An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis.

Daptomycin is a last-line antibiotic commonly used to treat vancomycin-resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol-modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here, we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH, resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the ΔmprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced extracellular acidification and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies.

Purine and carbohydrate availability drive Enterococcus faecalis fitness during wound and urinary tract infections.

IMPORTANCE: Although E. faecalis is a common wound pathogen, its pathogenic mechanisms during wound infection are unexplored. Here, combining a mouse wound infection model with in vivo transposon and RNA sequencing approaches, we identified the E. faecalis purine biosynthetic pathway and galactose/mannose MptABCD phosphotransferase system as essential for E. faecalis acute replication and persistence during wound infection, respectively. The essentiality of purine biosynthesis and the MptABCD PTS is driven by the consumption of purine metabolites by E. faecalis during acute replication and changing carbohydrate availability during the course of wound infection. Overall, our findings reveal the importance of the wound microenvironment in E. faecalis wound pathogenesis and how these metabolic pathways can be targeted to better control wound infections.

Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis.

The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human ß-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild-type and mprF mutant strains and show that while ΔmprF2 and ΔmprF1 ΔmprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the ΔmprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in ΔmprF2 and ΔmprF1 ΔmprF2. In the mprF mutants, particularly ΔmprF1 ΔmprF2, the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the ΔmprF1 ΔmprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both ΔmprF2 and ΔmprF1 ΔmprF2, compared to the wild type and ΔmprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. IMPORTANCE The cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.

Lipid-Polymer Hybrid Nanoparticles Enhance the Potency of Ampicillin against Enterococcus faecalis in a Protozoa Infection Model.

Enterococcus faecalis (E. faecalis) biofilms are implicated in endocarditis, urinary tract infections, and biliary tract infections. Coupled with E. faecalis internalization into host cells, this opportunistic pathogen poses great challenges to conventional antibiotic therapy. The inability of ampicillin (Amp) to eradicate bacteria hidden in biofilms and intracellular niches greatly reduces its efficacy against complicated E. faecalis infections. To enhance the potency of Amp against different forms of E. faecalis infections, Amp was loaded into Lipid-Polymer hybrid Nanoparticles (LPNs), a highly efficient nano delivery platform consisting of a unique combination of DOTAP lipid shell and PLGA polymeric core. The antibacterial activity of these nanoparticles (Amp-LPNs) was investigated in a protozoa infection model, achieving a much higher multiplicity of infection (MOI) compared with studies using animal phagocytes. A significant reduction of total E. faecalis was observed in all groups receiving 250 µg/mL Amp-LPNs compared with groups receiving the same concentration of free Amp during three different interventions, simulating acute and chronic infections and prophylaxis. In early intervention, no viable E. faecalis was observed after 3 h LPNs treatment whereas free Amp did not clear E. faecalis after 24 h treatment. Amp-LPNs also greatly enhanced the antibacterial activity of Amp at late intervention and boosted the survival rate of protozoa approaching 400%, where no viable protozoa were identified in the free Amp groups at the 40 h postinfection treatment time point. Prophylactic effectiveness with Amp-LPNs at a concentration of 250 µg/mL was exhibited in both bacteria elimination and protozoa survival toward subsequent infections. Using protozoa as a surrogate model for animal phagocytes to study high MOI infections, this study suggests that LPN-formulated antibiotics hold the potential to significantly improve the therapeutic outcome in highly complicated bacterial infections.

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.

Enterococcus faecalis Modulates Immune Activation and Slows Healing During Wound Infection.

Enterococcus faecalis is one of the most frequently isolated bacterial species in wounds yet little is known about its pathogenic mechanisms in this setting. Here, we used a mouse wound excisional model to characterize the infection dynamics of E faecalis and show that infected wounds result in 2 different states depending on the initial inoculum. Low-dose inocula were associated with short-term, low-titer colonization whereas high-dose inocula were associated with acute bacterial replication and long-term persistence. High-dose infection and persistence were also associated with immune cell infiltration, despite suppression of some inflammatory cytokines and delayed wound healing. During high-dose infection, the multiple peptide resistance factor, which is involved in resisting immune clearance, contributes to E faecalis fitness. These results comprehensively describe a mouse model for investigating E faecalis wound infection determinants, and suggest that both immune modulation and resistance contribute to persistent, nonhealing wounds.

Enterococcus faecalis Promotes Innate Immune Suppression and Polymicrobial Catheter-Associated Urinary Tract Infection.

Enterococcus faecalis, a member of the human gastrointestinal microbiota, is an opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. E. faecalis can subvert or evade immune-mediated clearance, although the mechanisms are poorly understood. In this study, we examined E. faecalis-mediated subversion of macrophage activation. We observed that E. faecalis actively prevents NF-κB signaling in mouse RAW264.7 macrophages in the presence of Toll-like receptor agonists and during polymicrobial infection with Escherichia coliE. faecalis and E. coli coinfection in a mouse model of catheter-associated urinary tract infection (CAUTI) resulted in a suppressed macrophage transcriptional response in the bladder compared to that with E. coli infection alone. Finally, we demonstrated that coinoculation of E. faecalis with a commensal strain of E. coli into catheterized bladders significantly augmented E. coli CAUTI. Taken together, these results support the hypothesis that E. faecalis suppression of NF-κB-driven responses in macrophages promotes polymicrobial CAUTI pathogenesis, especially during coinfection with less virulent or commensal E. coli strains.

Model systems for the study of Enterococcal colonization and infection.

Enterococcus faecalis and Enterococcus faecium are common inhabitants of the human gastrointestinal tract, as well as frequent opportunistic pathogens. Enterococci cause a range of infections including, most frequently, infections of the urinary tract, catheterized urinary tract, bloodstream, wounds and surgical sites, and heart valves in endocarditis. Enterococcal infections are often biofilm-associated, polymicrobial in nature, and resistant to antibiotics of last resort. Understanding Enterococcal mechanisms of colonization and pathogenesis are important for identifying new ways to manage and intervene with these infections. We review vertebrate and invertebrate model systems applied to study the most common E. faecalis and E. faecium infections, with emphasis on recent findings examining Enterococcal-host interactions using these models. We discuss strengths and shortcomings of each model, propose future animal models not yet applied to study mono- and polymicrobial infections involving E. faecalis and E. faecium, and comment on the significance of anti-virulence strategies derived from a fundamental understanding of host-pathogen interactions in model systems.

Polymicrobial-Host Interactions during Infection.

Microbial pathogenesis research has, historically, focused on the study of infections as monomicrobial events. However, the advent of next generation sequencing and culture-independent identification methods has revealed that many, if not most, infections are polymicrobial either in origin or in manifestation. Polymicrobial infections are often associated with increased infection severity and poorer patient outcome. Multiple infecting microbes can interact synergistically to induce virulence traits, alter the infected niche, or modulate the host immune response, all of which can promote polymicrobial infection. Importantly, a polymicrobial environment at the time of inoculation, consisting of multiple pathogens or pathogens in combination with the native microbiota, can contribute to the pathogenic progression of a single predominant organism at the time of diagnosis. Hence, in order to completely understand and elucidate the impact of these polymicrobial interactions on infection outcomes, a thorough examination of the entire microbial community present throughout the pathogenic cascade is required: from the time of inoculation to symptomology to resolution. In this review, we highlight the themes of metabolite exploitation, immune modulation, niche optimization, and virulence induction that contribute to polymicrobial infections. We focus on recent literature about microbe-microbe and microbe-host interactions that promote polymicrobial infections with an emphasis on understanding these interactions to identify better interventions for these sometimes complex infections.

Accessory proteins of SARS-CoV and other coronaviruses.

The huge RNA genome of SARS coronavirus comprises a number of open reading frames that code for a total of eight accessory proteins. Although none of these are essential for virus replication, some appear to have a role in virus pathogenesis. Notably, some SARS-CoV accessory proteins have been shown to modulate the interferon signaling pathways and the production of pro-inflammatory cytokines. The structural information on these proteins is also limited, with only two (p7a and p9b) having their structures determined by X-ray crystallography. This review makes an attempt to summarize the published knowledge on SARS-CoV accessory proteins, with an emphasis on their involvement in virus-host interaction. The accessory proteins of other coronaviruses are also briefly discussed. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses" (see Introduction by Hilgenfeld and Peiris (2013)).