Utilization of glycoprotein-derived N-acetylglucosamine-L-asparagine during Enterococcus faecalis infection depends on catabolic and transport enzymes of the glycosylasparaginase locus.

Enterococcus faecalis is a Gram-positive clinical pathogen causing severe infections. Its survival during infection depends on its ability to utilize host-derived metabolites, such as protein-deglycosylation products. We have identified in E. faecalis OG1RF a locus (ega) involved in the catabolism of the glycoamino acid N-acetylglucosamine-L-asparagine. This locus is separated into two transcription units, genes egaRP and egaGBCD1D2, respectively. RT-qPCR experiments revealed that the expression of the ega locus is regulated by the transcriptional repressor EgaR. Electromobility shift assays evidenced that N-acetylglucosamine-L-asparagine interacts directly with the EgaR protein, which leads to the transcription of the ega genes. Growth studies with egaG, egaB and egaC mutants confirmed that the encoded proteins are necessary for N-acetylglucosamine-L-asparagine catabolism. This glycoamino acid is transported and phosphorylated by a specific phosphotransferase system EIIABC components (OG1RF_10751, EgaB, EgaC) and subsequently hydrolyzed by the glycosylasparaginase EgaG, which generates aspartate and 6-P-N-acetyl-ß-d-glucosaminylamine. The latter can be used as a fermentable carbon source by E. faecalis. Moreover, Galleria mellonella larvae had a significantly higher survival rate when infected with ega mutants compared to the wild-type strain, suggesting that the loss of N-acetylglucosamine-L-asparagine utilization affects enterococcal virulence.

The enigmatic physiological roles of AhpCF, Gpx, Npr and Kat in peroxide stress response of Enterococcus faecium.

In this work, the physiological roles of the primary peroxide scavenging activities of Enterococcus faecium AUS0004 strain were analysed. This healthcare-associated pathogen harbours genes encoding putative NADH peroxidase (Npr), alkyl hydroperoxide reductase (AhpCF), glutathione peroxidase (Gpx) and manganese-dependent catalase (Mn-Kat). Gene expression analyses showed that npr and kat genes are especially and significantly induced in cells treated with hydrogen peroxide (H2O2) and cumene hydroperoxide (CuOOH), which suggested an important function of these enzymes to protect E. faecium against peroxide stress. Mutants affected in one or several predicted anti-oxidative activities mentioned above showed that neither the peroxidases nor the catalase are implicated in the defence against peroxide challenges. However, our investigations allowed us to show that Npr is responsible for the degradation of approximately 45% of metabolically derived H2O2 which avoids accumulation of the peroxide to lethal concentrations.

Alternatives to Fight Vancomycin-Resistant Staphylococci and Enterococci.

Gram positive pathogens are a significant cause of healthcare-associated infections, with Staphylococci and Enterococci being the most prevalent ones. Vancomycin, a last resort glycopeptide, is used to fight these bacteria but the emergence of resistance against this drug leaves some patients with few therapeutic options. To counter this issue, new generations of antibiotics have been developed but resistance has already been reported. In this article, we review the strategies in place or in development to counter vancomycin-resistant pathogens. First, an overview of traditional antimicrobials already on the market or in the preclinical or clinical pipeline used individually or in combination is summarized. The second part focuses on the non-traditional antimicrobials, such as antimicrobial peptides, bacteriophages and nanoparticles. The conclusion is that there is hitherto no substitute equivalent to vancomycin. However, promising strategies based on drugs with multiple mechanisms of action and treatments based on bacteriophages possibly combined with conventional antibiotics are hoped to provide treatment options for vancomycin-resistant Gram-positive pathogens.

Characterisation of the manganese superoxide dismutase of Enterococcus faecium.

The manganese superoxide dismutase (SodA) of E. faecium strain AUS0004 has been characterised. It is most closely related to Enterococcus hirae, Enterococcus durans, Enterococcus villorium, and Enterococcus mundtii with 100%, 91,55%, 90,85%, and 90,58% homology, respectively, but more distant from SodA of E. faecalis (81.68%). A sodA deletion mutant has been constructed. Compared to the parental strain, the ΔsodA mutant was affected in aerobic growth and more sensitive to hydrogen peroxide (H2O2), cumene hydroperoxide (CuOOH), and the superoxide anion (O2•-) generator menadione. The E. faecium strain AUS0004 is part of those bacteria accumulating H2O2 to high concentrations (around 5 mM) starting from late exponential growth phase. Accumulation of the peroxide was around 25% less in the mutant suggesting that this part of H2O2 is due to the dismutation of O2•- by SodA. The sodA gene of E. faecium AUS0004 was induced by oxygen, peroxides and menadione but the corresponding regulator remains hitherto unknown. Finally, we showed that SodA activity is important for virulence in the Galleria mellonella model.

Inhibition of d-alanylation of teichoic acids overcomes resistance of methicillin-resistant Staphylococcus aureus.

BACKGROUND: MRSA are high-priority multidrug-resistant pathogens. Although there are still some antibiotics active against MRSA, continuous efforts to discover new antibiotics and treatment strategies are needed because resistance to these new drugs has already been reported. OBJECTIVES: Here we explore if d-alanylation of teichoic acids (TAs) mediated by the dlt operon gene products might be a druggable target to overcome ß-lactam-resistance of MRSA. METHODS: MICs and bactericidal effects of several ß-lactam antibiotics were monitored in a panel of clinical MRSA strains with genetic or chemically induced deficiency in d-alanylation of TAs. Efficiency of the chemical inhibitor to rescue MRSA-infected larvae of Galleria mellonella as well as its ability to prevent or eradicate biofilms of S. aureus were analysed. RESULTS: Genetic inactivation of the Dlt system or its chemical inhibition re-sensitizes MRSA to ß-lactams. Among the 13 strains, the most pronounced effect was obtained using the inhibitor with imipenem, reducing the median MIC from 16 to 0.25 mg/L. This combination was also bactericidal in some strains and significantly protected G. mellonella larvae from lethal MRSA infections. Finally, inactivation of d-alanylation potentiated the effect of imipenem on inhibition and/or eradication of biofilm. CONCLUSIONS: Our combined results show that highly efficient inhibitors of d-alanylation of TAs targeting enzymes of the Dlt system should be promising therapeutic adjuvants, especially in combination with carbapenems, for restoring the therapeutic efficacy of this class of antibiotics against MRSA.

Analysis of glycerol and dihydroxyacetone metabolism in Enterococcus faecium.

Glycerol (Gly) can be dissimilated by two pathways in bacteria. Either this sugar alcohol is first oxidized to dihydroxyacetone (DHA) and then phosphorylated or it is first phosphorylated to glycerol-3-phosphate (GlyP) followed by oxidation. Oxidation of GlyP can be achieved by NAD-dependent dehydrogenases or by a GlyP oxidase. In both cases, dihydroxyacetone phosphate is the product. Genomic analysis showed that Enterococcus faecium harbors numerous genes annotated to encode activities for the two pathways. However, our physiological analyses of growth on glycerol showed that dissimilation is limited to aerobic conditions and that despite the presence of genes encoding presumed GlyP dehydrogenases, the GlyP oxidase is essential in this process. Although E. faecium contains an operon encoding the phosphotransfer protein DhaM and DHA kinase, which are required for DHA phosphorylation, it is unable to grow on DHA. This operon is highly expressed in stationary phase but its physiological role remains unknown. Finally, data obtained from sequencing of a transposon mutant bank of E. faecium grown on BHI revealed that the GlyP dehydrogenases and a major intrinsic family protein have important but hitherto unknown physiological functions.

Enterococcus faecalis Maltodextrin Gene Regulation by Combined Action of Maltose Gene Regulator MalR and Pleiotropic Regulator CcpA.

Enterococci are Gram-positive bacteria present in the healthy human microbiota, but they are also a leading cause of nosocomial infections. Maltodextrin utilization by Enterococcus faecalis has been identified as an important factor for colonization of mammalians hosts. Here, we show that the LacI/GalR transcriptional regulator MalR, the maltose gene regulator, is also the main regulator of the operons encoding an ABC transporter (mdxEFG) and three metabolic enzymes (mmdH-gmdH-mmgT) required for the uptake and catabolism of maltotetraose and longer maltodextrins. The utilization of maltose and maltodextrins is consequently coordinated and induced by the disaccharide maltose, which binds to MalR. Carbon catabolite repression of the mdxEFG and mmdH-gmdH-mmgT operons is mediated by both P-Ser-HPr/MalR and P-Ser-HPr/CcpA. The latter complex exerts only moderate catabolite repression, which became visible when comparing maltodextrin operon expression levels of a malR- mutant (with a mutant allele for the malR gene) and a malR- ΔccpA double mutant grown in the presence of maltose, which is transported via a phosphotransferase system and, thus, favors the formation of P-Ser-HPr. Moreover, maltodextrin transport via MdxEFG slows rapidly when glucose is added, suggesting an additional regulation via inducer exclusion. This complex regulation of metabolic operons likely allows E. faecalis to fine-tune gene expression in response to changing environmental conditions.IMPORTANCEEnterococcus faecalis represents a leading cause of hospital-acquired infections worldwide. Several studies highlighted the importance of carbohydrate metabolism in the infection process of this bacterium. The genes required for maltodextrin metabolism are particularly induced during mouse infection and, therefore, should play an important role for pathogenesis. Since no data were hitherto available concerning the regulation of expression of the maltodextrin operons, we have conducted experiments to study the underlying mechanisms.

Opposite effect of vancomycin and D-Cycloserine combination in both vancomycin resistant Staphylococcus aureus and enterococci.

The increasing spread of antibiotic resistant bacteria is a major human health concern. The challenging development of new effective antibiotics has led to focus on seeking synergistic antibiotic combinations. Vancomycin (VAN) is a glycopeptide antibiotic used to treat Staphylococcus aureus and enterococci infections. It is targeting D-Alanyl-D-Alanine dimers during peptidoglycan biosynthesis. D-cycloserine (DCS) is a D-Alanine analogue that targets peptidoglycan biosynthesis by inhibiting D-Alanine:D-Alanine ligase (Ddl). The VAN-DCS combination was found to be synergistic in VAN resistant S. aureus strains lacking van genes cluster. We hypothesize that this combination leads to opposite effects in S. aureus and enterococci strains harboring van genes cluster where VAN resistance is conferred by the synthesis of modified peptidoglycan precursors ending in D-Alanyl-D-Lactate. The calculated Fractional Inhibitory Concentration of VAN-DCS combination in a van- vancomycin-intermediate, VanA type, and VanB type strains were 0.5, 5 and 3, respectively. As a result, VAN-DCS combination leads to synergism in van-lacking strains, and to antagonism in strains harboring van genes cluster. The VAN-DCS antagonism is due to a mechanism that we named van-mediated Ddl inhibition bypass. Our results show that antibiotic combinations can lead to opposite effects depending on the genetic backgrounds.

Study of key RNA metabolism proteins in Enterococcus faecalis.

The control of mRNA turnover is essential in bacteria to allow rapid adaptation, especially in opportunistic pathogen like Enterococcus faecalis. This mechanism involves RNase and DEAD-box helicases that are key elements in RNA processing and their associations form the degradosome with accessory proteins. In this study, we investigated the function of four RNases (J1, J2, Y and III) and three DEAD-box helicases (CshA, CshB, CshC) present in most Enterococci. The interactions of all these RNA metabolism actors were investigated in vitro, and the results are in accordance with a degradosome structure close to the one of Bacillus subtilis. At the physiological level, we showed that RNase J1 is essential, whereas RNases J2 and III have a role in cold, oxidative and bile salts stress response, and RNase Y in general fitness. Furthermore, RNases J2, Y and III mutants are affected in virulence in the Galleria mellonella infection model. Concerning DEAD-box helicases, all of them are involved in cold shock response. Since the ΔcshA mutant was the most stress impacted strain, we studied this DEAD-box helicase CshA in more detail. This showed that CshA autoregulates its own expression by binding to its mRNA 5'Unstranslated Region. Interestingly, CshC is also involved in the expression control of CshA by a hitherto unprecedented mechanism.

Diversity of volatile organic compound production from leucine and citrate in Enterococcus faecium.

Enterococcus faecium is frequently isolated from fermented food; in particular, they positively contribute to the aroma compound generation in traditional cheese. Citrate fermentation is a desirable property in these bacteria, but this feature is not uniformly distributed among E. faecium strains. In the present study, three selected E. faecium strains, IQ110 (cit-), GM70 (cit+ type I), and Com12 (cit+ type II), were analyzed in their production of aroma compounds in milk. End products and volatile organic compounds (VOCs) were determined by solid-phase micro-extraction combined with gas chromatography mass spectrometry (SPME-GC-MS). Principal component analysis (PCA) of aroma compound profiles revealed a different VOC composition for the three strains. In addition, resting cell experiments of E. faecium performed in the presence of leucine, citrate, or pyruvate as aroma compound precursors allowed us to determine metabolic differences between the studied strains. GM70 (cit+ type I) showed an active citrate metabolism, with increased levels of diacetyl and acetoin generation relative to Com12 or to citrate defective IQ110 strains. In addition, in the experimental conditions tested, a defective citrate-fermenting phenotype for the Com12 strain was found, while its leucine degradation and pyruvate metabolism were conserved. In conclusion, rational selection of E. faecium strains could be performed based on genotypic and phenotypic analyses. This would result in a performing strain, such as GM70, that could positively contribute to flavor, with typical notes of diacetyl, acetoin, 3-methyl butanal, and 3-methyl butanol in an adjuvant culture.

Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl-phosphorylated-HPr.

Enterococci are gram-positive pathogens and lead to cause hospital-acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α-polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA-independent carbon catabolite repression. The central PTS protein P-Ser-HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions.

ß-Lactam Exposure Triggers Reactive Oxygen Species Formation in Enterococcus faecalis via the Respiratory Chain Component DMK.

Whereas the primary actions of ß-lactams are well characterized, their downstream effects are less well understood. Although their targets are extracellular, ß-lactams stimulate respiration in Escherichia coli leading to increased intracellular accumulation of reactive oxygen species (ROS). Here, we show that ß-lactams over a large concentration range trigger a strong increase in ROS production in Enterococcus faecalis under aerobic, but not anaerobic, conditions. Both amoxicillin, to which the bacterium is susceptible, and cefotaxime, to which E. faecalis is resistant, triggers this response. This stimulation of ROS formation depends mainly on demethylmenaquinone (DMK), a component of the E. faecalis respiratory chain, but in contrast to E. coli is observed only in the absence of respiration. Our results suggest that in E. faecalis, ß-lactams increase electron flux through the respiratory chain, thereby stimulating the auto-oxidation of reduced DMK in the absence of respiration, which triggers increased extracellular ROS production.

Characterization of the gen locus involved in ß-1,6-oligosaccharide utilization by Enterococcus faecalis.

The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6-phospho-ß-glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of ß-1,6- rather than ß-1,4-linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose-6'-P with a Kd of 0.04 mM and with lower affinity also other phospho-sugars. The GenR/gentiobiose-6'-P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel-shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750-OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter.

Genetic and pharmacological inactivation of d-alanylation of teichoic acids sensitizes pathogenic enterococci to ß-lactams.

BACKGROUND: Enterococci intrinsically resistant to cephalosporins represent a major cause of healthcare-associated infections, and the emergence of MDR makes therapeutic approaches particularly challenging. OBJECTIVES: Teichoic acids are cell wall glycopolymers present in Gram-positive bacteria. Teichoic acids can be modified by d-alanylation, which requires four proteins encoded by the dltABCD operon. Our objective was to evaluate the Dlt system as a druggable target to treat enterococcal infections. METHODS: The susceptibility of a d-alanylation-deficient strain of Enterococcus faecalis to ß-lactam antibiotics individually and/or in combination was analysed. Moreover, a DltA inhibitor was synthesized to test pharmacological inhibition of d-alanylation in vivo and in host using the animal model Galleria mellonella with different clinical isolates of E. faecalis and Enterococcus faecium. RESULTS: Most cephalosporins used as mono treatment had no impact on survival of the parental strain, but were slightly lethal for the dltA mutant of E. faecalis. Addition of a very low concentration of amoxicillin significantly increased killing of the dltA mutant under these conditions. The most spectacular effect was obtained with a combination of cefotaxime (1 mg/L) and amoxicillin (0.03 mg/L). In the presence of the inhibitor, the WT strain was as susceptible to this combination treatment as the dltA mutant. This molecule associated with the antibiotics was also effective in killing other E. faecalis clinical isolates and successfully prevented death of Galleria infected with either E. faecalis or E. faecium. CONCLUSIONS: The combined results support the potential usefulness of the Dlt system as a target to potentiate antibiotic combination therapies for the treatment of drug-resistant enterococci.

Expression profiling in a mammalian host reveals the strong induction of genes encoding LysM domain-containing proteins in Enterococcus faecium.

Enterococcus faecium is an important health care-associated pathogen that is difficult to treat due to the high level of antibiotic resistance of clinical isolates. The identification of new potential therapeutic targets or vaccination strategies is therefore urgently needed. In this regard, we carried out a transcriptomic analysis of the E. faecium vancomycin-resistant strain AUS0004, comparing the gene expression of bacteria grown under laboratory conditions and bacteria isolated from an infection site. This analysis highlighted more than 360 genes potentially induced under infection conditions. Owing to their expression profiles, four LysM domain-containing proteins were characterized in more detail. The EFAU004_01059, 1150 and 494 proteins are highly homologous, whereas EFAU004_01209 has a unique domain-architecture and sequence. The analysis of corresponding mutants showed that all LysM proteins played relevant roles in the infection process of E. faecium in mice. The EFAU004_01209 mutant also displayed profound morphological modifications, suggesting it has a role in cell wall synthesis or cell division. Furthermore, the adhesion to kidney cells and growth of the mutant was affected in human urine. All these phenotypes and the surface exposure of EFAU004_01209 identify this protein as an interesting new drug target in E. faecium.

Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities.

Maltose and maltodextrins are formed during the degradation of starch or glycogen. Maltodextrins are composed of a mixture of maltooligosaccharides formed by α-1,4- but also some α-1,6-linked glucosyl residues. The α-1,6-linked glucosyl residues are derived from branching points in the polysaccharides. In Enterococcus faecalis, maltotriose is mainly transported and phosphorylated by a phosphoenolpyruvate:carbohydrate phosphotransferase system. The formed maltotriose-6″-phosphate is intracellularly dephosphorylated by a specific phosphatase, MapP. In contrast, maltotetraose and longer maltooligosaccharides up to maltoheptaose are taken up without phosphorylation via the ATP binding cassette transporter MdxEFG-MsmX. We show that the maltose-producing maltodextrin hydrolase MmdH (GenBank accession no. EFT41964) in strain JH2-2 catalyzes the first catabolic step of α-1,4-linked maltooligosaccharides. The purified enzyme converts even-numbered α-1,4-linked maltooligosaccharides (maltotetraose, etc.) into maltose and odd-numbered (maltotriose, etc.) into maltose and glucose. Inactivation of mmdH therefore prevents the growth of E. faecalis on maltooligosaccharides ranging from maltotriose to maltoheptaose. Surprisingly, MmdH also functions as a maltogenic α-1,6-glucosidase, because it converts the maltotriose isomer isopanose into maltose and glucose. In addition, E. faecalis contains a glucose-producing α-1,6-specific maltodextrin hydrolase (GenBank accession no. EFT41963, renamed GmdH). This enzyme converts panose, another maltotriose isomer, into glucose and maltose. A gmdH mutant had therefore lost the capacity to grow on panose. The genes mmdH and gmdH are organized in an operon together with GenBank accession no. EFT41962 (renamed mmgT). Purified MmgT transfers glucosyl residues from one α-1,4-linked maltooligosaccharide molecule to another. For example, it catalyzes the disproportionation of maltotriose by transferring a glucosyl residue to another maltotriose molecule, thereby forming maltotetraose and maltose together with a small amount of maltopentaose.IMPORTANCE The utilization of maltodextrins by Enterococcus faecalis has been shown to increase the virulence of this nosocomial pathogen. However, little is known about how this organism catabolizes maltodextrins. We identified two enzymes involved in the metabolism of various α-1,4- and α-1,6-linked maltooligosaccharides. We found that one of them functions as a maltose-producing α-glucosidase with relaxed linkage specificity (α-1,4 and α-1,6) and exo- and endoglucosidase activities. A third enzyme, which resembles amylomaltase, exclusively transfers glucosyl residues from one maltooligosaccharide molecule to another. Similar enzymes are present in numerous other Firmicutes, such as streptococci and lactobacilli, suggesting that these organisms follow the same maltose degradation pathway as E. faecalis.

Enterococcus faecalis Uses a Phosphotransferase System Permease and a Host Colonization-Related ABC Transporter for Maltodextrin Uptake.

Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection.IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two different transporters is used: the maltose-PTS transporter MalT, or the MdxEFG-MsmX ABC transporter. MdxEFG-MsmX takes up longer maltodextrins as well as complex molecules, such as panose and isopanose.

Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections.

BACKGROUND: Enterococcus faecium and faecalis are Gram-positive opportunistic pathogens that have become leading causes of nosocomial infections over the last decades. Especially multidrug resistant enterococci have become a challenging clinical problem worldwide. Therefore, new treatment options are needed and the identification of alternative targets for vaccine development has emerged as a feasible alternative to fight the infections caused by these pathogens. RESULTS: We extrapolate the transcriptomic data from a mice peritonitis infection model in E. faecalis to identify putative up-regulated surface proteins under infection conditions in E. faecium. After the bionformatic analyses two metal binding lipoproteins were identified to have a high homology (>72%) between the two species, the manganese ABC transporter substrate-binding lipoprotein (PsaAfm,) and the zinc ABC transporter substrate-binding lipoprotein (AdcAfm). These candidate lipoproteins were overexpressed in Escherichia coli and purified. The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Mice were passively immunized with the antibodies raised against recombinant lipoproteins, showing significant reduction of colony counts in mice livers after the bacterial challenge and demonstrating the efficacy of these metal binding lipoproteins as promising vaccine candidates to treat infections caused by these enterococcal pathogens. CONCLUSION: Overall, our results demonstrate that these two metal binding lipoproteins elicited specific, opsonic and protective antibodies, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens. Pointing these two protein antigens as promising immunogens, that can be used as single components or as carrier proteins together with polysaccharide antigens in vaccine development against enterococcal infections.

Characterization of Sviceucin from Streptomyces Provides Insight into Enzyme Exchangeability and Disulfide Bond Formation in Lasso Peptides.

Lasso peptides are bacterial ribosomally synthesized and post-translationally modified peptides. They have sparked increasing interest in peptide-based drug development because of their compact, interlocked structure, which offers superior stability and protein-binding capacity. Disulfide bond-containing lasso peptides are rare and exhibit highly sought-after activities. In an effort to expand the repertoire of such molecules, we heterologously expressed, in Streptomyces coelicolor, the gene cluster encoding sviceucin, a type I lasso peptide with two disulfide bridges originating from Streptomyces sviceus, which allowed it to be fully characterized. Sviceucin and its reduced forms were characterized by mass spectrometry and peptidase digestion. The three-dimensional structure of sviceucin was determined using NMR. Sviceucin displayed antimicrobial activity selectively against Gram-positive bacteria and inhibition of fsr quorum sensing in Enterococcus faecalis. This study adds sviceucin to the type I lasso peptide family as a new representative. Moreover, new clusters encoding disulfide-bond containing lasso peptides from Actinobacteria were identified by genome mining. Genetic and functional analyses revealed that the formation of disulfide bonds in sviceucin does not require a pathway-encoded thiol-disulfide oxidoreductase. Most importantly, we demonstrated the functional exchangeability of the sviceucin and microcin J25 (a non-disulfide-bridged lasso peptide) macrolactam synthetases in vitro, highlighting the potential of hybrid lasso synthetases in lasso peptide engineering.

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci.

UNLABELLED: Enterococci are naturally tolerant to typically bactericidal cell wall-active antibiotics, meaning that their growth is inhibited but they are not killed even when exposed to a high concentration of the drug. The molecular reasons for this extraordinary tolerance are still incompletely understood. Previous work showed that resistance to killing collapsed specifically in mutants affected in superoxide dismutase (Sod) activity, arguing that bactericidal antibiotic treatment led to induction of a superoxide burst. In the present work, we show that loss of antibiotic tolerance in ΔsodA mutants of pathogenic enterococci is dependent on the energy source present during antibiotic treatment. Hexoses induce greater killing than the pentose ribose, and no killing was observed with glycerol as the energy source. These results point to glycolytic reactions as crucial for antibiotic-mediated killing of ΔsodA mutants. A transposon mutant library was constructed in Enterococcus faecalis ΔsodA mutants and screened for restored tolerance of vancomycin. Partially restored tolerance was observed in mutants with transposon integrations into intergenic regions upstream of regulators implicated in arginine catabolism. In these mutants, the arginine deiminase operon was highly upregulated. A model for the action of cell wall-active antibiotics in tolerant and nontolerant bacteria is proposed. IMPORTANCE: Antibiotic tolerance is a serious clinical concern, since tolerant bacteria have considerably increased abilities to resist killing by bactericidal drugs. Using enterococci as models for highly antibiotic-tolerant pathogens, we showed that tolerance of these bacteria is linked to their superoxide dismutase (Sod), arguing that bactericidal antibiotics induce generation of reactive oxygen species inside cells. Wild-type strains are tolerant because they detoxify these deleterious molecules by the activity of Sod, whereas Sod-deficient strains are killed. This study showed that killing depends on the energy source present during treatment and that an increase in arginine catabolism partially restored tolerance of the Sod mutants. These results are used to propose a mode-of-action model of cell wall-active antibiotics in tolerant and nontolerant bacteria.