Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance. - Related Documents




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70901.0000Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance. Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria. Interestingly, several clinically relevant and human pathogenic strains are inherently resistant towards lantibiotics. The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators. Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding.201626930060
821310.9996The Extracellular Domain of Two-component System Sensor Kinase VanS from Streptomyces coelicolor Binds Vancomycin at a Newly Identified Binding Site. The glycopeptide antibiotic vancomycin has been widely used to treat infections of Gram-positive bacteria including Clostridium difficile and methicillin-resistant Staphylococcus aureus. However, since its introduction, high level vancomycin resistance has emerged. The genes responsible require the action of the two-component regulatory system VanSR to induce expression of resistance genes. The mechanism of detection of vancomycin by this two-component system has yet to be elucidated. Diverging evidence in the literature supports activation models in which the VanS protein binds either vancomycin, or Lipid II, to induce resistance. Here we investigated the interaction between vancomycin and VanS from Streptomyces coelicolor (VanS(SC)), a model Actinomycete. We demonstrate a direct interaction between vancomycin and purified VanS(SC), and traced these interactions to the extracellular region of the protein, which we reveal adopts a predominantly α-helical conformation. The VanS(SC)-binding epitope within vancomycin was mapped to the N-terminus of the peptide chain, distinct from the binding site for Lipid II. In targeting a separate site on vancomycin, the effective VanS ligand concentration includes both free and lipid-bound molecules, facilitating VanS activation. This is the first molecular description of the VanS binding site within vancomycin, and could direct engineering of future therapeutics.202032235931
821520.9996Insight into Two ABC Transporter Families Involved in Lantibiotic Resistance. Antimicrobial peptides, which contain (methyl)-lanthionine-rings are called lantibiotics. They are produced by several Gram-positive bacteria and are mainly active against these bacteria. Although these are highly potent antimicrobials, some human pathogenic bacteria express specific ABC transporters that confer resistance and counteract their antimicrobial activity. Two distinct ABC transporter families are known to be involved in this process. These are the Cpr- and Bce-type ABC transporter families, named after their involvement in cationic peptide resistance in Clostridium difficile, and bacitracin efflux in Bacillus subtilis, respectively. Both resistance systems differentiate to each other in terms of the proteins involved. Here, we summarize the current knowledge and describe the divergence as well as the common features present in both the systems to confer lantibiotic resistance.201729404338
70630.9996Effect of PhoP-PhoQ activation by broad repertoire of antimicrobial peptides on bacterial resistance. Pathogenic bacteria can resist their microenvironment by changing the expression of virulence genes. In Salmonella typhimurium, some of these genes are controlled by the two-component system PhoP-PhoQ. Studies have shown that activation of the system by cationic antimicrobial peptides (AMPs) results, among other changes, in outer membrane remodeling. However, it is not fully clear what characteristics of AMPs are required to activate the PhoP-PhoQ system and whether activation can induce resistance to the various AMPs. For that purpose, we investigated the ability of a broad repertoire of AMPs to traverse the inner membrane, to activate the PhoP-PhoQ system, and to induce bacterial resistance. The AMPs differ in length, composition, and net positive charge, and the tested bacteria include two wild-type (WT) Salmonella strains and their corresponding PhoP-PhoQ knock-out mutants. A lacZ-reporting system was adapted to follow PhoP-PhoQ activation. The data revealed that: (i) a good correlation exists among the extent of the positive charge, hydrophobicity, and amphipathicity of an AMP and its potency to activate PhoP-PhoQ; (ii) a +1 charged peptide containing histidines was highly potent, suggesting the existence of an additional mechanism independent of the peptide charge; (iii) the WT bacteria are more resistant to AMPs that are potent activators of PhoP-PhoQ; (iv) only a subset of AMPs, independent of their potency to activate the system, is more toxic to the mutated bacteria compared with the WT strains; and (v) short term exposure of WT bacteria to these AMPs does not enhance resistance. Overall, this study advances our understanding of the molecular mechanism by which AMPs activate PhoP-PhoQ and induce bacterial resistance. It also reveals that some AMPs can overcome such a resistance mechanism.201222158870
70540.9996First structure of the polymyxin resistance proteins. PmrA/PmrB and PhoP/PhoQ are a pair of two-component systems (TCSs) that allow the Gram-negative bacteria to survive the cationic antimicrobial peptide polymyxin B. The two TCSs are linked by the polymyxin resistance protein, PmrD. The PhoP-activated PmrD protects the phosphorylated response regulator PmrA from dephosphorylation, and promotes the transcription of PmrA-activated genes responsible for polymyxin resistance. PmrD is the first protein identified to mediate the connectivity between two TCSs by protecting the phosphorylated response regulator of the downstream TCS. PmrD shows no homology to proteins with known structures. We present here the solution structure of PmrD from Escherichia coli, the first three-dimensional structure of the PmrD family. Our study provides the structural basis of the novel interacting mechanism of bacterial two-component signal-transduction systems.200717686460
71050.9995The L box regulon: lysine sensing by leader RNAs of bacterial lysine biosynthesis genes. Expression of amino acid biosynthesis genes in bacteria is often repressed when abundant supplies of the cognate amino acid are available. Repression of the Bacillus subtilis lysC gene by lysine was previously shown to occur at the level of premature termination of transcription. In this study we show that lysine directly promotes transcription termination during in vitro transcription with B. subtilis RNA polymerase and causes a structural shift in the lysC leader RNA. We find that B. subtilis lysC is a member of a large family of bacterial lysine biosynthesis genes that contain similar leader RNA elements. By analogy with related regulatory systems, we designate this leader RNA pattern the "L box." Genes in the L box family from Gram-negative bacteria appear to be regulated at the level of translation initiation rather than transcription termination. Mutations of B. subtilis lysC that disrupt conserved leader features result in loss of lysine repression in vivo and loss of lysine-dependent transcription termination in vitro. The identification of the L box pattern also provides an explanation for previously described mutations in both B. subtilis and Escherichia coli lysC that result in lysC overexpression and resistance to the lysine analog aminoethylcysteine. The L box regulatory system represents an example of gene regulation using an RNA element that directly senses the intracellular concentration of a small molecule.200314523230
68460.9995Transcriptome analysis reveals mechanisms by which Lactococcus lactis acquires nisin resistance. Nisin, a posttranslationally modified antimicrobial peptide produced by Lactococcus lactis, is widely used as a food preservative. Yet, the mechanisms leading to the development of nisin resistance in bacteria are poorly understood. We used whole-genome DNA microarrays of L. lactis IL1403 to identify the factors underlying acquired nisin resistance mechanisms. The transcriptomes of L. lactis IL1403 and L. lactis IL1403 Nis(r), which reached a 75-fold higher nisin resistance level, were compared. Differential expression was observed in genes encoding proteins that are involved in cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal and/or peptide transport and binding. These results were further substantiated by showing that several knockout and overexpression mutants of these genes had strongly altered nisin resistance levels and that some knockout strains could no longer become resistant to the same level of nisin as that of the wild-type strain. The acquired nisin resistance mechanism in L. lactis is complex, involving various different mechanisms. The four major mechanisms are (i) preventing nisin from reaching the cytoplasmic membrane, (ii) reducing the acidity of the extracellular medium, thereby stimulating the binding of nisin to the cell wall, (iii) preventing the insertion of nisin into the membrane, and (iv) possibly transporting nisin across the membrane or extruding nisin out of the membrane.200616641446
70270.9995Cutting edge: the toll pathway is required for resistance to gram-positive bacterial infections in Drosophila. In Drosophila, the response against various microorganisms involves different recognition and signaling pathways, as well as distinct antimicrobial effectors. On the one hand, the immune deficiency pathway regulates the expression of antimicrobial peptides that are active against Gram-negative bacteria. On the other hand, the Toll pathway is involved in the defense against filamentous fungi and controls the expression of antifungal peptide genes. The gene coding for the only known peptide with high activity against Gram-positive bacteria, Defensin, is regulated by both pathways. So far, survival experiments to Gram-positive bacteria have been performed with Micrococcus luteus and have failed to reveal the involvement of one or the other pathway in host defense against such infections. In this study, we report that the Toll pathway, but not that of immune deficiency, is required for resistance to other Gram-positive bacteria and that this response does not involve Defensin.200211823479
69680.9995Identification of a two-component regulatory system involved in antimicrobial peptide resistance in Streptococcus pneumoniae. Two-component regulatory systems (TCS) are among the most widespread mechanisms that bacteria use to sense and respond to environmental changes. In the human pathogen Streptococcus pneumoniae, a total of 13 TCS have been identified and many of them have been linked to pathogenicity. Notably, TCS01 strongly contributes to pneumococcal virulence in several infection models. However, it remains one of the least studied TCS in pneumococci and its functional role is still unclear. In this study, we demonstrate that TCS01 cooperates with a BceAB-type ABC transporter to sense and induce resistance to structurally-unrelated antimicrobial peptides of bacterial origin that all target undecaprenyl-pyrophosphate or lipid II, which are essential precursors of cell wall biosynthesis. Even though tcs01 and bceAB genes do not locate in the same gene cluster, disruption of either of them equally sensitized the bacterium to the same set of antimicrobial peptides. We show that the key function of TCS01 is to upregulate the expression of the transporter, while the latter appears the main actor in resistance. Electrophoretic mobility shift assays further demonstrated that the response regulator of TCS01 binds to the promoter region of the bceAB genes, implying a direct control of these genes. The BceAB transporter was overexpressed and purified from E. coli. After reconstitution in liposomes, it displayed substantial ATPase and GTPase activities that were stimulated by antimicrobial peptides to which it confers resistance to, revealing new functional features of a BceAB-type transporter. Altogether, this inducible defense mechanism likely contributes to the survival of the opportunistic microorganism in the human host, in which competition among commensal microorganisms is a key determinant for effective host colonization and invasive path.202235395062
821090.9995Bacterial sensing of antimicrobial peptides. Antimicrobial peptides (AMPs) form a crucial part of human innate host defense, especially in neutrophil phagosomes and on epithelial surfaces. Bacteria have a variety of efficient resistance mechanisms to human AMPs, such as efflux pumps, secreted proteases, and alterations of the bacterial cell surface that are aimed to minimize attraction of the typically cationic AMPs. In addition, bacteria have specific sensors that activate AMP resistance mechanisms when AMPs are present. The prototypical Gram-negative PhoP/PhoQ and the Gram-positive Aps AMP-sensing systems were first described and investigated in Salmonella typhimurium and Staphylococcus epidermidis, respectively. Both include a classical bacterial two-component sensor/regulator system, but show many structural, mechanistic, and functional differences. The PhoP/PhoQ regulon controls a variety of genes not necessarily limited to AMP resistance mechanisms, but apparently aimed to combat innate host defense on a broad scale. In contrast, the staphylococcal Aps system predominantly upregulates AMP resistance mechanisms, namely the D-alanylation of teichoic acids, inclusion of lysyl-phosphati-dylglycerol in the cytoplasmic membrane, and expression of the putative VraFG AMP efflux pump. Notably, both systems are crucial for virulence and represent possible targets for antimicrobial therapy.200919494583
704100.9995Aminoarabinose is essential for lipopolysaccharide export and intrinsic antimicrobial peptide resistance in Burkholderia cenocepacia(†). One common mechanism of resistance against antimicrobial peptides in Gram-negative bacteria is the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipopolysaccharide (LPS) molecule. Burkholderia cenocepacia exhibits extraordinary intrinsic resistance to antimicrobial peptides and other antibiotics. We have previously discovered that unlike other bacteria, B. cenocepacia requires L-Ara4N for viability. Here, we describe the isolation of B. cenocepacia suppressor mutants that remain viable despite the deletion of genes required for L-Ara4N synthesis and transfer to the LPS. The absence of L-Ara4N is the only structural difference in the LPS of the mutants compared with that of the parental strain. The mutants also become highly sensitive to polymyxin B and melittin, two different classes of antimicrobial peptides. The suppressor phenotype resulted from a single amino acid replacement (aspartic acid to histidine) at position 31 of LptG, a protein component of the multi-protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that L-Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly at the outer membrane of B. cenocepacia, and is the most critical determinant for the intrinsic resistance of this bacterium to antimicrobial peptides.201222742453
685110.9995Implication of a Key Region of Six Bacillus cereus Genes Involved in Siroheme Synthesis, Nitrite Reductase Production and Iron Cluster Repair in the Bacterial Response to Nitric Oxide Stress. Bacterial response to nitric oxide (NO) is of major importance for bacterial survival. NO stress is a main actor of the eukaryotic immune response and several pathogenic bacteria have developed means for detoxification and repair of the damages caused by NO. However, bacterial mechanisms of NO resistance by Gram-positive bacteria are poorly described. In the opportunistic foodborne pathogen Bacillus cereus, genome sequence analyses did not identify homologs to known NO reductases and transcriptional regulators, such as NsrR, which orchestrate the response to NO of other pathogenic or non-pathogenic bacteria. Using a transcriptomic approach, we investigated the adaptation of B. cereus to NO stress. A cluster of 6 genes was identified to be strongly up-regulated in the early phase of the response. This cluster contains an iron-sulfur cluster repair enzyme, a nitrite reductase and three enzymes involved in siroheme biosynthesis. The expression pattern and close genetic localization suggest a functional link between these genes, which may play a pivotal role in the resistance of B. cereus to NO stress during infection.202134064887
598120.9995Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving the acyl carrier protein-SpoT interaction. Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation.200918996989
8214130.9995The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile. The dlt operon in Gram-positive bacteria encodes proteins that are necessary for the addition of d-alanine to teichoic acids of the cell wall. The addition of d-alanine to the cell wall results in a net positive charge on the bacterial cell surface and, as a consequence, can decrease the effectiveness of antimicrobials, such as cationic antimicrobial peptides (CAMPs). Although the roles of the dlt genes have been studied for some Gram-positive organisms, the arrangement of these genes in Clostridium difficile and the life cycle of the bacterium in the host are markedly different from those of other pathogens. In the current work, we determined the contribution of the putative C. difficile dlt operon to CAMP resistance. Our data indicate that the dlt operon is necessary for full resistance of C. difficile to nisin, gallidermin, polymyxin B and vancomycin. We propose that the d-alanylation of teichoic acids provides protection against antimicrobial peptides that may be essential for growth of C. difficile in the host.201121330441
291140.9994Deregulation of translation due to post-transcriptional modification of rRNA explains why erm genes are inducible. A key mechanism of bacterial resistance to macrolide antibiotics is the dimethylation of a nucleotide in the large ribosomal subunit by erythromycin resistance methyltransferases. The majority of erm genes are expressed only when the antibiotic is present and the erythromycin resistance methyltransferase activity is critical for the survival of bacteria. Although these genes were among the first discovered inducible resistance genes, the molecular basis for their inducibility has remained unknown. Here we show that erythromycin resistance methyltransferase expression reduces cell fitness. Modification of the nucleotide in the ribosomal tunnel skews the cellular proteome by deregulating the expression of a set of proteins. We further demonstrate that aberrant translation of specific proteins results from abnormal interactions of the nascent peptide with the erythromycin resistance methyltransferase-modified ribosomal tunnel. Our findings provide a plausible explanation why erm genes have evolved to be inducible and underscore the importance of nascent peptide recognition by the ribosome for generating a balanced cellular proteome.201323749080
727150.9994Bacillus subtilis extracytoplasmic function (ECF) sigma factors and defense of the cell envelope. Bacillus subtilis provides a model for investigation of the bacterial cell envelope, the first line of defense against environmental threats. Extracytoplasmic function (ECF) sigma factors activate genes that confer resistance to agents that threaten the integrity of the envelope. Although their individual regulons overlap, σ(W) is most closely associated with membrane-active agents, σ(X) with cationic antimicrobial peptide resistance, and σ(V) with resistance to lysozyme. Here, I highlight the role of the σ(M) regulon, which is strongly induced by conditions that impair peptidoglycan synthesis and includes the core pathways of envelope synthesis and cell division, as well as stress-inducible alternative enzymes. Studies of these cell envelope stress responses provide insights into how bacteria acclimate to the presence of antibiotics.201626901131
595160.9994Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes. Survival in aerobic conditions is critical to the pathogenicity of many bacteria. To investigate the means of aerotolerance and resistance to oxidative stress in the catalase-negative organism Streptococcus pyogenes, we used a genomics-based approach to identify and inactivate homologues of two peroxidase genes, encoding alkyl hydroperoxidase (ahpC) and glutathione peroxidase (gpoA). Single and double mutants survived as well as the wild type under aerobic conditions. However, they were more susceptible than the wild type to growth suppression by paraquat and cumene hydroperoxide. In addition, we show that S. pyogenes demonstrates an inducible peroxide resistance response when treated with sublethal doses of peroxide. This resistance response was intact in ahpC and gpoA mutants but not in mutants lacking PerR, a repressor of several genes including ahpC and catalase (katA) in Bacillus subtilis. Because our data indicate that these peroxidase genes are not essential for aerotolerance or induced resistance to peroxide stress in S. pyogenes, genes for a novel mechanism of managing peroxide stress may be regulated by PerR in streptococci.200010986229
686170.9994SigB-dependent general stress response in Bacillus subtilis and related gram-positive bacteria. One of the strongest and most noticeable responses of Bacillus subtilis cells to a range of stress and starvation stimuli is the dramatic induction of about 150 SigB-dependent general stress genes. The activity of SigB itself is tightly regulated by a complex signal transduction cascade with at least three main signaling pathways that respond to environmental stress, energy depletion, or low temperature. The SigB-dependent response is conserved in related gram-positive bacteria but is missing in strictly anaerobic or in some facultatively anaerobic gram-positive bacteria. It covers functions from nonspecific and multiple stress resistance to the control of virulence in pathogenic bacteria. A comprehensive understanding of this crucial stress response is essential not only for bacterial physiology but also for applied microbiology, including pathogenicity and pathogen control.200718035607
694180.9994The role of sigmaB in the stress response of Gram-positive bacteria -- targets for food preservation and safety. The alternative sigma factor sigmaB modulates the stress response of several Gram-positive bacteria, including Bacillus subtilis and the food-borne human pathogens Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. In all these bacteria, sigmaB is responsible for the transcription of genes that can confer stress resistance to the vegetative cell. Recent findings indicate that sigmaB also plays an important role in antibiotic resistance, pathogenesis and cellular differentiation processes such as biofilm formation and sporulation. Although there are important differences in the regulation of sigmaB and in the set of genes regulated by sigmaB in B. subtilis, B. cereus, L. monocytogenes and S. aureus, there are also some conserved themes. A mechanistic understanding of the sigmaB activation processes and assessment of its regulon could provide tools for pathogen control and inactivation both in the food industry and clinical settings.200515831390
701190.9994Antimicrobial Peptide Resistance Genes in the Plant Pathogen Dickeya dadantii. Modification of teichoic acid through the incorporation of d-alanine confers resistance in Gram-positive bacteria to antimicrobial peptides (AMPs). This process involves the products of the dltXABCD genes. These genes are widespread in Gram-positive bacteria, and they are also found in a few Gram-negative bacteria. Notably, these genes are present in all soft-rot enterobacteria (Pectobacterium and Dickeya) whose dltDXBAC operons have been sequenced. We studied the function and regulation of these genes in Dickeya dadantii dltB expression was induced in the presence of the AMP polymyxin. It was not regulated by PhoP, which controls the expression of some genes involved in AMP resistance, but was regulated by ArcA, which has been identified as an activator of genes involved in AMP resistance. However, arcA was not the regulator responsible for polymyxin induction of these genes in this bacterium, which underlines the complexity of the mechanisms controlling AMP resistance in D. dadantii Two other genes involved in resistance to AMPs have also been characterized, phoS and phoH dltB, phoS, phoH, and arcA but not dltD mutants were more sensitive to polymyxin than the wild-type strain. Decreased fitness of the dltB, phoS, and phoH mutants in chicory leaves indicates that their products are important for resistance to plant AMPs. IMPORTANCE: Gram-negative bacteria can modify their lipopolysaccharides (LPSs) to resist antimicrobial peptides (AMPs). Soft-rot enterobacteria (Dickeya and Pectobacterium spp.) possess homologues of the dlt genes in their genomes which, in Gram-positive bacteria, are involved in resistance to AMPs. In this study, we show that these genes confer resistance to AMPs, probably by modifying LPSs, and that they are required for the fitness of the bacteria during plant infection. Two other new genes involved in resistance were also analyzed. These results show that bacterial resistance to AMPs can occur in bacteria through many different mechanisms that need to be characterized.201627565623