RESUMEN
Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faecalis, the endocarditis and biofilm-associated pilus (Ebp) is polymerized on the membrane by sortase C (SrtC) and attached to the cell wall by sortase A (SrtA). In the absence of SrtA, polymerized pili remain anchored to the membrane (i.e. off-pathway). Here we show that the high temperature requirement A (HtrA) bifunctional chaperone/protease of E. faecalis is a quality control system that clears aberrant off-pathway pili from the cell membrane. In the absence of HtrA and SrtA, accumulation of membrane-bound pili leads to cell envelope stress and partially induces the regulon of the ceftriaxone resistance-associated CroRS two-component system, which in turn causes hyper-piliation and cell morphology alterations. Inactivation of croR in the OG1RF ΔsrtAΔhtrA background partially restores the observed defects of the ΔsrtAΔhtrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal resistance of E. faecalis against cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into the E. faecalis response to endogenous membrane perturbations.
Asunto(s)
Aminoaciltransferasas , Proteínas Bacterianas , Biopelículas , Cisteína Endopeptidasas , Enterococcus faecalis , Fimbrias Bacterianas , Chaperonas Moleculares , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Enterococcus faecalis/genética , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Biopelículas/crecimiento & desarrollo , Membrana Celular/metabolismo , Regulación Bacteriana de la Expresión Génica , Virulencia/genética , Antibacterianos/farmacología , Ceftriaxona/farmacologíaRESUMEN
Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.
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Compuestos de Anilina , Antibacterianos , Supervivencia Celular , Macrófagos , Fagocitosis , Animales , Humanos , Ratones , Compuestos de Anilina/farmacología , Antibacterianos/farmacología , Supervivencia Celular/efectos de los fármacos , Enterococcus faecalis/efectos de los fármacos , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Nitrilos/farmacología , Fagocitosis/efectos de los fármacos , Quinolinas/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
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Daptomicina , Enterococcus faecalis , Péptido Hidrolasas , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Daptomicina/farmacología , Farmacorresistencia Bacteriana/genética , Enterococcus faecalis/genética , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/metabolismo , Enterococcus faecalis/enzimología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/genéticaRESUMEN
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.
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Infecciones Urinarias , Infección de Heridas , Animales , Ratones , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Carbohidratos , PurinasRESUMEN
Enterococcus faecalis is an opportunistic pathogen that is frequently co-isolated with other microbes in wound infections. While E. faecalis can subvert the host immune response and promote the survival of other microbes via interbacterial synergy, little is known about the impact of E. faecalis-mediated immune suppression on co-infecting microbes. We hypothesized that E. faecalis can attenuate neutrophil-mediated responses in mixed-species infection to promote survival of the co-infecting species. We found that neutrophils control E. faecalis infection via phagocytosis, ROS production, and degranulation of azurophilic granules, but it does not trigger neutrophil extracellular trap formation (NETosis). However, E. faecalis attenuates Staphylococcus aureus-induced NETosis in polymicrobial infection by interfering with citrullination of histone, suggesting E. faecalis can actively suppress NETosis in neutrophils. Residual S. aureus-induced NETs that remain during co-infection do not impact E. faecalis, further suggesting that E. faecalis possess mechanisms to evade or survive NET-associated killing mechanisms. E. faecalis-driven reduction of NETosis corresponds with higher S. aureus survival, indicating that this immunomodulating effect could be a risk factor in promoting the virulence polymicrobial infection. These findings highlight the complexity of the immune response to polymicrobial infections and suggest that attenuated pathogen-specific immune responses contribute to pathogenesis in the mammalian host.
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Antibiotic resistance critically limits treatment options for infection caused by opportunistic pathogens such as enterococci. Here, we investigate the antibiotic and immunological activity of the anticancer agent mitoxantrone (MTX) in vitro and in vivo against vancomycin-resistant Enterococcus faecalis (VRE). We show that, in vitro, MTX is a potent antibiotic against Gram-positive bacteria through induction of reactive oxygen species and DNA damage. MTX also synergizes with vancomycin against VRE, rendering the resistant strains more permeable to MTX. In a murine wound infection model, single-dose MTX treatment effectively reduces VRE numbers, with further reduction when combined with vancomycin. Multiple MTX treatments accelerate wound closure. MTX also promotes macrophage recruitment and proinflammatory cytokine induction at the wound site and augments intracellular bacterial killing in macrophages by up-regulating the expression of lysosomal enzymes. These results show that MTX represents a promising bacterium- and host-targeted therapeutic for overcoming vancomycin resistance.
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Enterococcus faecalis , Enterococos Resistentes a la Vancomicina , Animales , Ratones , Enterococcus faecalis/genética , Resistencia a la Vancomicina/genética , Vancomicina/farmacología , Mitoxantrona/farmacología , Antibacterianos/farmacología , Enterococos Resistentes a la Vancomicina/genéticaRESUMEN
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.
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Antibacterianos , Antiinfecciosos , Humanos , Antibacterianos/farmacología , Antibacterianos/metabolismo , Enterococcus faecalis/metabolismo , Farmacorresistencia Bacteriana , Fosfolípidos/metabolismo , Antiinfecciosos/metabolismo , Ácidos Grasos/metabolismo , Fosfatidilgliceroles/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/metabolismo , Cationes/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
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.
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Aminoaciltransferasas , Proteínas Fimbrias , Proteínas Fimbrias/metabolismo , Enterococcus faecalis/metabolismo , Proteínas Bacterianas/metabolismo , Fimbrias Bacterianas/metabolismo , Pared Celular/metabolismo , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismoRESUMEN
Wound infections are often polymicrobial in nature, biofilm associated and therefore tolerant to antibiotic therapy, and associated with delayed healing. Escherichia coli and Staphylococcus aureus are among the most frequently cultured pathogens from wound infections. However, little is known about the frequency or consequence of E. coli and S. aureus polymicrobial interactions during wound infections. Here we show that E. coli kills Staphylococci, including S. aureus, both in vitro and in a mouse excisional wound model via the genotoxin, colibactin. Colibactin biosynthesis is encoded by the pks locus, which we identified in nearly 30% of human E. coli wound infection isolates. While it is not clear how colibactin is released from E. coli or how it penetrates target cells, we found that the colibactin intermediate N-myristoyl-D-Asn (NMDA) disrupts the S. aureus membrane. We also show that the BarA-UvrY two component system (TCS) senses the environment created during E. coli and S. aureus mixed species interaction, leading to upregulation of pks island genes. Further, we show that BarA-UvrY acts via the carbon storage global regulatory (Csr) system to control pks expression. Together, our data demonstrate the role of colibactin in interspecies competition and show that it is regulated by BarA-UvrY TCS during interspecies competition.
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Infecciones por Escherichia coli , Proteínas de Escherichia coli , Proteínas de la Membrana , Fosfotransferasas , Policétidos , Staphylococcus aureus , Factores de Transcripción , Animales , Antibacterianos/metabolismo , Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Mutágenos/metabolismo , N-Metilaspartato/metabolismo , Péptidos , Fosfotransferasas/genética , Policétidos/metabolismo , Staphylococcus/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Factores de Transcripción/metabolismo , Infección de Heridas/microbiologíaRESUMEN
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.
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Enterococcus faecalis , Pseudomonas aeruginosa , Biopelículas , Enterococcus faecalis/metabolismo , Hierro/metabolismo , Ácido Láctico/metabolismo , Pseudomonas aeruginosa/metabolismoRESUMEN
The contribution of biofilms to virulence and as a barrier to treatment is well-established for Staphylococcus aureus and Enterococcus faecalis, both nosocomial pathogens frequently isolated from biofilm-associated infections. Despite frequent co-isolation, their interactions in biofilms have not been well-characterized. We report that in combination, these two species can give rise to augmented biofilms biomass that is dependent on the activation of E. faecalis aerobic respiration. In E. faecalis, respiration requires both exogenous heme to activate the cydAB-encoded heme-dependent cytochrome bd, and the availability of O2. We determined that the ABC transporter encoded by cydDC contributes to heme import. In dual species biofilms, S. aureus provides the heme to activate E. faecalis respiration. S. aureus mutants deficient in heme biosynthesis were unable to augment biofilms whereas heme alone is sufficient to augment E. faecalis mono-species biofilms. Our results demonstrate that S. aureus-derived heme, likely in the form of released hemoproteins, promotes E. faecalis biofilm formation, and that E. faecalis gelatinase activity facilitates heme extraction from hemoproteins. This interspecies interaction and metabolic cross-feeding may explain the frequent co-occurrence of these microbes in biofilm-associated infections.
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Enterococcus faecalis , Staphylococcus aureus , Biopelículas , Enterococcus faecalis/genética , Hemo , Staphylococcus aureus/genética , VirulenciaRESUMEN
Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using in vitro keratinocyte and macrophage infection models, we show that E. faecalis becomes internalized and a subpopulation of bacteria can survive and replicate intracellularly. E. faecalis are internalized into keratinocytes primarily via macropinocytosis into single membrane-bound compartments and can persist in late endosomes up to 24 h after infection in the absence of colocalization with the lysosomal protease Cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in heterotypic intracellular trafficking with partial or absent labelling of E. faecalis-containing compartments with Rab5 and Rab7, small GTPases required for the endosome-lysosome trafficking. In addition, E. faecalis infection results in marked reduction of Rab5 and Rab7 protein levels which may also contribute to attenuated Rab incorporation into E. faecalis-containing compartments. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection.
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Enterococcus faecalis , Proteínas de Unión al GTP rab , Animales , Endosomas/metabolismo , Enterococcus faecalis/metabolismo , Lisosomas/metabolismo , Mamíferos , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Biofilm formation is a multifactorial process and often a multi-species endeavour that involves complex signalling networks, chemical gradients, bacterial adhesion, and production or acquisition of matrix components. Antibiotics remain the main choice when treating bacterial biofilm-associated infections despite their intrinsic tolerance to antimicrobials, and propensity for acquisition and rapid dissemination of antimicrobial resistance within the biofilm. Eliminating hard to treat biofilm-associated infections that are antibiotic resistant will demand a holistic and multi-faceted approach, targeting multiple stages of biofilm formation, many of which are already in development. This mini review will highlight the current approaches that are employed to treat bacterial biofilm infections and discuss new approaches in development that have promise to reach clinical practice.
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Antiinfecciosos , Infecciones Bacterianas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Biopelículas , Humanos , Estudios ProspectivosRESUMEN
[This corrects the article DOI: 10.1093/femsml/uqab002.].
RESUMEN
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.
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Staphylococcus aureus (SA) is the most common bacterial species in chronic wounds. However, there is a lack of understanding of how SA secretions affect the cell biology during the healing process. We studied the effects of biofilm-secretions from SA strain SA29213 on 3T3 fibroblasts. SA29213 is a chronic wound isolate and widely used as a reference strain. We used a series of concentrations of biofilm-conditioned media (BCM) and found 100% BCM is lethal within 10 h. Cells survived in ≤75% BCM but the rate of closure in scratch wound assays was reduced. Treatment with 75% and 50% BCM caused fibroblasts to change shape and develop dendrite like processes. Prolonged treatment with 75% and 50% BCM reduced cell proliferation and increased the 4n deoxyribonucleic acid cell population with cell cycle arrest. There was also an elevation in the senescence marker beta galactosidase and the number of multinucleated cells. Shorter treatments with 75% and 50% SA BCM caused an increase in cell-cell adhesion and a redistribution of ß-catenin from the cell membrane to the cytoplasm along with a change in the appearance and decrease in size of ZO-1, vinculin and paxillin structures. Fibroblasts in the edge of chronic wounds exposed to the secretions of SA may suffer similar effects such as induction of senescence, reduced proliferation and migration, which may contribute to the delayed healing of these chronic infected wounds.
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The emergence of multidrug-resistant bacteria has made minor bacterial infections incurable with many existing antibiotics. Lysins are phage-encoded peptidoglycan hydrolases that have demonstrated therapeutic potential as a novel class of antimicrobials. The modular architecture of lysins enables the functional domains - catalytic domain (CD) and cell wall binding domain (CBD) - to be shuffled to create novel lysins. The CD is classically thought to be only involved in peptidoglycan hydrolysis whereas the CBD dictates the lytic spectrum of a lysin. While there are many studies that extended the lytic spectrum of a lysin by domain swapping, few have managed to introduce species specificity in a chimeric lysin. In this work, we constructed two chimeric lysins by swapping the CBDs of two parent lysins with different lytic spectra against enterococci and staphylococci. We showed that these chimeric lysins exhibited customized lytic spectra distinct from the parent lysins. Notably, the chimeric lysin P10N-V12C, which comprises a narrow-spectrum CD fused with a broad-spectrum CBD, displayed species specificity not lysing Enterococcus faecium while targeting Enterococcus faecalis and staphylococci. Such species specificity can be attributed to the narrow-spectrum CD of the chimeric lysin. Using flow cytometry and confocal microscopy, we found that the E. faecium cells that were treated with P10N-V12C are less viable with compromised membranes yet remained morphologically intact. Our results suggest that while the CBD is a major determinant of the lytic spectrum of a lysin, the CD is also responsible in the composition of the final lytic spectrum, especially when it pertains to species-specificity.
RESUMEN
Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication.IMPORTANCEEnterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.
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Biopelículas/crecimiento & desarrollo , Sistemas CRISPR-Cas , Enterococcus faecalis/genética , Enterococcus faecalis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genes EsencialesRESUMEN
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.
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Proteínas Bacterianas/metabolismo , Enterococcus faecalis/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Manganeso/metabolismo , Animales , Biopelículas , Transporte Biológico , Modelos Animales de Enfermedad , Tracto Gastrointestinal/microbiología , Homeostasis , Espacio Intracelular/metabolismo , Manganeso/toxicidad , Metales/metabolismo , RatonesRESUMEN
Characterisation of protein function based solely on homology searches may overlook functions under specific environmental conditions, or the possibility of a protein having multiple roles. In this study we investigated the role of YtfB, a protein originally identified in a genome-wide screen to cause inhibition of cell division, and has demonstrated to localise to the Escherichia coli division site with some degree of glycan specificity. Interestingly, YtfB also shows homology to the virulence factor OapA from Haemophilus influenzae, which is important for adherence to epithelial cells, indicating the potential of additional function(s) for YtfB. Here we show that E. coli YtfB binds to N'acetylglucosamine and mannobiose glycans with high affinity. The loss of ytfB results in a reduction in the ability of the uropathogenic E. coli strain UTI89 to adhere to human kidney cells, but not to bladder cells, suggesting a specific role in the initial adherence stage of ascending urinary tract infections. Taken together, our results suggest a role for YtfB in adhesion to specific eukaryotic cells, which may be additional, or complementary, to its role in cell division. This study highlights the importance of understanding the possible multiple functions of proteins based on homology, which may be specific to different environmental conditions.