RESUMO
Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.
Assuntos
Toxinas Bacterianas/metabolismo , Enterococcus , Leucócitos Mononucleares , Fatores de Virulência/metabolismo , Animais , Bovinos , Enterococcus/metabolismo , Enterococcus/patogenicidade , Cavalos , Camundongos , Testes de Sensibilidade Microbiana , SuínosRESUMO
The microbiota shields the host against infections in a process known as colonization resistance. How infections themselves shape this fundamental process remains largely unknown. Here, we show that gut microbiota from previously infected hosts display enhanced resistance to infection. This long-term functional remodeling is associated with altered bile acid metabolism leading to the expansion of taxa that utilize the sulfonic acid taurine. Notably, supplying exogenous taurine alone is sufficient to induce this alteration in microbiota function and enhance resistance. Mechanistically, taurine potentiates the microbiota's production of sulfide, an inhibitor of cellular respiration, which is key to host invasion by numerous pathogens. As such, pharmaceutical sequestration of sulfide perturbs the microbiota's composition and promotes pathogen invasion. Together, this work reveals a process by which the host, triggered by infection, can deploy taurine as a nutrient to nourish and train the microbiota, promoting its resistance to subsequent infection.
Assuntos
Microbioma Gastrointestinal , Interações Hospedeiro-Patógeno , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Contagem de Colônia Microbiana , Microbioma Gastrointestinal/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Imunidade , Camundongos Endogâmicos C57BL , Sulfetos/metabolismo , Taurina/farmacologiaRESUMO
We examined the evolutionary history of leading multidrug resistant hospital pathogens, the enterococci, to their origin hundreds of millions of years ago. Our goal was to understand why, among the vast diversity of gut flora, enterococci are so well adapted to the modern hospital environment. Molecular clock estimation, together with analysis of their environmental distribution, phenotypic diversity, and concordance with host fossil records, place the origins of the enterococci around the time of animal terrestrialization, 425-500 mya. Speciation appears to parallel the diversification of hosts, including the rapid emergence of new enterococcal species following the End Permian Extinction. Major drivers of speciation include changing carbohydrate availability in the host gut. Life on land would have selected for the precise traits that now allow pathogenic enterococci to survive desiccation, starvation, and disinfection in the modern hospital, foreordaining their emergence as leading hospital pathogens.
Assuntos
Evolução Biológica , Enterococcus/genética , Animais , Doenças Transmissíveis Emergentes/microbiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana , Enterococcus/classificação , Enterococcus/citologia , Enterococcus/efeitos dos fármacos , Especiação Genética , Interações Hospedeiro-Patógeno , Larva/microbiologia , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.
Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Animais , Humanos , Enterococcus/genética , Antibacterianos/farmacologia , Enterococcus faecium/genética , Enterococcus faecalis/genética , Filogenia , Testes de Sensibilidade Microbiana , Farmacorresistência BacterianaRESUMO
SUMMARYEnterococci are a diverse group of Gram-positive bacteria that are typically found as commensals in humans, animals, and the environment. Occasionally, they may cause clinically relevant diseases such as endocarditis, septicemia, urinary tract infections, and wound infections. The majority of clinical infections in humans are caused by two species: Enterococcus faecium and Enterococcus faecalis. However, there is an increasing number of clinical infections caused by non-faecium non-faecalis (NFF) enterococci. Although NFF enterococcal species are often overlooked, studies have shown that they may harbor antimicrobial resistance (AMR) genes and virulence factors that are found in E. faecium and E. faecalis. In this review, we present an overview of the NFF enterococci with a particular focus on human clinical manifestations, epidemiology, virulence genes, and AMR genes.
Assuntos
Infecções por Bactérias Gram-Positivas , Fatores de Virulência , Humanos , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Fatores de Virulência/genética , Animais , Farmacorresistência Bacteriana , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Enterococcus/patogenicidade , Enterococcus/efeitos dos fármacos , Enterococcus/genética , VirulênciaRESUMO
Enterococcus faecalis is an opportunistic pathogen frequently causing nosocomial infections. The virulence of this organism is underpinned by its capacity to evade phagocytosis, allowing dissemination in the host. Immune evasion requires a surface polysaccharide produced by all enterococci, known as the enterococcal polysaccharide antigen (EPA). EPA consists of a cell wall-anchored rhamnose backbone substituted by strain-specific polysaccharides called 'decorations', essential for the biological activity of this polymer. However, the structural determinants required for innate immune evasion remain unknown, partly due to a lack of suitable validated assays. Here, we describe a quantitative, in vitro assay to investigate how EPA decorations alter phagocytosis. Using the E. faecalis model strain OG1RF, we demonstrate that a mutant with a deletion of the locus encoding EPA decorations can be used as a platform strain to express heterologous decorations, thereby providing an experimental system to investigate the inhibition of phagocytosis by strain-specific decorations. We show that the aggregation of cells lacking decorations is increasing phagocytosis and that this process does not involve the recognition of lipoproteins by macrophages. Collectively, our work provides novel insights into innate immune evasion by enterococci and paves the way for further studies to explore the structure/function relationship of EPA decorations.
Assuntos
Enterococcus faecalis , Evasão da Resposta Imune , Lipoproteínas , Macrófagos , Fagocitose , Enterococcus faecalis/imunologia , Enterococcus faecalis/metabolismo , Enterococcus faecalis/genética , Lipoproteínas/metabolismo , Lipoproteínas/genética , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/imunologia , Humanos , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Imunidade Inata , Virulência , Animais , CamundongosRESUMO
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.
Assuntos
Daptomicina , Enterococcus faecalis , Peptídeo Hidrolases , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Farmacorresistência Bacteriana/genética , Enterococcus faecalis/genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/metabolismo , Enterococcus faecalis/enzimologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genéticaRESUMO
Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.
RESUMO
BACKGROUND: Admission and discharge screening of patients for asymptomatic gut colonization with multidrug-resistant organisms (MDROs) is a traditional approach to active surveillance, but its sensitivity for detecting colonization is uncertain. METHODS: Daily rectal or fecal swab samples and clinical data were collected over 12 months from patients in one 25-bed intensive care unit (ICU) in Chicago, IL USA and tested for the following multidrug-resistant organisms (MDROs): vancomycin-resistant enterococci (VRE); third-generation cephalosporin-resistant Enterobacterales, including extended-spectrum ß-lactamase-producing Enterobacterales (ESBL); and carbapenem-resistant Enterobacterales (CRE). MDRO detection by (1) admission/discharge surveillance cultures or (2) clinical cultures were compared to daily surveillance cultures. Samples underwent 16S rRNA gene sequencing to measure the relative abundance of operational taxonomic units (OTUs) corresponding to each MDRO. RESULTS: Compared with daily surveillance cultures, admission/discharge cultures detected 91% of prevalent MDRO colonization and 63% of incident MDRO colonization among medical ICU patients. Only a minority (7%) of MDRO carriers were identified by clinical cultures. Higher relative abundance of MDRO-associated OTUs and specific antibiotic exposures were independently associated with higher probability of MDRO detection by culture. CONCLUSION: Admission and discharge surveillance cultures underestimated MDRO acquisitions in an ICU. These limitations should be considered when designing sampling strategies for epidemiologic studies that use culture-based surveillance.
RESUMO
Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.
Assuntos
Daptomicina , Enterococcus faecium , Transferases , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Daptomicina/metabolismo , Daptomicina/farmacologia , Farmacorresistência Bacteriana , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/metabolismo , Testes de Sensibilidade Microbiana , Peptidoglicano/metabolismo , Transferases/metabolismoRESUMO
Enterococci are common commensal bacteria that colonize the gastrointestinal tracts of most mammals, including humans. Importantly, these bacteria are one of the leading causes of nosocomial infections. This study examined the role of colonic macrophages in facilitating Enterococcus faecalis infections in mice. We determined that depletion of colonic phagocytes resulted in the reduction of E. faecalis dissemination to the gut-draining mesenteric lymph nodes. Furthermore, we established that trafficking of monocyte-derived CX3CR1-expressing macrophages contributed to E. faecalis dissemination in a manner that was not reliant on CCR7, the conventional receptor involved in lymphatic migration. Finally, we showed that E. faecalis mutants with impaired intracellular survival exhibited reduced dissemination, suggesting that E. faecalis can exploit host immune cell migration to disseminate systemically and cause disease. Our findings indicate that modulation of macrophage trafficking in the context of antibiotic therapy could serve as a novel approach for preventing or treating opportunistic infections by disseminating enteric pathobionts like E. faecalis.
Assuntos
Receptor 1 de Quimiocina CX3C , Colo , Enterococcus faecalis , Macrófagos , Receptores CCR2 , Receptores de Quimiocinas , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Macrófagos/microbiologia , Macrófagos/imunologia , Camundongos , Colo/microbiologia , Colo/imunologia , Receptores CCR2/metabolismo , Receptores CCR2/genética , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/genética , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/microbiologia , Camundongos Endogâmicos C57BL , Linfonodos/microbiologia , Linfonodos/imunologia , Receptores CCR7/metabolismo , Receptores CCR7/genéticaRESUMO
Enterococcus faecalis is a common cause of healthcare-acquired bloodstream infections and catheter-associated urinary tract infections (CAUTIs) 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 the 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, ΔhylA exhibited defects in bladder colonization and dissemination to the bloodstream, and ΔhylB exhibited a defect in kidney colonization. Furthermore, a ΔhylAΔhylB 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 hyaluronic acid (HA) and chondroitin sulfate 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 the stationary phase and also contributed to dampening of lipopolysaccharide-mediated NF-κB activation. Overall, these data demonstrate that glycosaminoglycan degradation is important for E. faecalis pathogenesis in the urinary tract and during bloodstream infection.
Assuntos
Bacteriemia , Infecções Relacionadas a Cateter , Enterococcus faecalis , Glicosaminoglicanos , Infecções por Bactérias Gram-Positivas , Infecções Urinárias , Enterococcus faecalis/genética , Enterococcus faecalis/enzimologia , Enterococcus faecalis/metabolismo , Infecções Urinárias/microbiologia , Bacteriemia/microbiologia , Infecções Relacionadas a Cateter/microbiologia , Animais , Infecções por Bactérias Gram-Positivas/microbiologia , Camundongos , Glicosaminoglicanos/metabolismo , Hialuronoglucosaminidase/metabolismo , Hialuronoglucosaminidase/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Feminino , Humanos , Ácido Hialurônico/metabolismoRESUMO
BACKGROUND: Current guidelines recommend adjunctive gentamicin for the treatment of Enterococcus faecalis infective endocarditis (EFIE) despite a risk of toxicity. We sought to revisit the evidence for adjunctive therapy in EFIE and to synthesize the comparative safety and effectiveness of adjunctive use of the aminoglycosides versus ceftriaxone by systematic review and meta-analysis. METHODS: For historical context, we reviewed the seminal case series and in vitro studies informing the evolution from penicillin monotherapy to modern-day regimens for EFIE. Next, we searched MEDLINE and Embase from inception to January 16, 2024 for studies of EFIE comparing 1) adjunctive aminoglycosides versus ceftriaxone or 2) adjunctive therapy versus monotherapy. Where possible, clinical outcomes were compared between regimens by random-effects meta-analysis. Otherwise, data were narratively summarized. RESULTS: Results for the systematic review and meta-analysis were limited to 10 observational studies totaling 911 patients. All studies were at high risk of bias. Relative to adjunctive ceftriaxone, gentamicin had similar all-cause mortality (Risk Difference [RD]=-0.8%, 95% Confidence interval [95%CI]=-5.0, 3.5), relapse (RD=-0.1%, 95%CI=-2.4, 2.3), and treatment failure (RD=1.1%, 95%CI=-1.6, 3.7), but higher discontinuation due to toxicity (RD=26.3%, 95%CI=19.8, 32.7). The 3 studies comparing adjunctive therapy to monotherapy included only 30 monotherapy patients and heterogeneity precluded meta-analysis. CONCLUSION: Adjunctive therapy with ceftriaxone appeared to be equally effective and less toxic than gentamicin for the treatment of EFIE. The existing evidence does not clearly establish the superiority of either adjunctive therapy or monotherapy. Pending randomized evidence, if adjunctive therapy is to be used, ceftriaxone appears to be a reasonable option.
RESUMO
BACKGROUND: Enterococcus faecium and E. lactis are phylogenetically closely related lactic acid bacteria that are ubiquitous in nature and are known to be beneficial or pathogenic. Despite their considerable industrial and clinical importance, comprehensive studies on their evolutionary relationships and genomic, metabolic, and pathogenic traits are still lacking. Therefore, we conducted comparative pangenome analyses using all available dereplicated genomes of these species. RESULTS: E. faecium was divided into two subclades: subclade I, comprising strains derived from humans, animals, and food, and the more recent phylogenetic subclade II, consisting exclusively of human-derived strains. In contrast, E. lactis strains, isolated from diverse sources including foods, humans, animals, and the environment, did not display distinct clustering based on their isolation sources. Despite having similar metabolic features, noticeable genomic differences were observed between E. faecium subclades I and II, as well as E. lactis. Notably, E. faecium subclade II strains exhibited significantly larger genome sizes and higher gene counts compared to both E. faecium subclade I and E. lactis strains. Furthermore, they carried a higher abundance of antibiotic resistance, virulence, bacteriocin, and mobile element genes. Phylogenetic analysis of antibiotic resistance and virulence genes suggests that E. faecium subclade II strains likely acquired these genes through horizontal gene transfer, facilitating their effective adaptation in response to antibiotic use in humans. CONCLUSIONS: Our study offers valuable insights into the adaptive evolution of E. faecium strains, enabling their survival as pathogens in the human environment through horizontal gene acquisitions.
Assuntos
Enterococcus faecium , Animais , Humanos , Filogenia , Enterococcus , Genômica , AntibacterianosRESUMO
Enterococcus faecalis, a formidable nosocomial and community-acquired opportunistic pathogen, can persist a wide range of extreme environments, including low pH and nutrient deficiency. Clarifying the survival mechanism of E. faecalis in low-pH conditions is the key to combating the infectious diseases caused by E. faecalis. In this study, we combined transcriptome profiling (RNA-seq) and transposon insertion sequencing (TIS) to comprehensively understand the genes that confer these features on E. faecalis. The metadata showed that genes whose products are involved in cation transportation and amino acid biosynthesis were predominantly differentially expressed under acid conditions. The products of genes such as opp1C and copY reduced the hydrion concentration in the cell, whereas those of gldA2, gnd2, ubiD, and ubiD2 mainly participated in amino metabolism, increasing matters to neutralize excess acid. These, together with the folE and hexB genes, which are involved in mismatch repair, form a network of E. faecalis genes necessary for its survival under acid conditions.
IMPORTANCE: As a serious nosocomial pathogen, Enterococcus faecalis was considered responsible for large numbers of infections. Its ability to survive under stress conditions, such as acid condition and nutrient deficiency was indispensable for its growth and infection. Therefore, understanding how E. faecalis survives acid stress is necessary for the prevention and treatment of related diseases. RNA-seq and TIS provide us a way to analyze the changes in gene expression under such conditions.
Assuntos
Enterococcus faecalis , Perfilação da Expressão Gênica , RNA-Seq , Enterococcus faecalis/genética , GenomaRESUMO
Enterococcus faecalis virulence requires cell wall-associated proteins, including the sortase-assembled endocarditis and biofilm associated pilus (Ebp), important for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that sortases attach substrates to lipid II peptidoglycan (PG) precursors, prior to their incorporation into the growing cell wall. Contrary to prevailing dogma, by following the distribution of Ebp and PG throughout the E. faecalis cell cycle, we found that cell surface Ebp do not co-localize with newly synthesized PG. Instead, surface-exposed Ebp are localized to the older cell hemisphere and excluded from sites of new PG synthesis at the septum. Moreover, Ebp deposition on the younger hemisphere of the E. faecalis diplococcus appear as foci adjacent to the nascent septum. We propose a new model whereby sortase substrate deposition can occur on older PG rather than at sites of new cell wall synthesis. Consistent with this model, we demonstrate that sequestering lipid II to block PG synthesis via ramoplanin, does not impact new Ebp deposition at the cell surface. These data support an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto uncrosslinked cell wall, independent of new PG synthesis.
Assuntos
Aminoaciltransferases , Proteínas de Fímbrias , Proteínas de Fímbrias/metabolismo , Enterococcus faecalis/metabolismo , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Parede Celular/metabolismo , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismoRESUMO
The bacterial PASTA kinase, IreK, is required for intrinsic cephalosporin resistance in the Gram-positive opportunistic pathogen, Enterococcus faecalis. IreK activity is enhanced in response to cell wall stress, such as cephalosporin exposure. The downstream consequences of IreK activation are not well understood in E. faecalis, but recent work in other low-GC Gram-positive bacteria demonstrated PASTA kinase-dependent regulation of MurAA, an enzyme that performs the first committed step in the peptidoglycan synthesis pathway. Here, we used genetic suppressor selections to identify MurAA as a downstream target of IreK signaling in E. faecalis. Using complementary genetic and biochemical approaches, we demonstrated that MurAA abundance is regulated by IreK signaling in response to physiologically relevant cell wall stress to modulate substrate flux through the peptidoglycan synthesis pathway. Specifically, the IreK substrate, IreB, promotes proteolysis of MurAA through a direct physical interaction in a manner responsive to phosphorylation by IreK. MurAB, a homolog of MurAA, also promotes MurAA proteolysis and interacts directly with IreB. Our results therefore establish a connection between the cell wall stress sensor IreK and one critical physiological output to modulate peptidoglycan synthesis and drive cephalosporin resistance.
Assuntos
Enterococcus faecalis , Peptidoglicano , Enterococcus faecalis/metabolismo , Peptidoglicano/metabolismo , Resistência às Cefalosporinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fosfotransferases/metabolismo , Parede Celular/metabolismoRESUMO
Antimicrobial tolerance is the ability of a microbial population to survive, but not proliferate, during antimicrobial exposure. Significantly, it has been shown to precede the development of bona fide antimicrobial resistance. We have previously identified the two-component system CroRS as a critical regulator of tolerance to antimicrobials like teixobactin in the bacterial pathogen Enterococcus faecalis. To understand the molecular mechanism of this tolerance, we have carried out RNA-seq analyses in the E. faecalis wild-type and isogenic ∆ croRS mutant to determine the teixobactin-induced CroRS regulon. We identified a 132 gene CroRS regulon and demonstrate that CroRS upregulates biosynthesis of all major components of the enterococcal cell envelope in response to teixobactin. This suggests a coordinating role of this regulatory system in maintaining integrity of the multiple layers of the enterococcal envelope during antimicrobial stress, likely contributing to bacterial survival. Using experimental evolution, we observed that truncation of HppS, a key enzyme in the synthesis of the quinone electron carrier demethylmenaquinone, was sufficient to rescue tolerance in the croRS deletion strain. This highlights a key role for isoprenoid biosynthesis in antimicrobial tolerance in E. faecalis. Here, we propose a model of CroRS acting as a master regulator of cell envelope biogenesis and a gate-keeper between isoprenoid biosynthesis and respiration to ensure tolerance against antimicrobial challenge.
Assuntos
Antibacterianos , Anti-Infecciosos , Antibacterianos/farmacologia , Enterococcus faecalis/genética , Proteínas de Bactérias/genética , Homeostase , Terpenos , Testes de Sensibilidade MicrobianaRESUMO
Daptomycin (DAP) is often used as a first-line therapy to treat vancomycin-resistant Enterococcus faecium infections, but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system, and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface-exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium with an activated LiaFSR system. Unlike E. faecalis, E. faecium LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX enzyme-linked immunosorbent assay (ELISA). We then assessed 86 clinical E. faecium bloodstream isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-resistant clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-susceptible isolates by standard MIC determination also had elevated LiaX ELISAs compared to a well-characterized DAP-susceptible strain. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many E. faecium isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.
Assuntos
Membrana Celular , Daptomicina , Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Humanos , Antibacterianos/uso terapêutico , Biomarcadores/metabolismo , Daptomicina/farmacologia , Daptomicina/uso terapêutico , Farmacorresistência Bacteriana/genética , Enterococcus faecalis , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/metabolismo , Testes de Sensibilidade Microbiana , Filogenia , Reprodutibilidade dos TestesRESUMO
Infective endocarditis (IE) caused by Enterococcus spp. represents the third most common cause of IE, with high rates of relapse compared with other bacteria. Interestingly, late relapses (>6 months) have only been described in Enterococcus faecalis, but here we describe the first reported IE relapse with Enterococcus faecium more than a year (17 months) after the initial endocarditis episode. Firstly, by multi locus sequence typing (MLST), we demonstrated that both isolates (EF646 and EF641) belong to the same sequence type (ST117). Considering that EF641 was able to overcome starvation and antibiotic treatment conditions surviving for a long period of time, we performed bioinformatic analysis in identifying potential genes involved in virulence and stringent response. Our results showed a 13-nucleotide duplication (positions 1638-1650) in the gene relA, resulting in a premature stop codon, with a loss of 167 amino acids from the C-terminal domains of the RelA enzyme. RelA mediates the stringent response in bacteria, modulating levels of the alarmone guanosine tetraphosphate (ppGpp). The relA mutant (EF641) was associated with lower growth capacity, the presence of small colony variants, and higher capacity to produce biofilms (compared with the strain EF646), but without differences in antimicrobial susceptibility patterns according to standard procedures during planktonic growth. Instead, EF641 demonstrated tolerance to high doses of teicoplanin when growing in a biofilm. We conclude that all these events would be closely related to the long-term survival of the E. faecium and the late relapse of the IE. These data represent the first clinical evidence of mutations in the stringent response (relA gene) related with E. faecium IE relapse.