RESUMO
The renowned antimicrobial activity of copper stems in part from its ability to undergo redox cycling between Cu1+/2+ oxidation states. Bacteria counter copper toxicity with a network of sensors that often include two-component signaling systems to direct transcriptional responses. As in typical two-component systems, ligand binding by the extracellular domain of the membrane bound copper sensor component leads to phosphorylation and activation of the cognate response regulator transcription factor. In Listeria monocytogenes, the plasmid-borne CopRS two-component system upregulates both copper resistance and lipoprotein remodeling genes upon copper challenge, but the oxidation state of copper bound by CopS is unknown. Herein, we show CopS utilizes a triad of key residues (His-His-Phe) that are predicted to be at the dimerization interface and that are analogous with the Escherichia coli CusS copper sensor to specifically bind Cu1+/Ag1+ and activate CopR transcription. We demonstrate Cu2+ only induces CopRS if first reduced by electron transport systems, as strains lacking menaquinone carriers were unable to respond to Cu2+. The flavin-dependent extracellular electron transport system (EET) was the main mechanism for metal reduction, capable of either generating inducing ligand (Cu2+ to Cu1+) or removing it by precipitation (Ag1+ to Ag0). We show that EET flux is directly proportional to the rate of Cu2+ reduction and that since EET activity is low under oxygenated conditions when a competing respiratory chain is operating, CopRS signaling in turn is activated only under anaerobic conditions. EET metal reduction thus sensitizes cells to copper while providing resistance to silver under anaerobic growth. IMPORTANCE Two-component extracellular copper sensing from the periplasm of Gram-negative bacteria has been well studied, but copper detection at the cell surface of the Gram-positive L. monocytogenes is less understood. Collectively, our results show that EET is most active under anaerobic conditions and reduces Cu2+ and Ag1+ to, respectively, generate or remove the monovalent ligands that directly bind to CopS and lead to the induction of lipoprotein remodeling genes. This reducing activity regulates CopRS signaling and links the upregulation of copper resistance genes with increasing EET flux. Our studies provide insight into how a two-component copper sensing system is integrated into a model monoderm Firmicute to take cues from the electron transport chain activity.
Assuntos
Cobre , Listeria monocytogenes , Cobre/metabolismo , Transporte de Elétrons , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Histidina Quinase/metabolismo , Anaerobiose , Ligantes , Escherichia coli/metabolismo , PrataRESUMO
Bacterial lipoproteins are membrane-associated proteins with a characteristic acylated N-terminal cysteine residue anchoring C-terminal globular domains to the membrane surface. While all lipoproteins are modified with acyl chains, the number, length, and position can vary depending on host. The acylation pattern also alters ligand recognition by the Toll-like receptor 2 (TLR2) protein family, a signaling system that is central to bacterial surveillance and innate immunity. In select Listeria monocytogenes isolates carrying certain plasmids, copper exposure converts the lipoprotein chemotype into a weak TLR2 ligand through expression of the enzyme lipoprotein intramolecular acyltransferase (Lit). In this study, we identify the response regulator (CopR) from a heavy metal-sensing two-component system as the transcription factor that integrates external copper levels with lipoprotein structural modifications. We show that phosphorylated CopR controls the expression of three distinct transcripts within the plasmid cassette encoding Lit2, prolipoprotein diacylglyceryl transferase (Lgt2), putative copper resistance determinants, and itself (the CopRS two-component system). CopR recognizes a direct repeat half-site consensus motif (TCTACACA) separated by 3 bp that overlaps the -35 promoter element. Target gene expression and lipoprotein conversion were not observed in the absence of the response regulator, indicating that CopR phosphorylation is the dominant mechanism of regulation. IMPORTANCE Copper is a frontline antimicrobial used to limit bacterial growth in multiple settings. Here, we demonstrate how the response regulator CopR from a plasmid-borne two-component system in the opportunistic pathogen L. monocytogenes directly induces lipoprotein remodeling in tandem with copper resistance genes due to extracellular copper stress. Activation of CopR by phosphorylation converts the lipoprotein chemotype from a high- to low-immunostimulatory TLR2 ligand. The two-component system-mediated coregulation of copper resistance determinants, in tandem with lipoprotein biosynthesis demonstrated here in L. monocytogenes, may be a common feature of transmissible copper resistance cassettes found in other Firmicutes.
Assuntos
Listeria monocytogenes , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Receptor 2 Toll-Like , Cobre/metabolismo , Ligantes , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Synthetic lethality occurs when inactivation of two genes is lethal but inactivation of either single gene is not. This phenomenon provides an opportunity for efficient compound discovery. Using differential growth screens, one can identify biologically active compounds that selectively inhibit proteins within the synthetic lethal network of any inactivated gene. Here, based purely on synthetic lethalities, we identified two compounds as the only possible inhibitors of Staphylococcus aureus lipoteichoic acid (LTA) biosynthesis from a screen of â¼230,000 compounds. Both compounds proved to inhibit the glycosyltransferase UgtP, which assembles the LTA glycolipid anchor. UgtP is required for ß-lactam resistance in methicillin-resistant S. aureus (MRSA), and the inhibitors restored sensitivity to oxacillin in a highly resistant S. aureus strain. As no other compounds were pursued as possible LTA glycolipid assembly inhibitors, this work demonstrates the extraordinary efficiency of screens that exploit synthetic lethality to discover compounds that target specified pathways. The general approach should be applicable not only to other bacteria but also to eukaryotic cells.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Glicolipídeos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Mutações Sintéticas LetaisRESUMO
Surface proteins of Staphylococcus aureus play vital roles in bacterial physiology and pathogenesis. Recent work suggests that surface proteins are spatially regulated by a YSIRK/GXXS signal peptide that promotes cross-wall targeting at the mid-cell, though the mechanisms remain unclear. We previously showed that protein A (SpA), a YSIRK/GXXS protein and key staphylococcal virulence factor, mis-localizes in a ltaS mutant deficient in lipoteichoic acid (LTA) production. Here, we identified that SpA contains another cross-wall targeting signal, the LysM domain, which, in addition to the YSIRK/GXXS signal peptide, significantly enhances SpA cross-wall targeting. We show that LTA synthesis, but not LtaS, is required for SpA septal anchoring and cross-wall deposition. Interestingly, LTA is predominantly found at the peripheral cell membrane and is diminished at the septum of dividing staphylococcal cells, suggesting a restriction mechanism for SpA septal localization. Finally, we show that D-alanylation of LTA abolishes SpA cross-wall deposition by disrupting SpA distribution in the peptidoglycan layer without altering SpA septal anchoring. Our study reveals that multiple factors contribute to the spatial regulation and cross-wall targeting of SpA via different mechanisms, which coordinately ensures efficient incorporation of surface proteins into the growing peptidoglycan during the cell cycle.
Assuntos
Lipopolissacarídeos/biossíntese , Peptidoglicano/biossíntese , Sinais Direcionadores de Proteínas/fisiologia , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/biossíntese , Ciclo Celular/fisiologia , Membrana Celular/metabolismo , Parede Celular/metabolismo , Proteínas de Membrana/metabolismo , Domínios ProteicosRESUMO
All bacterial lipoproteins share a variably acylated N-terminal cysteine residue. Gram-negative bacterial lipoproteins are triacylated with a thioether-linked diacylglycerol moiety and an N-acyl chain. The latter is transferred from a membrane phospholipid donor to the α-amino terminus by the enzyme lipoprotein N-acyltransferase (Lnt), using an active-site cysteine thioester covalent intermediate. Many Gram-positive Firmicutes also have N-acylated lipoproteins, but the enzymes catalyzing N-acylation remain uncharacterized. The integral membrane protein Lit (lipoprotein intramolecular transacylase) from the opportunistic nosocomial pathogen Enterococcus faecalis synthesizes a specific lysoform lipoprotein (N-acyl S-monoacylglycerol) chemotype by an unknown mechanism that helps this bacterium evade immune recognition by the Toll-like receptor 2 family complex. Here, we used a deuterium-labeled lipoprotein substrate with reconstituted Lit to investigate intramolecular acyl chain transfer. We observed that Lit transfers the sn-2 ester-linked lipid from the diacylglycerol moiety to the α-amino terminus without forming a covalent thioester intermediate. Utilizing Mut-Seq to analyze an alanine scan library of Lit alleles, we identified two stretches of functionally important amino acid residues containing two conserved histidines. Topology maps based on reporter fusion assays and cysteine accessibility placed both histidines in the extracellular half of the cytoplasmic membrane. We propose a general acid base-promoted catalytic mechanism, invoking direct nucleophilic attack by the substrate α-amino group on the sn-2 ester to form a cyclic tetrahedral intermediate that then collapses to produce lyso-lipoprotein. Lit is a unique example of an intramolecular transacylase differentiated from that catalyzed by Lnt, and provides insight into the heterogeneity of bacterial lipoprotein biosynthetic systems.
Assuntos
Proteínas de Bactérias/biossíntese , Enterococcus faecalis/metabolismo , Lipoproteínas/biossíntese , Acilação , Proteínas de Bactérias/genética , Enterococcus faecalis/genética , Lipoproteínas/genéticaRESUMO
Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. We treated the libraries with daptomycin to assess whether the strain-dependent differences impact pathways important for survival. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, consistently promote survival under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis/genética , Daptomicina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Infecções Estafilocócicas/genética , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacosRESUMO
New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway in Acinetobacter We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Acinetobacter baumannii/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Lipopolissacarídeos/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/genéticaRESUMO
Lipopolysaccharide (LPS) from the Gram-negative bacterial outer membrane potently activates the human innate immune system. LPS is recognized by the Toll-like receptor 4/myeloid differentiation factor-2 (TLR4/MD2) complex, leading to the release of pro-inflammatory cytokines. Alkaline phosphatase (AP) is currently being investigated as an anti-inflammatory agent for detoxifying LPS through dephosphorylating lipid A, thus providing a potential treatment for managing both acute (sepsis) and chronic (metabolic endotoxemia) pathologies wherein aberrant TLR4/MD2 activation has been implicated. Endogenous LPS preparations are chemically heterogeneous, and little is known regarding the LPS chemotype substrate range of AP. Here, we investigated the activity of AP on a panel of structurally defined LPS chemotypes isolated from Escherichia coli and demonstrate that calf intestinal AP (cIAP) has only minimal activity against unmodified enteric LPS chemotypes. Pi was only released from a subset of LPS chemotypes harboring spontaneously labile phosphoethanolamine (PEtN) modifications connected through phosphoanhydride bonds. We demonstrate that the spontaneously hydrolyzed O-phosphorylethanolamine is the actual substrate for AP. We found that the 1- and 4'-lipid A phosphate groups critical in TLR4/MD2 signaling become susceptible to hydrolysis only after de-O-acylation of ester linked primary acyl chains on lipid A. Furthermore, PEtN modifications on lipid A specifically enhanced hTLR4 agonist activity of underacylated LPS preparations. Computational binding models are proposed to explain the limitation of AP substrate specificity imposed by the acylation state of lipid A, and the mechanism of PEtN in enhancing hTLR4/MD2 signaling.
Assuntos
Fosfatase Alcalina/metabolismo , Intestinos/enzimologia , Lipopolissacarídeos/metabolismo , Animais , Bovinos , Escherichia coli/química , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Identifying targets of antibacterial compounds remains a challenging step in the development of antibiotics. We have developed a two-pronged functional genomics approach to predict mechanism of action that uses mutant fitness data from antibiotic-treated transposon libraries containing both upregulation and inactivation mutants. We treated a Staphylococcus aureus transposon library containing 690,000 unique insertions with 32 antibiotics. Upregulation signatures identified from directional biases in insertions revealed known molecular targets and resistance mechanisms for the majority of these. Because single-gene upregulation does not always confer resistance, we used a complementary machine-learning approach to predict the mechanism from inactivation mutant fitness profiles. This approach suggested the cell wall precursor Lipid II as the molecular target of the lysocins, a mechanism we have confirmed. We conclude that docking to membrane-anchored Lipid II precedes the selective bacteriolysis that distinguishes these lytic natural products, showing the utility of our approach for nominating the antibiotic mechanism of action.
Assuntos
Antibacterianos/química , Biblioteca Gênica , Lipídeos/química , Staphylococcus aureus/química , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Parede Celular , Biologia Computacional , Elementos de DNA Transponíveis , Genoma Bacteriano , Lysobacter , Aprendizado de Máquina , Mutação , Peptídeos Cíclicos/química , Regulação para Cima , Uridina Difosfato Ácido N-Acetilmurâmico/químicaRESUMO
Bacterial lipoproteins are globular proteins anchored to the extracytoplasmic surfaces of cell membranes through lipidation at a conserved N-terminal cysteine. Lipoproteins contribute to an array of important cellular functions for bacteria, as well as being a focal point for innate immune system recognition through binding to Toll-like receptor 2 (TLR2) heterodimer complexes. Although lipoproteins are conserved among nearly all classes of bacteria, the presence and type of α-amino-linked acyl chain are highly variable and even strain specific within a given bacterial species. The reason for lyso-lipoprotein formation and N-acylation variability in general is presently not fully understood. In Enterococcus faecalis, lipoproteins are anchored by an N-acyl-S-monoacyl-glyceryl cysteine (lyso form) moiety installed by a chromosomally encoded lipoprotein intramolecular transacylase (Lit). Here, we describe a mobile genetic element common to environmental isolates of Listeria monocytogenes and Enterococcus spp. encoding a functional Lit ortholog (Lit2) that is cotranscribed with several well-established copper resistance determinants. Expression of Lit2 is tightly regulated, and induction by copper converts lipoproteins from the diacylglycerol-modified form characteristic of L. monocytogenes type strains to the α-amino-modified lyso form observed in E. faecalis Conversion to the lyso form through either copper addition to media or constitutive expression of lit2 decreases TLR2 recognition when using an activated NF-κB secreted embryonic alkaline phosphatase reporter assay. While lyso formation significantly diminishes TLR2 recognition, lyso-modified lipoprotein is still predominantly recognized by the TLR2/TLR6 heterodimer.IMPORTANCE The induction of lipoprotein N-terminal remodeling in response to environmental copper in Gram-positive bacteria suggests a more general role in bacterial cell envelope physiology. N-terminal modification by lyso formation, in particular, simultaneously modulates the TLR2 response in direct comparison to their diacylglycerol-modified precursors. Thus, use of copper as a frontline antimicrobial control agent and ensuing selection raises the potential of diminished innate immune sensing and enhanced bacterial virulence.
Assuntos
Aciltransferases/genética , Cobre/metabolismo , Lipoproteínas/metabolismo , Listeria monocytogenes/genética , Receptor 2 Toll-Like/imunologia , Acilação , Enterococcus faecalis/enzimologia , Enterococcus faecalis/genética , Células HEK293 , Humanos , Listeria monocytogenes/enzimologia , Óperon , Transdução de SinaisRESUMO
Lipoteichoic acid (LTA) in Staphylococcus aureus is a poly-glycerophosphate polymer anchored to the outer surface of the cell membrane. LTA has numerous roles in cell envelope physiology, including regulating cell autolysis, coordinating cell division, and adapting to environmental growth conditions. LTA is often further modified with substituents, including d-alanine and glycosyl groups, to alter cellular function. While the genetic determinants of d-alanylation have been largely defined, the route of LTA glycosylation and its role in cell envelope physiology have remained unknown, in part due to the low levels of basal LTA glycosylation in S. aureus We demonstrate here that S. aureus utilizes a membrane-associated three-component glycosylation system composed of an undecaprenol (Und) N-acetylglucosamine (GlcNAc) charging enzyme (CsbB; SAOUHSC_00713), a putative flippase to transport loaded substrate to the outside surface of the cell (GtcA; SAOUHSC_02722), and finally an LTA-specific glycosyltransferase that adds α-GlcNAc moieties to LTA (YfhO; SAOUHSC_01213). We demonstrate that this system is specific for LTA with no cross recognition of the structurally similar polyribitol phosphate containing wall teichoic acids. We show that while wild-type S. aureus LTA has only a trace of GlcNAcylated LTA under normal growth conditions, amounts are raised upon either overexpressing CsbB, reducing endogenous d-alanylation activity, expressing the cell envelope stress responsive alternative sigma factor SigB, or by exposure to environmental stress-inducing culture conditions, including growth media containing high levels of sodium chloride.IMPORTANCE The role of glycosylation in the structure and function of Staphylococcus aureus lipoteichoic acid (LTA) is largely unknown. By defining key components of the LTA three-component glycosylation pathway and uncovering stress-induced regulation by the alternative sigma factor SigB, the role of N-acetylglucosamine tailoring during adaptation to environmental stresses can now be elucidated. As the dlt and glycosylation pathways compete for the same sites on LTA and induction of glycosylation results in decreased d-alanylation, the interplay between the two modification systems holds implications for resistance to antibiotics and antimicrobial peptides.
Assuntos
Glicosiltransferases/metabolismo , Lipopolissacarídeos/metabolismo , Cloreto de Sódio/metabolismo , Staphylococcus aureus/enzimologia , Ácidos Teicoicos/metabolismo , Acetilglucosamina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glicosilação , Glicosiltransferases/genética , Lipopolissacarídeos/química , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/química , Terpenos/metabolismoRESUMO
The majority of bacterial proteins are dispensable for growth in the laboratory but nevertheless have important physiological roles. There are no systematic approaches to identify cell-permeable small-molecule inhibitors of these proteins. We demonstrate a strategy to identify such inhibitors that exploits synthetic lethal relationships both for small-molecule discovery and for target identification. Applying this strategy in Staphylococcus aureus, we have identified a compound that inhibits DltB, a component of the teichoic acid D-alanylation machinery that has been implicated in virulence. This D-alanylation inhibitor sensitizes S. aureus to aminoglycosides and cationic peptides and is lethal in combination with a wall teichoic acid inhibitor. We conclude that DltB is a druggable target in the D-alanylation pathway. More broadly, the work described demonstrates a systematic method to identify biologically active inhibitors of major bacterial processes that can be adapted to numerous organisms.
Assuntos
Amsacrina/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Staphylococcus aureus/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Amsacrina/química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Bibliotecas de Moléculas Pequenas/farmacologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Ácidos Teicoicos/metabolismoRESUMO
Bacterial lipoproteins are embedded in the cell membrane of both Gram-positive and Gram-negative bacteria, where they serve numerous functions central to cell envelope physiology. Lipoproteins are tethered to the membrane by an N-acyl-S-(mono/di)-acyl-glyceryl-cysteine anchor that is variously acylated depending on the genus. In several low-GC, Gram-positive firmicutes, a monoacyl-glyceryl-cysteine with an N-terminal fatty acid (known as the lyso form) has been reported, though how it is formed is unknown. Here, through an intergenic complementation rescue assay in Escherichia coli, we report the identification of a common orthologous transmembrane protein in both Enterococcus faecalis and Bacillus cereus that is capable of forming lyso-form lipoproteins. When deleted from the native host, lipoproteins remain diacylated with a free N terminus, as maturation to the N-acylated lyso form is abolished. Evidence is presented suggesting that the previously unknown gene product functions through a novel intramolecular transacylation mechanism, transferring a fatty acid from the diacylglycerol moiety to the α-amino group of the lipidated cysteine. As such, the discovered gene has been named lipoprotein intramolecular transacylase (lit), to differentiate it from the gene for the intermolecular N-acyltransferase (lnt) involved in triacyl lipoprotein biosynthesis in Gram-negative organisms.IMPORTANCE This study identifies a new enzyme, conserved among low-GC, Gram-positive bacteria, that is involved in bacterial lipoprotein biosynthesis and synthesizes lyso-form lipoproteins. Its discovery is an essential first step in determining the physiological role of N-terminal lipoprotein acylation in Gram-positive bacteria and how these modifications impact bacterial cell envelope function.
Assuntos
Aciltransferases/metabolismo , Bacillus cereus/enzimologia , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/enzimologia , Lipoproteínas/metabolismo , Acilação , Aciltransferases/química , Aciltransferases/genética , Bacillus cereus/química , Bacillus cereus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cisteína/análise , Cisteína/metabolismo , Enterococcus faecalis/química , Enterococcus faecalis/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Lipoproteínas/química , Lipoproteínas/genéticaRESUMO
Antibiotic-resistant strains of Staphylococcus aureus pose a major threat to human health and there is an ongoing need for new antibiotics to treat resistant infections. In a high throughput screen (HTS) of 230â¯000 small molecules designed to identify bioactive wall teichoic acid (WTA) inhibitors, we identified one hit, which was expanded through chemical synthesis into a small panel of potent compounds. We showed that these compounds target TarG, the transmembrane component of the two-component ATP-binding cassette (ABC) transporter TarGH, which exports WTA precursors to the cell surface for attachment to peptidoglycan. We purified, for the first time, a WTA transporter and have reconstituted ATPase activity in proteoliposomes. We showed that this new compound series inhibits TarH-catalyzed ATP hydrolysis even though the binding site maps to TarG near the opposite side of the membrane. These are the first ABC transporter inhibitors shown to block ATPase activity by binding to the transmembrane domain. The compounds have potential as therapeutic agents to treat S. aureus infections, and purification of the transmembrane transporter will enable further development.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Ácidos Teicoicos/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Sítios de Ligação , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Modelos Biológicos , Estrutura Molecular , Peptidoglicano/química , Peptidoglicano/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
Since the introduction of penicillin into the clinic in 1942, antibiotics have saved the lives of millions of people around the world. While penicillin and other traditional broad spectrum antibiotics were effective as monotherapies, the inexorable spread of antibiotic resistance has made alternative therapeutic approaches necessary. Compound combinations are increasingly seen as attractive options. Such combinations may include: lethal compounds; synthetically lethal compounds; or administering a lethal compound with a nonlethal compound that targets a virulence factor or a resistance factor. Regardless of the therapeutic strategy, high throughput screening is a key approach to discover potential leads. Unfortunately, the discovery of biologically active compounds that inhibit a desired pathway can be a very slow process, and an inordinate amount of time is often spent following up on compounds that do not have the desired biological activity. Here we describe a pathway-directed high throughput screening paradigm that combines the advantages of target-based and whole cell screens while minimizing the disadvantages. By exploiting this paradigm, it is possible to rapidly identify biologically active compounds that inhibit a pathway of interest. We describe some previous successful applications of this paradigm and report the discovery of a new class of d-alanylation inhibitors that may be useful as components of compound combinations to treat methicillin-resistant Staphylococcus aureus (MRSA).
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Antibacterianos/química , Staphylococcus aureus Resistente à Meticilina/citologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. High throughput, parallel-sequencing methods for analyzing transposon mutant libraries have the potential to revolutionize studies of S. aureus, but the genetic tools to take advantage of the power of next generation sequencing have not been fully developed. RESULTS: Here we report a phage-based transposition system to make ultra-high density transposon libraries for genome-wide analysis of mutant fitness in any Φ11-transducible S. aureus strain. The high efficiency of the delivery system has made it possible to multiplex transposon cassettes containing different regulatory elements in order to make libraries in which genes are over- or under-expressed as well as deleted. By incorporating transposon-specific barcodes into the cassettes, we can evaluate how null mutations and changes in gene expression levels affect fitness in a single sequencing data set. Demonstrating the power of the system, we have prepared a library containing more than 690,000 unique insertions. Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress. We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures. CONCLUSIONS: The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.
Assuntos
Bacteriófagos/genética , Elementos de DNA Transponíveis , Biblioteca Gênica , Vetores Genéticos/metabolismo , Staphylococcus aureus/genética , Expressão Gênica , Genes Essenciais , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Análise de Sequência de DNA , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , TemperaturaRESUMO
Staphylococcus aureus peptidoglycan (PG) is densely functionalized with anionic polymers called wall teichoic acids (WTAs). These polymers contain three tailoring modifications: d-alanylation, α-O-GlcNAcylation, and ß-O-GlcNAcylation. Here we describe the discovery and biochemical characterization of a unique glycosyltransferase, TarS, that attaches ß-O-GlcNAc (ß-O-N-acetyl-D-glucosamine) residues to S. aureus WTAs. We report that methicillin resistant S. aureus (MRSA) is sensitized to ß-lactams upon tarS deletion. Unlike strains completely lacking WTAs, which are also sensitive to ß-lactams, ΔtarS strains have no growth or cell division defects. Because neither α-O-GlcNAc nor ß-O-Glucose modifications can confer resistance, the resistance phenotype requires a highly specific chemical modification of the WTA backbone, ß-O-GlcNAc residues. These data suggest ß-O-GlcNAcylated WTAs scaffold factors required for MRSA resistance. The ß-O-GlcNAc transferase identified here, TarS, is a unique target for antimicrobials that sensitize MRSA to ß-lactams.
Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Glicosiltransferases/metabolismo , Resistência a Meticilina/fisiologia , Staphylococcus aureus Resistente à Meticilina/enzimologia , Ácidos Teicoicos/metabolismo , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Parede Celular/genética , Deleção de Genes , Glicosilação , Glicosiltransferases/antagonistas & inibidores , Glicosiltransferases/genética , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Ácidos Teicoicos/genética , beta-Lactamas/farmacologiaRESUMO
The ß-acetoacetyl-acyl carrier protein synthase FabY is a key enzyme in the initiation of fatty acid biosynthesis in Pseudomonas aeruginosa. Deletion of fabY results in an increased susceptibility of P. aeruginosa in vitro to a number of antibiotics, including vancomycin and cephalosporins. Because antibiotic susceptibility can be influenced by changes in membrane lipid composition, we determined the total fatty acid profile of the ΔfabY mutant, which suggested alterations in the lipid A region of the lipopolysaccharide. The majority of lipid A species in the ΔfabY mutant lacked a single secondary lauroyl group, resulting in hypoacylated lipid A. Adding exogenous fatty acids to the growth media restored the wild-type antibiotic susceptibility profile and the wild-type lipid A fatty acid profile. We suggest that incorporation of hypoacylated lipid A species into the outer membrane contributes to the shift in the antibiotic susceptibility profile of the ΔfabY mutant.
Assuntos
Aciltransferases/metabolismo , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Aciltransferases/genética , Proteínas de Bactérias/genética , Pseudomonas aeruginosa/genéticaRESUMO
Polymyxins [colistin and polymyxin B (PMB)] comprise an important class of natural product lipopeptide antibiotics used to treat multidrug-resistant Gram-negative bacterial infections. These positively charged lipopeptides interact with lipopolysaccharide (LPS) located in the outer membrane and disrupt the permeability barrier, leading to increased uptake and bacterial cell death. Many bacteria counter polymyxins by upregulating genes involved in the biosynthesis and transfer of amine-containing moieties to increase positively charged residues on LPS. Although 4-deoxy-l-aminoarabinose (Ara4N) and phosphoethanolamine (PEtN) are highly conserved LPS modifications in Escherichia coli, different lineages exhibit variable PMB susceptibilities and frequencies of resistance for reasons that are poorly understood. Herein, we describe a mechanism prevalent in E. coli B strains that depends on specific insertion sequence 1 (IS1) elements that flank genes involved in the biosynthesis and transfer of Ara4N to LPS. Spontaneous and transient chromosomal amplifications mediated by IS1 raise the frequency of PMB resistance by 10- to 100-fold in comparison to strains where a single IS1 element located 90 kb away from the end of the arn operon has been deleted. Amplification involving IS1 becomes the dominant resistance mechanism in the absence of PEtN modification. Isolates with amplified arn operons gradually lose their PMB-resistant phenotype with passaging, consistent with classical PMB heteroresistance behavior. Analysis of the whole genome transcriptome profile showed altered expression of genes residing both within and outside of the duplicated chromosomal segment, suggesting complex phenotypes including PMB resistance can result from tandem amplification events.IMPORTANCEPhenotypic variation in susceptibility and the emergence of resistant subpopulations are major challenges to the clinical use of polymyxins. While a large database of genes and alleles that can confer polymyxin resistance has been compiled, this report demonstrates that the chromosomal insertion sequence (IS) content and distribution warrant consideration as well. Amplification of large chromosomal segments containing the arn operon by IS1 increases the Ara4N content of the lipopolysaccharide layer in Escherichia coli B lineages using a mechanism that is orthogonal to transcriptional upregulation through two-component regulatory systems. Altogether, our work highlights the importance of IS elements in modulating gene expression and generating diverse subpopulations that can contribute to phenotypic polymyxin B heteroresistance.
Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Escherichia coli , Lipopolissacarídeos , Óperon , Polimixina B , Polimixina B/farmacologia , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Lipopolissacarídeos/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão GênicaRESUMO
Connecting bacterial growth inhibitors to molecular targets at the whole-cell level is a major impediment to antibacterial development. Herein we report the design of a highly efficient and versatile bacteriophage-based mariner transposon delivery system in Staphylococcus aureus for determining inhibitor mode of action. Using bacteriophage-mediated delivery of concatameric minitransposon cassettes, we generated nonclonal transposant libraries with genome-wide insertion-site coverage in either laboratory or methicillin-resistant strain backgrounds and screened for drug resistance in situ on a single agar plate in one step. A gradient of gene-target expression levels, along with a correspondingly diverse assortment of drug-resistant phenotypes, was achieved by fitting the transposon cassette with a suite of outward-facing promoters. Using a panel of antibiotics, we demonstrate the ability to unveil not only an inhibitor's molecular target but also its route of cellular entry, efflux susceptibility and other off-target resistance mechanisms.