In contrast, infection with the panel of NTHi mutants showed significant decrease in lung bacterial load. Comparable results were observed in bactericidal assays with neutrophil extracellular traps in vitro. Thus, we conclude that thiol mediated redox homeostasis promotes persistence of NTHi within biofilm communities. ImportanceChronic bacterial respiratory infections are a significant problem for smoke exposed individuals, especially those with chronic obstructive pulmonary disease (COPD). These infections often persist despite antibiotic use. Thus, the bacteria remain and contribute to the development of inflammation and other respiratory problems. Respiratory bacteria often form biofilms within the lungs, while growing in a biofilm their antibiotic and oxidative stress resistance is incredibly heightened. It is well documented that redox homeostasis genes are upregulated during this phase of growth. Many common respiratory pathogens such as NTHi and Streptococcus pneumoniae are reliant on scavenging from the host the necessary components they need to maintain these redox systems. This work here begins to lay down the foundation for exploiting this requirement and thiol redox homeostasis pathways of these bacteria as a therapeutic target for managing chronic respiratory bacterial infections, which are resistant to traditional antibiotic treatments alone.

microbiology↗

bioRxiv2021

Nontypeable Haemophilus influenzae infection impedes Pseduomonas aeruginosa colonization and persistence in mouse respiratory tract

Patients with cystic fibrosis (CF) experience lifelong respiratory infections which are a significant cause of morbidity and mortality. These infections are polymicrobial in nature, and the predominant bacterial species undergo a predictable series of changes as patients age. Young patients have populations dominated by opportunists that are typically found within the microbiome of the human nasopharynx, such as nontypeable Haemophilus influenzae (NTHi); these are eventually supplanted and the population within the CF lung is later dominated by pathogens such as Pseudomonas aeruginosa (Pa). In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of Pa in the respiratory tract. Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater levels of Pa biofilm volume and diffusion. However, sequential respiratory infection of mice with NTHi followed by Pa resulted in significantly lower Pa as compared to infection with Pa alone. Coinfected mice also had reduced airway tissue damage and lower levels of inflammatory cytokines as compared with Pa infected mice. Similar results were observed after instillation of heat-inactivated NTHi bacteria or purified NTHi lipooligosaccharide (LOS) endotoxin prior to Pa introduction. Based on these results, we conclude that NTHi significantly reduces susceptibility to subsequent Pa infection, most likely due to priming of host innate immunity rather than a direct competitive interaction between species. These findings have potential significance with regard to therapeutic management of early life infections in patients with CF.

microbiology↗