Biology subjects

Brunson, D. N.

Publications and source records attributed to Brunson, D. N..

5 recordsLinked to original sources

bioRxiv2025

Metal Uptake Systems Underpin Enterococcus faecalis Virulence in Both Non-Diabetic and Diabetic Wound Infection Models

Wound infections remain an important medical problem, which is aggravated by the prevalence of multidrug resistant bacteria. Among them, Enterococcus faecalis is a major pathogen of surgical site incisional and of diabetic chronic wounds, but factors driving its colonization and persistence in wounds remain poorly understood. Iron, manganese, and zinc are essential cofactors in cellular processes, prompting the host to restrict their availability through mobilization of metal-sequestering proteins, a defense known as nutritional immunity. Previously, we showed that E. faecalis strains lacking key iron ({Delta}5Fe), manganese ({Delta}3Mn), or zinc ({Delta}2Zn) uptake systems have impaired virulence. Here, we used an excisional wound model in normoglycemic (C57Bl/6J or B6) and diabetic (C57Bl/6J lepR-/- or DB) mice to examine the role of these metal import systems in wounds. The strong upregulation of metal import genes and reduced wound colonization by {Delta}3Mn, {Delta}5Fe, and {Delta}2Zn strains in B6 mice indicate that iron, manganese, and zinc are limited during wound infection. While {Delta}2Zn and {Delta}3Mn strains showed no improved colonization in diabetic wounds, the {Delta}5Fe exhibited a temporary colonization advantage over non-diabetic mice. Quantifications of metal-sequestering proteins lactoferrin, transferrin, calprotectin and psoriasin from intact skin and infected wounds indicated that nutritional immunity, especially iron restriction, is delayed in diabetes. In conclusion, this study underscores the crucial role of trace metal acquisition in E. faecalis wound colonization and suggests differences in metal bioavailability between diabetic and non-diabetic wounds, helping to explain the increased susceptibility of diabetic wounds to chronic infection.

microbiology↗

bioRxiv2025

Characterization of a Novel Cell Wall-Associated Nucleotidase of Enterococcus faecalis that Degrades Extracellular c-di-AMP

Enterococcus faecalis is a prolific opportunistic pathogen responsible for a range of life-threatening infections for which treatment options are increasingly limited due to the high prevalence of multidrug-resistant isolates. Cyclic di-AMP has emerged as an essential bacterial signaling molecule due to its impact on physiological processes, including osmotic adaptation, cell wall homeostasis, antibiotic tolerance, and virulence. In addition, c-di-AMP is a potent pathogen-associated molecular pattern (PAMP) molecule recognized by the host immune system to trigger protective responses. In previous work, we identified and characterized the enzymes responsible for the synthesis and degradation of intracellular c-di-AMP in E. faecalis, demonstrating that maintaining c-di-AMP homeostasis is vital for bacterial fitness and virulence. In addition to the intracellular enzymes that regulate c-di-AMP levels, a limited number of bacteria encode surface-associated nucleotidases capable of cleaving extracellular c-di-AMP, potentially facilitating immune evasion. Here, we characterize a novel and unique cell wall-anchored phosphodiesterase, termed EecP (E. faecalis extracellular c-di-AMP phosphodiesterase), which features duplicated catalytic domains and specifically degrades extracellular c-di-AMP. Deletion of eecP ({Delta}eecP) resulted in a marked accumulation of extracellular c-di-AMP. Although the {Delta}eecP strain exhibited comparable growth and behavior to the parent strain in vitro, it displayed increased susceptibility to killing by phagocytic cells. Using two murine infection models, we show that the impact of eecP deletion and the consequent buildup of extracellular c-di-AMP on E. faecalis pathogenesis may be site-specific. Notably, disseminated infection was more severe in mice infected with the {Delta}eecP strain, suggesting that extracellular c-di-AMP influences infection outcomes, likely through modulation of host immune responses. Author SummaryEnterococcus faecalis is a major opportunistic pathogen and a leading cause of several life-threatening hospital-associated infections. Cyclic di-AMP is a bacterial second messenger nucleotide that regulates essential cellular processes and plays key roles in bacterial pathogenesis and host immune activation. We previously characterized the enzymes responsible for the synthesis and degradation of c-di-AMP in E. faecalis, demonstrating that this signaling molecule is crucial for bacterial fitness and virulence. In this study, we describe the characterization of EecP, a novel cell wall-associated enzyme that degrades c-di-AMP extracellularly. Our findings identify EecP as a new virulence factor in E. faecalis, capable of modulating infection outcomes.

microbiology↗

bioRxiv2025

A Novel Heme-Degrading Enzyme that Regulates Heme and Iron Homeostasis and Promotes Virulence in Enterococcus faecalis

Enterococcus faecalis, a gut commensal, is a leading cause of opportunistic infections. Its virulence is linked to its ability to thrive in hostile environments, which includes host-imposed metal starvation. We recently showed that E. faecalis evades iron starvation using five dedicated transporters that collectively scavenge iron from host tissues and other iron-deprived conditions. Interestingly, heme, the most abundant source of iron in the human body, supported growth of a strain lacking all five iron transporters ({Delta}5Fe). To release iron from heme, many bacterial pathogens utilize heme oxygenase enzymes to degrade the porphyrin that coordinates the iron ion of heme. Although E. faecalis lacks these enzymes, bioinformatics revealed a potential ortholog of the anaerobic heme-degrading enzyme anaerobilin synthase, found in Escherichia coli and a few other Gram-negative bacteria. Here, we demonstrated that deletion of OG1RF_RS05575 in E. faecalis ({Delta}RS05575) or in the {Delta}5Fe background ({Delta}5Fe{Delta}RS05575) led to intracellular heme accumulation and hypersensitivity under anaerobic conditions, suggesting RS05575 encodes an anaerobilin synthase, the first of its kind described in Gram- positive bacteria. Additionally, deletion of RS05575, either alone or in the {Delta}5Fe background, impaired E. faecalis colonization in the mouse gastrointestinal tract and virulence in mouse peritonitis and rabbit infective endocarditis models. These results reveal that RS05575 is responsible for anaerobic degradation of heme and identify this relatively new enzyme class as a novel factor in bacterial pathogenesis. Findings from this study are likely to have broad implications, as homologues of RS05575 are found in other Gram-positive facultative anaerobes. IMPORTANCEHeme is an important nutrient for bacterial pathogens, mainly for its ability to serve as an iron source during infection. While bacteria are known to release iron from heme using enzymes called heme oxygenases, a new family of anaerobic heme-degrading enzymes has been described recently in Gram-negative bacteria. Here, we report the first description of anaerobic heme degradation by a Gram-positive bacterium, the opportunistic pathogen Enterococcus faecalis, and link activity of this enzyme to their ability to colonize and infect the host. We also show that homologues of this enzyme are found in many Gram-positive facultative anaerobes, implying that the ability to degrade heme under anaerobic conditions may be an overlooked fitness and virulence factor of bacterial pathogens.

microbiology↗

bioRxiv2023

Optimized replication of arrayed bacterial mutant libraries increase access to biological resources

Biological collections, including arrayed libraries of single transposon or deletion mutants, greatly accelerate the pace of bacterial genetics research. Despite the importance of these resources, few protocols exist for the replication and distribution of these materials. Here, we describe a protocol for creating multiple replicates of an arrayed bacterial Tn library consisting of approximately 6,800 mutants in 73 x 96-well plates. Our protocol provides multiple checkpoints to guard against contamination and minimize genetic drift caused by freeze/thaw cycles. This approach can also be scaled for arrayed culture collections of various sizes. Overall, this protocol is a valuable resource for other researchers considering the construction and distribution of arrayed culture collection resources for the benefit of the greater scientific community. ImportanceArrayed mutant collections drive robust genetic screens, yet few protocols exist for replication of these resources and subsequent quality control. Increasing distribution of arrayed biological collections will increase accessibility to and use of these resources. Developing standardized techniques for replication of these resources is essential for ensuring their quality and usefulness to the scientific community.

microbiology↗

bioRxiv2022

Identification of Multiple Iron Uptake Mechanisms in Enterococcus faecalis and Their Relationship to Virulence

Among the unfavorable conditions bacteria encounter within the host is restricted access to essential trace metals such as iron. To overcome iron deficiency, bacteria deploy multiple strategies to scavenge iron from host tissues with abundant examples of iron acquisition systems being implicated in bacterial pathogenesis. Yet, the mechanisms utilized by the major nosocomial pathogen Enterococcus faecalis to maintain intracellular iron balance are poorly understood. In this report, we conducted a systematic investigation to identify and characterize the iron acquisition mechanisms of E. faecalis and to determine their contribution to virulence. Bioinformatic analysis and literature surveys revealed that E. faecalis possesses three conserved iron uptake systems. Through transcriptomics, we discovered two novel ABC-type transporters that mediate iron uptake. While inactivation of a single transporter had minimal impact on the ability of E. faecalis to maintain iron homeostasis, inactivation of all five systems ({Delta}5Fe strain) disrupted intracellular iron homeostasis and considerably impaired cell growth under iron-deficiency. Virulence of the {Delta}5Fe strain was generally impaired in different animal models but showed niche-specific variations in mouse models, leading us to suspect that heme can serve as an iron source to E. faecalis during mammalian infections. Indeed, heme supplementation restored growth of {Delta}5Fe under iron-depletion and virulence in an invertebrate infection model. Collectively, this study reveals that the collective contribution of five iron transporters promotes E. faecalis virulence and that the ability to acquire and utilize heme as an iron source is critical to the systemic dissemination of E. faecalis.

microbiology↗