An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis.

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyze the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identify large decreases in N2-methyladenosine (m2A) in both 23 S ribosomal RNA and transfer RNA. This we determine to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gives rise to a proteome that mimics the oxidative stress response, with an increase in levels of superoxide dismutase and decrease in virulence proteins. While tRNA modifications were established to be dynamic for fine-tuning translation, here we report the discovery of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to a model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, adding a different paradigm in which RNA modifications can directly regulate the proteome.

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 study, 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 (Δ5Fe strain) disrupted intracellular iron homeostasis and considerably impaired cell growth under iron deficiency. Virulence of the Δ5Fe 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 Δ5Fe under iron depletion and virulence in an invertebrate infection model. This study revealed 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.

Dual species biofilms are enhanced by metabolite cross-feeding.

Enterococcus faecalis and Staphylococcus aureus are frequently co-isolated from biofilm-associated infections. A new study by Ch'ng et al. revealed that S. aureus-released heme feeds E. faecalis respiration, augmenting E. faecalis growth and overall biofilm biomass. Their finding further supports the theory that metabolite cross-feeding is a critical aspect shaping polymicrobial biofilm interactions.

Enterococcus faecalis Antagonizes Pseudomonas aeruginosa Growth in Mixed-Species Interactions.

Enterococcus faecalis is often coisolated with Pseudomonas aeruginosa in polymicrobial biofilm-associated infections of wounds and the urinary tract. As a defense strategy, the host innately restricts iron availability at infection sites. Despite their coprevalence, the polymicrobial interactions of these two species in biofilms and under iron-restricted conditions remain unexplored. Here, we show that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase (ldh1) gives rise to l-lactate production during fermentative growth. We find that an E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced under iron-restricted conditions, resulting in increased lactic acid exported and, consequently, a reduction in local environmental pH. Together, our results suggest that E. faecalis synergistically inhibits P. aeruginosa growth by decreasing environmental pH and l-lactate-mediated iron chelation. Overall, this study emphasizes the importance of the microenvironment in polymicrobial interactions and how manipulating the microenvironment can impact the growth trajectory of bacterial communities. IMPORTANCE Many infections are polymicrobial and biofilm-associated in nature. Iron is essential for many metabolic processes and plays an important role in controlling infections, where the host restricts iron as a defense mechanism against invading pathogens. However, polymicrobial interactions between pathogens are underexplored under iron-restricted conditions. Here, we explore the polymicrobial interactions between commonly coisolated E. faecalis and P. aeruginosa within biofilms. We find that E. faecalis modulates the microenvironment by exporting lactic acid which further chelates already limited iron and also lowers the environmental pH to antagonize P. aeruginosa growth under iron-restricted conditions. Our findings provide insights into polymicrobial interactions between bacteria and how manipulating the microenvironment can be taken advantage of to better control infections.

Methods for Using the Galleria mellonella Invertebrate Model to Probe Enterococcus faecalis Pathogenicity.

The Enterococci, mainly Enterococcus faecalis and E. faecium, are ubiquitous members of the human gastrointestinal tract consortia but also a leading cause of opportunistic infections. The global rise in human-associated enterococcal infections, often caused by multidrug resistant strains, highlights an urgent need to identify the bacterial factors contributing to its pathogenicity such that new therapies can be devised. The use of the Galleria mellonella (greater wax moth) larvae, commonly known as wax worm, as a model to study host-pathogen interactions has allowed the identification and characterization of numerous bacterial factors that contribute to disease in humans, serving both as an alternative and complementary approach to mammalian models. Here, we describe the methods for using G. mellonella to characterize the virulence factors of E. faecalis.

The AdcACB/AdcAII system is essential for zinc homeostasis and an important contributor of Enterococcus faecalis virulence.

Bacterial pathogens require a variety of micronutrients for growth, including trace metals such as iron, manganese, and zinc (Zn). Despite their relative abundance in host environments, access to these metals is severely restricted during infection due to host-mediated defense mechanisms collectively known as nutritional immunity. Despite a growing appreciation of the importance of Zn in host-pathogen interactions, the mechanisms of Zn homeostasis and the significance of Zn to the pathophysiology of E. faecalis, a major pathogen of nosocomial and community-associated infections, have not been thoroughly investigated. Here, we show that E. faecalis encoded ABC-type transporter AdcACB and an orphan substrate-binding lipoprotein AdcAII that work cooperatively to maintain Zn homeostasis. Simultaneous inactivation of adcA and adcAII or the entire adcACB operon led to a significant reduction in intracellular Zn under Zn-restricted conditions and heightened sensitivity to Zn-chelating agents including human calprotectin, aberrant cell morphology, and impaired fitness in serum ex vivo. Additionally, inactivation of adcACB and adcAII significantly reduced bacterial tolerance toward cell envelope-targeting antibiotics. Finally, we showed that the AdcACB/AdcAII system contributes to E. faecalis virulence in a Galleria mellonella invertebrate infection model and in two catheter-associated mouse infection models that recapitulate many of the host conditions associated with enterococcal human infections. Collectively, this report reveals that high-affinity Zn import is important for the pathogenesis of E. faecalis establishing the surface-associated AdcA and AdcAII lipoproteins as potential therapeutic targets.

c-di-AMP Is Essential for the Virulence of Enterococcus faecalis.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP, and ΔdhhP ΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the ΔcdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

The composition and function of Enterococcus faecalis membrane vesicles.

Membrane vesicles (MVs) contribute to various biological processes in bacteria, including virulence factor delivery, antimicrobial resistance, host immune evasion and cross-species communication. MVs are frequently released from the surface of both Gram-negative and Gram-positive bacteria during growth. In some Gram-positive bacteria, genes affecting MV biogenesis have been identified, but the mechanism of MV formation is unknown. In Enterococcus faecalis, a causative agent of life-threatening bacteraemia and endocarditis, neither mechanisms of MV formation nor their role in virulence has been examined. Since MVs of many bacterial species are implicated in host-pathogen interactions, biofilm formation, horizontal gene transfer, and virulence factor secretion in other species, we sought to identify, describe and functionally characterize MVs from E. faecalis. Here, we show that E. faecalis releases MVs that possess unique lipid and protein profiles, distinct from the intact cell membrane and are enriched in lipoproteins. MVs of E. faecalis are specifically enriched in unsaturated lipids that might provide membrane flexibility to enable MV formation, providing the first insights into the mechanism of MV formation in this Gram-positive organism.

Enterococcus faecalis Manganese Exporter MntE Alleviates Manganese Toxicity and Is Required for Mouse Gastrointestinal Colonization.

Bacterial pathogens encounter a variety of nutritional environments in the human host, including nutrient metal restriction and overload. Uptake of manganese (Mn) is essential for Enterococcus faecalis growth and virulence; however, it is not known how this organism prevents Mn toxicity. In this study, we examine the role of the highly conserved MntE transporter in E. faecalis Mn homeostasis and virulence. We show that inactivation of mntE results in growth restriction in the presence of excess Mn, but not other metals, demonstrating its specific role in Mn detoxification. Upon growth in the presence of excess Mn, an mntE mutant accumulates intracellular Mn, iron (Fe), and magnesium (Mg), supporting a role for MntE in Mn and Fe export and a role for Mg in offsetting Mn toxicity. Growth of the mntE mutant in excess Fe also results in increased levels of intracellular Fe, but not Mn or Mg, providing further support for MntE in Fe efflux. Inactivation of mntE in the presence of excess iron also results in the upregulation of glycerol catabolic genes and enhanced biofilm growth, and addition of glycerol is sufficient to augment biofilm growth for both the mntE mutant and its wild-type parental strain, demonstrating that glycerol availability significantly enhances biofilm formation. Finally, we show that mntE contributes to colonization of the antibiotic-treated mouse gastrointestinal (GI) tract, suggesting that E. faecalis encounters excess Mn in this niche. Collectively, these findings demonstrate that the manganese exporter MntE plays a crucial role in E. faecalis metal homeostasis and virulence.

Author Correction: Biofilm-associated infection by enterococci.

In the section on initial attachment and in Figure 1 it was erroneously indicated that enterococcal surface protein (Esp) binds collagen and fibrinogen. The text and figure were changed to remove this binding interaction both online and in the pdf. The authors apologize for any confusion caused.

Biofilm-associated infection by enterococci.

Enterococci are ubiquitous members of the human gut microbiota and frequent causes of biofilm-associated opportunistic infections. Enterococci cause 25% of all catheter-associated urinary tract infections, are frequently isolated in wounds and are increasingly found in infective endocarditis, and all of these infections are associated with biofilms. Enterococcal biofilms are intrinsically tolerant to antimicrobials and thus are a serious impediment for treating infections. In this Review, we describe the spatiotemporal development of enterococcal biofilms and the factors that promote or inhibit biofilm formation. We discuss how the environment, including the host and other co-colonizing microorganisms, affects biofilm development. Finally, we provide an overview of current and future interventions to limit enterococcal biofilm-associated infections. Overall, enterococcal biofilms remain a pressing clinical problem, and there is an urgent need to better understand their development and persistence and to identify novel treatments.

Extracellular Electron Transfer Powers Enterococcus faecalis Biofilm Metabolism.

Enterococci are important human commensals and significant opportunistic pathogens. Biofilm-related enterococcal infections, such as endocarditis, urinary tract infections, wound and surgical site infections, and medical device-associated infections, often become chronic upon the formation of biofilm. The biofilm matrix establishes properties that distinguish this state from free-living bacterial cells and increase tolerance to antimicrobial interventions. The metabolic versatility of the enterococci is reflected in the diversity and complexity of environments and communities in which they thrive. Understanding metabolic factors governing colonization and persistence in different host niches can reveal factors influencing the transition to biofilm pathogenicity. Here, we report a form of iron-dependent metabolism for Enterococcus faecalis where, in the absence of heme, extracellular electron transfer (EET) and increased ATP production augment biofilm growth. We observe alterations in biofilm matrix depth and composition during iron-augmented biofilm growth. We show that the ldh gene encoding l-lactate dehydrogenase is required for iron-augmented energy production and biofilm formation and promotes EET.IMPORTANCE Bacterial metabolic versatility can often influence the outcome of host-pathogen interactions, yet causes of metabolic shifts are difficult to resolve. The bacterial biofilm matrix provides the structural and functional support that distinguishes this state from free-living bacterial cells. Here, we show that the biofilm matrix can immobilize iron, providing access to this growth-promoting resource which is otherwise inaccessible in the planktonic state. Our data show that in the absence of heme, Enterococcus faecalis l-lactate dehydrogenase promotes EET and uses matrix-associated iron to carry out EET. Therefore, the presence of iron within the biofilm matrix leads to enhanced biofilm growth.