RESUMO
New inhibitors of the bacterial tranferase MraY are described. Their structure is based on an aminoribosyl uridine scaffold, which is known to be important for the biological activity of natural MraY inhibitors. A decyl alkyl chain was introduced onto this scaffold through various linkers. The synthesized compounds were tested against the MraYAA transferase activity, and the most active compound with an original (S,S)-tartaric diamide linker inhibits MraY activity with an IC50 equal to 0.37 µM. Their antibacterial activity was also evaluated on a panel of Gram-positive and Gram-negative strains; however, the compounds showed no antibacterial activity. Docking and molecular dynamics studies revealed that this new linker established two stabilizing key interactions with N190 and H325, as observed for the highly potent inhibitors carbacaprazamycin, muraymycin D2 and tunicamycin.
Assuntos
Diamida , Transferases , Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Transferases/química , Transferases (Outros Grupos de Fosfato Substituídos) , Uridina/química , Uridina/farmacologiaRESUMO
The straightforward synthesis of aminoribosyl uridines substituted by a 5'-methylene-urea is described. Their convergent synthesis involves the urea formation from various activated amides and an azidoribosyl uridine substituted at the 5' position by an aminomethyl group. This common intermediate resulted from the diastereoselective glycosylation of a phthalimido uridine derivative with a ribosyl fluoride as a ribosyl donor. The inhibition of the MraY transferase activity by the synthetized 11 urea-containing inhibitors was evaluated and 10 compounds revealed MraY inhibition with IC50 ranging from 1.9 µM to 16.7 µM. Their antibacterial activity was also evaluated on a panel of Gram-positive and Gram-negative bacteria. Four compounds exhibited a good activity against Gram-positive bacterial pathogens with MIC ranging from 8 to 32 µg mL-1, including methicillin resistant Staphylococcus aureus (MRSA) and Enterococcus faecium. Interestingly, one compound also revealed antibacterial activity against Pseudomonas aeruginosa with MIC equal to 64 µg mL-1. Docking experiments predicted two modes of positioning of the active compounds urea chain in different hydrophobic areas (HS2 and HS4) within the MraY active site from Aquifex aeolicus. However, molecular dynamics simulations showed that the urea chain adopts a binding mode similar to that observed in structural model and targets the hydrophobic area HS2.
Assuntos
AntibacterianosRESUMO
Innovative monocyclic ß-lactam entities create opportunities in the battle against resistant bacteria because of their PBP acylation potential, intrinsically high ß-lactamase stability and compact scaffold. α-Benzylidene-substituted 3-amino-1-carboxymethyl-ß-lactams were recently shown to be potent PBP inhibitors and constitute eligible anchor points for synthetic elaboration of the chemical space around the central ß-lactam ring. The present study discloses a 12-step synthesis of ten α-arylmethylidenecarboxylates using a microwave-assisted Wittig olefination as the crucial reaction step. The library was designed aiming at enhanced ß-lactam electrophilicity and extended electron flow after enzymatic attack. Additionally, increased ß-lactamase stability and intermolecular target interaction were envisioned by tackling both the substitution pattern of the aromatic ring and the ß-lactam C4-position. The significance of α-unsaturation was validated and the R39/PBP3 inhibitory potency shown to be augmented the most through decoration of the aromatic ring with electron-withdrawing groups. Furthermore, ring cleavage by representative ß-lactamases was ruled out, providing new insights in the SAR landscape of monocyclic ß-lactams as eligible PBP or ß-lactamase inhibitors.
RESUMO
As a complement to the renowned bicyclic ß-lactam antibiotics, monocyclic analogues provide a breath of fresh air in the battle against resistant bacteria. In that framework, the present study discloses the in silico design and unprecedented ten-step synthesis of eleven nocardicin-like enantiomerically pure 2-{3-[2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido]-2-oxoazetidin-1-yl}acetic acids starting from serine as a readily accessible precursor. The capability of this novel class of monocyclic 3-amino-ß-lactams to inhibit penicillin-binding proteins (PBPs) of various (resistant) bacteria was assessed, revealing the potential of α-benzylidenecarboxylates as interesting leads in the pursuit of novel PBP inhibitors. No deactivation by representative enzymes belonging to the four ß-lactamase classes was observed, while weak inhibition of class C ß-lactamase P99 was demonstrated.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Enterococcus faecium/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , beta-Lactamas/química , beta-Lactamas/farmacologia , Aminação , Antibacterianos/síntese química , Infecções Bacterianas/tratamento farmacológico , Simulação por Computador , Desenho Assistido por Computador , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Enterococcus faecium/metabolismo , Escherichia coli/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Humanos , Simulação de Acoplamento Molecular , Proteínas de Ligação às Penicilinas/metabolismo , beta-Lactamas/síntese químicaRESUMO
BACKGROUND: Proteins from the LytR-CpsA-Psr family are found in almost all Gram-positive bacteria. Although LCP proteins have been studied in other pathogens, their functions in enterococci remain uncharacterized. The Psr protein from Enterococcus hirae, here renamed LcpA, previously associated with the regulation of the expression of the low-affinity PBP5 and ß-lactam resistance, has been characterized. RESULTS: LcpA protein of E. hirae ATCC 9790 has been produced and purified with and without its transmembrane helix. LcpA appears, through different methods, to be localized in the membrane, in agreement with in silico predictions. The interaction of LcpA with E. hirae cell wall indicates that LcpA binds enterococcal peptidoglycan, regardless of the presence of secondary cell wall polymers. Immunolocalization experiments showed that LcpA and PBP5 are localized at the division site of E. hirae. CONCLUSIONS: LcpA belongs to the LytR-CpsA-Psr family. Its topology, localization and binding to peptidoglycan support, together with previous observations on defective mutants, that LcpA plays a role related to the cell wall metabolism, probably acting as a phosphotransferase catalyzing the attachment of cell wall polymers to the peptidoglycan.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Streptococcus faecium ATCC 9790/metabolismo , Peptidoglicano/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Membrana Celular/metabolismo , Parede Celular/metabolismo , Clonagem Molecular , DNA Bacteriano , Streptococcus faecium ATCC 9790/citologia , Streptococcus faecium ATCC 9790/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Fosfotransferases/metabolismo , Mapas de Interação de Proteínas , Proteínas Recombinantes , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Resistência beta-LactâmicaRESUMO
Human L-ficolin is a soluble protein of the innate immune system able to sense pathogens through its fibrinogen (FBG) recognition domains and to trigger activation of the lectin complement pathway through associated serine proteases. L-Ficolin has been previously shown to recognize pneumococcal clinical isolates, but its ligands and especially its molecular specificity remain to be identified. Using solid-phase binding assays, serum and recombinant L-ficolins were shown to interact with serotype 2 pneumococcal strain D39 and its unencapsulated R6 derivative. Incubation of both strains with serum triggered complement activation, as measured by C4b and C3b deposition, which was decreased by using ficolin-depleted serum. Recombinant L-ficolin and its FBG-like recognition domain bound to isolated pneumococcal cell wall extracts, whereas binding to cell walls depleted of teichoic acid (TA) was decreased. Both proteins were also shown to interact with two synthetic TA compounds, each comprising part structures of the complete lipoteichoic acid molecule with two PCho residues. Competition studies and direct interaction measurements by surface plasmon resonance identified PCho as a novel L-ficolin ligand. Structural analysis of complexes of the FBG domain of L-ficolin and PCho revealed that the phosphate moiety interacts with amino acids previously shown to define an acetyl binding site. Consequently, binding of L-ficolin to immobilized acetylated BSA was inhibited by PCho and synthetic TA. Binding of serum L-ficolin to immobilized synthetic TA and PCho-conjugated BSA triggered activation of the lectin complement pathway, thus further supporting the hypothesis of L-ficolin involvement in host antipneumococcal defense.
Assuntos
Lectinas/metabolismo , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/metabolismo , Ácidos Teicoicos/metabolismo , Acetilação , Parede Celular/metabolismo , Ativação do Complemento , Complemento C3b/metabolismo , Complemento C4b/metabolismo , Fibrinogênio/genética , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Lectinas/genética , Fosforilcolina/química , Ligação Proteica , Estrutura Terciária de Proteína/genética , Streptococcus pneumoniae/imunologia , Ressonância de Plasmônio de Superfície , Ácidos Teicoicos/química , FicolinasRESUMO
The straightforward synthesis of 5'-methylene-[1,4]-triazole-substituted aminoribosyl uridines is described. Two families of compounds were synthesized from a unique epoxide which was regioselectively opened by acetylide ions (for compounds II) or azide ions (for compounds III). Sequential diastereoselective glycosylation with a ribosyl fluoride derivative, Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) with various complementary azide and alkyne partners afforded the targeted compounds after final deprotection. The biological activity of the 16 resulting compounds together with that of 14 previously reported compounds I, lacking the 5' methylene group, was evaluated on the MraY transferase activity. Out of the 30 tested compounds, 18 compounds revealed MraY inhibition with IC50 ranging from 15 to 150 µM. A molecular modeling study was performed to rationalize the observed structure-activity relationships (SAR), which allowed us to correlate the activity of the most potent compounds with an interaction involving Leu191 of MraYAA. The antibacterial activity was also evaluated and seven compounds exhibited a good activity against Gram-positive bacterial pathogens with MIC ranging from 8 to 32 µg mL(-1), including the methicillin resistant Staphylococcus aureus (MRSA).
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Modelos Moleculares , Transferases/antagonistas & inibidores , Triazóis/química , Uridina/química , Uridina/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Domínio Catalítico , Técnicas de Química Sintética , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Testes de Sensibilidade Microbiana , Transferases/química , Transferases (Outros Grupos de Fosfato Substituídos) , Uridina/síntese químicaRESUMO
To resist to ß-lactam antibiotics Eubacteria either constitutively synthesize a ß-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of ß-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a ß-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible ß-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation.
Assuntos
Bacillus/genética , Regulação Bacteriana da Expressão Gênica/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Peptidoglicano/metabolismo , Resistência beta-Lactâmica/genética , Acilação , Bacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Parede Celular/química , Parede Celular/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Dipeptídeos/química , Dipeptídeos/metabolismo , Indução Enzimática/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/efeitos dos fármacos , Penicilinas/metabolismo , Penicilinas/farmacologia , Peptidoglicano/química , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , beta-Lactamases/biossíntese , beta-Lactamases/genéticaRESUMO
The opportunistic human pathogen Enterococcus faecium overproduces the low-affinity PBP5. In clinical strains, mutations in PBP5 further reduce its acylation rate by ß-lactams. Previous studies have reported that ceftaroline had poor inhibitory activity against ß-lactam-resistant E. faecium strains. In this study, we show that ceftaroline exhibits killing activity against our laboratory-derived ampicillin-resistant E. faecium mutant that overproduces a wild-type PBP5 and that ceftaroline inactivates PBP5 much faster than benzylpenicillin and faster than ceftobiprole.
Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Resistência a Ampicilina/genética , Proteínas de Bactérias/genética , Resistência beta-Lactâmica/genética , CeftarolinaRESUMO
In response to the widespread use of ß-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent ß-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC(50): 1.3 µM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC(50) = 26 µM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC(50) = 138 µM) and a class C PBP (R39 from Actinomadura sp., IC(50) = 0.6 µM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species.
Assuntos
Ácidos Borônicos/síntese química , Ácidos Borônicos/farmacologia , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Proteínas de Ligação às Penicilinas/classificação , Actinomycetales/química , Ácidos Borônicos/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Streptococcus pneumoniae/química , Relação Estrutura-AtividadeRESUMO
Penicillin-binding proteins are a well established, validated and still a very promising target for the design and development of new antibacterial agents. Based on our previous discovery of several noncovalent small-molecule inhibitor hits for resistant PBPs we decided to additionally explore the chemical space around these compounds. In order to clarify their structure-activity relationships for PBP inhibition two new series of compounds were synthesized, characterized and evaluated biochemically: the derivatives of anthranilic acid and naphthalene-sulfonamide derivatives. The target compounds were tested for their inhibitory activities on three different transpeptidases: PBP2a from methicillin-resistant Staphylococcus aureus (MRSA) strains, PBP5fm from Enterococcus faecium strains, and PBP1b from Streptococcus pneumoniae strains. The most promising results for both of these series of compounds were obtained against the PBP2a enzyme with the IC50 values in the micromolar range. Although these results do not represent a significant breakthrough in the field of noncovalent PBP inhibitors, they do provide useful structure-activity relationship data, and thus a more solid basis for the design of potent and noncovalent inhibitors of resistant PBPs.
RESUMO
New inhibitors of the bacterial transferase MraY from Aquifex aeolicus (MraYAA), based on the aminoribosyl uridine central core of known natural MraY inhibitors, have been designed to generate interaction of their oxadiazole linker with the key amino acids (H324 or H325) of the enzyme active site, as observed for the highly potent inhibitors carbacaprazamycin, muraymycin D2 and tunicamycin. A panel of ten compounds was synthetized notably thanks to a robust microwave-activated one-step sequence for the synthesis of the oxadiazole ring that involved the O-acylation of an amidoxime and subsequent cyclization. The synthetized compounds, with various hydrophobic substituents on the oxadiazole ring, were tested against the MraYAA transferase activity. Although with poor antibacterial activity, nine out of the ten compounds revealed the inhibition of the MraYAA activity in the range of 0.8 µM to 27.5 µM.
RESUMO
We solved the crystal structure of a secreted protein, EXLX1, encoded by the yoaJ gene of Bacillus subtilis. Its structure is remarkably similar to that of plant beta-expansins (group 1 grass pollen allergens), consisting of 2 tightly packed domains (D1, D2) with a potential polysaccharide-binding surface spanning the 2 domains. Domain D1 has a double-psi beta-barrel fold with partial conservation of the catalytic site found in family 45 glycosyl hydrolases and in the MltA family of lytic transglycosylases. Domain D2 has an Ig-like fold similar to group 2/3 grass pollen allergens, with structural features similar to a type A carbohydrate-binding domain. EXLX1 bound to plant cell walls, cellulose, and peptidoglycan, but it lacked lytic activity against a variety of plant cell wall polysaccharides and peptidoglycan. EXLX1 promoted plant cell wall extension similar to, but 10 times weaker than, plant beta-expansins, which synergistically enhanced EXLX1 activity. Deletion of the gene encoding EXLX1 did not affect growth or peptidoglycan composition of B. subtilis in liquid medium, but slowed lysis upon osmotic shock and greatly reduced the ability of the bacterium to colonize maize roots. The presence of EXLX1 homologs in a small but diverse set of plant pathogens further supports a role in plant-bacterial interactions. Because plant expansins have proved difficult to express in active form in heterologous systems, the discovery of a bacterial homolog opens the door for detailed structural studies of expansin function.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Raízes de Plantas/microbiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Parede Celular/metabolismo , Cristalografia por Raios X , Genes Bacterianos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Alinhamento de Sequência , Zea mays/metabolismoRESUMO
The bacterial cell wall maintains a cell's integrity while allowing growth and division. It is made up of peptidoglycan (PG), a biopolymer forming a multigigadalton bag-like structure, and, additionally in gram-positive bacteria, of covalently linked anionic polymers collectively called teichoic acids. These anionic polymers are thought to play important roles in host-cell adhesion, inflammation, and immune activation. In this Article, we compare the flexibility and the organization of peptidoglycans from gram-negative bacteria (E. coli) with its counterpart from different gram-positive bacteria using solid-state nuclear magnetic resonance spectroscopy (NMR) under magic-angle sample spinning (MAS). The NMR fingerprints suggest an identical local conformation of the PG in all of these bacterial species. Dynamics in the peptidoglycan network decreases from E. coli to B. subtilis and from B. subtilis to S. aureus and correlates mainly with the degree of peptide cross-linkage. For intact bacterial cells and isolated cell walls, we show that (31)P solid-state NMR is particularly well adapted to characterize and differentiate wall teichoic acids of different species. We have further observed complexation with divalent ions, highlighting an important structural aspect of gram-positive cell wall architecture. We propose a new model for the interaction of divalent cations with both wall teichoic acids and carbonyl groups of peptidoglycan.
Assuntos
Bacillus subtilis/química , Parede Celular/química , Escherichia coli/química , Magnésio/química , Manganês/química , Staphylococcus aureus/química , Bacillus subtilis/citologia , Sítios de Ligação , Escherichia coli/citologia , Íons/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peptidoglicano/química , Staphylococcus aureus/citologia , Ácidos Teicoicos/química , Termodinâmica , Água/químicaRESUMO
Ceftobiprole is a new cephalosporin that exhibits a high level of affinity for methicillin-resistant Staphylococcus aureus PBP 2a. It was reported that ceftobiprole did not interact with a mutated form of the low-affinity protein Enterococcus faecium PBP 5 (PBP 5fm) that, when overexpressed, confers a beta-lactam resistance phenotype to the bacterium. Our results show that ceftobiprole binds to unmutated PBP 5fm to form a stable acyl-enzyme and that ceftobiprole is able to efficiently kill a penicillin-resistant Enterococcus faecium strain that produces this protein.
Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Cefalosporinas/metabolismo , Cefalosporinas/farmacologia , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/metabolismo , Proteínas de Bactérias/genética , Enterococcus faecium/genética , Testes de Sensibilidade Microbiana , Penicilina G/farmacologia , Ligação Proteica/genéticaRESUMO
Monocyclic ß-lactams revive the research field on antibiotics, which are threatened by the emergence of resistant bacteria. A six-step synthetic route was developed, providing easy access to new 3-amino-1-carboxymethyl-4-phenyl-ß-lactams, of which the penicillin-binding protein (PBP) inhibitory potency was demonstrated biochemically.
Assuntos
Aminoácidos/farmacologia , Antibacterianos/farmacologia , Dipeptídeos/farmacologia , Iminas/farmacologia , Lactamas/farmacologia , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Aminoácidos/química , Antibacterianos/síntese química , Antibacterianos/química , Dipeptídeos/síntese química , Dipeptídeos/química , Iminas/química , Lactamas/síntese química , Lactamas/química , Estrutura Molecular , Proteínas de Ligação às Penicilinas/metabolismoRESUMO
6-Beta-halogenopenicillanates are powerful, irreversible inhibitors of various beta-lactamases and penicillin-binding proteins. Upon acylation of these enzymes, the inhibitors are thought to undergo a structural rearrangement associated with the departure of the iodide and formation of a dihydrothiazine ring, but, to date, no structural evidence has proven this. 6-Beta-iodopenicillanic acid (BIP) is shown here to be an active antibiotic against various bacterial strains and an effective inhibitor of the class A beta-lactamase of Bacillus subtilis BS3 (BS3) and the D,D-peptidase of Actinomadura R39 (R39). Crystals of BS3 and of R39 were soaked with a solution of BIP and their structures solved at 1.65 and 2.2 A, respectively. The beta-lactam and the thiazolidine rings of BIP are indeed found to be fused into a dihydrothiazine ring that can adopt two stable conformations at these active sites. The rearranged BIP is observed in one conformation in the BS3 active site and in two monomers of the asymmetric unit of R39, and is observed in the other conformation in the other two monomers of the asymmetric unit of R39. The BS3 structure reveals a new mode of carboxylate interaction with a class A beta-lactamase active site that should be of interest in future inhibitor design.
Assuntos
Actinomycetales/enzimologia , Antibacterianos/química , Bacillus subtilis/enzimologia , Inibidores Enzimáticos/química , Ácido Penicilânico/análogos & derivados , Proteínas de Ligação às Penicilinas/química , beta-Lactamases/química , Cristalografia por Raios X , Modelos Moleculares , Ácido Penicilânico/química , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Estrutura Terciária de Proteína , Inibidores de beta-LactamasesRESUMO
In absence of beta-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in beta-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to beta-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.
Assuntos
Bacillus/genética , Proteínas de Bactérias/química , DNA Bacteriano/química , Regiões Operadoras Genéticas , Proteínas Repressoras/química , Staphylococcus aureus/genética , Resistência beta-Lactâmica/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Dimerização , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/metabolismo , TermodinâmicaRESUMO
Understanding the molecular mechanism by which the signal of the presence of an antibiotic is transduced from outside to inside the bacterial cell is of fundamental interest for the ß-lactam antibiotic resistance problem, but remains difficult to accomplish. No approach has ever addressed entire penicillin receptors in a membrane environment. Here we describe a method to investigate the purified Bacillus licheniformis BlaR1 receptor -a membrane-bound penicillin receptor involved in ß-lactam resistance- embedded into a lipid bilayer in absence or presence of penicillin. By selecting a mutated receptor blocked in its signal transduction pathway just after its activation by penicillin, we revealed the very first step of receptor signalling by unfolding the receptor from its C-terminal end by AFM-based single-molecule force spectroscopy. We showed that the presence of the antibiotic entails significant conformational changes within the receptor. Our approach opens an avenue to study signal-transduction pathways mediated by membrane-bound proteins in a membrane environment.
Assuntos
Proteínas de Bactérias/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Penicilinas/químicaRESUMO
The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background.