RESUMO
A series of gramicidin S derivatives 4-15 are presented that have four ornithine residues as polar protonated side chains and two central hydrophobic amino acids with unaltered turn regions. These peptides were screened against human erthrocytes and our standard panel of Gram negative- and Gram positive bacteria, including four MRSA strains. Based on the antibacterial- and hemolytic data, peptides 13 and 14 have an improved biological profile compared to the clinically applied topical antibiotic gramicidin S.
Assuntos
Antibacterianos/química , Gramicidina/análogos & derivados , Gramicidina/química , Antibacterianos/síntese química , Antibacterianos/farmacologia , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Gramicidina/síntese química , Gramicidina/farmacologia , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Monobenzylated sugar amino acids (SAAs) that differ in ether ring size (containing an oxetane, furanoid, and pyranoid ring) were synthesized and incorporated in one of the ß-turn regions of the cyclo-decapeptide gramicidin S (GS). CD, NMR spectroscopy, modeling, and X-ray diffraction reveal that the ring size of the incorporated SAA moieties determines the spatial positioning of their cis-oriented carboxyl and aminomethyl substituents, thereby subtly influencing the amide linkages with the adjacent amino acids in the sequence. Unlike GS itself, the conformational behavior of the SAA-containing peptides is solvent dependent. The derivative containing the pyranoid SAA is slightly less hydrophobic and displays a diminished haemolytic activity, but has similar antimicrobial properties as GS.
Assuntos
Aminoácidos/química , Anti-Infecciosos/química , Anti-Infecciosos/síntese química , Gramicidina/química , Oligopeptídeos/química , Oligopeptídeos/síntese química , Sequência de Aminoácidos , Amino Açúcares , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Difração de Raios XRESUMO
In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the ß-strand regions. This structure is in agreement with a previously reported modeling study of the same molecule. The polar side chain of the additional d-amino acid residue is positioned at the same face of the molecule as the hydrophobic side chains, and we believe that because of this compound 2 is considerably less hydrophobic than extended GS derivatives in which the strand regions are exclusively composed of l-amino acids. Using this backbone structure as our benchmark we prepared a small series of ring-extended GS analogues featuring sugar amino acid dipeptide isosteres of varied hydrophobicity at the turn region. We show that via this approach hydrophobicity of extended GS analogues can be tuned without affecting the secondary structure (as observed from NMR and CD spectra). Biological evaluation reveals that hydrophobicity correlates to cell toxicity, but still bacteriolysis is induced with GS analogues that are too hydrophilic to efficiently lyse human red blood cells.
Assuntos
Antibacterianos/síntese química , Gramicidina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Dicroísmo Circular , Cristalografia por Raios X , Eritrócitos/efeitos dos fármacos , Gramicidina/química , Gramicidina/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D-Phe-Pro two-residue turn motifs in the rigid cyclic beta-hairpin structure of GS was replaced with morpholine amino acids (MAA 2-5), differing in stereochemistry and length of the side-chain. The conformational properties of the thus obtained GS analogues (6-9) was assessed by using NMR spectroscopy and X-ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8, containing the dipeptide isostere trans-MAA 4, has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and -negative bacterial strains is better than the derivatives 6, 7 and 9.
Assuntos
Aminoácidos/química , Antibacterianos/química , Antibacterianos/farmacologia , Dipeptídeos/química , Gramicidina/química , Gramicidina/farmacologia , Morfolinas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The cyclic cationic antimicrobial peptide gramicidin S (GS) is an effective topical antibacterial agent that is toxic for human red blood cells (hemolysis). Herein, we present a series of amphiphilic derivatives of GS with either two or four positive charges and characteristics ranging between very polar and very hydrophobic. Screening of this series of peptide derivatives identified a compound that combines effective antibacterial activity with virtually no toxicity within the same concentration range. This peptide acts against both Gram-negative and Gram-positive bacteria, including several MRSA strains, and represents an interesting lead for the development of a broadly applicable antibiotic.
Assuntos
Adamantano/química , Adamantano/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Eritrócitos/efeitos dos fármacos , Gramicidina/química , Gramicidina/farmacologia , Hemólise/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Aminoácidos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Modelos Moleculares , Permeabilidade , Relação Estrutura-AtividadeRESUMO
Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial peptides are tetradecameric GS analogues containing six positives charges and two adamantane moieties.
Assuntos
Adamantano/química , Aminoácidos/química , Anti-Infecciosos/química , Gramicidina/análogos & derivados , Sequência de Aminoácidos , Anti-Infecciosos/farmacologia , Cátions/química , Dicroísmo Circular , Gramicidina/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacosRESUMO
Loloatin C is a cyclic cationic antimicrobial peptide which is active against gram positive as well as certain gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes. To probe the structure-activity relationship of this promising antibiotic peptide, amino acid substitution and/or incorporation of a constraint sugar amino acid dipeptide isoster has been applied. Six new derivatives have been synthesized using SPPS and their solution structure investigated using NMR studies. Finally, the antimicrobial and the hemolytic activities have been determined.
Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Antibacterianos/síntese química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Eritrócitos/efeitos dos fármacos , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Relação Estrutura-AtividadeRESUMO
Methicillin-resistant Staphylococcus aureus sequence type 398 (ST398 MRSA) was identified in Dutch pigs and pig farmers. ST398 methicillin-susceptible S. aureus circulates among humans at low frequency (0.2%) but was isolated in 3 human cases of bacteremia (2.1%; p = 0.026). Although its natural host is probably porcine, ST398 MRSA likely causes infections in humans.
Assuntos
Resistência a Meticilina , Meticilina/farmacologia , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacos , Suínos/microbiologia , Animais , Antibacterianos/farmacologia , Portador Sadio , Humanos , Países Baixos , Nariz/microbiologia , Exposição Ocupacional , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/genéticaRESUMO
BACKGROUND: Sporadic cases of CA-MRSA in persons without risk-factors for MRSA carriage are increasing. CASE PRESENTATION: We report a MRSA cluster among family members of a pig-farmer, his co-workers and his pigs. Initially a young mother was seen with mastitis due to MRSA. Six months later her baby daughter was admitted to the hospital with pneumococcal otitis. After staying five days in hospital, the baby was found to be MRSA positive. At that point it was decided to look for a possible source, such as other family members and house-hold animals, including pigs on the farm, since those were reported as a possible source of MRSA earlier. Swabs were taken from the throat and nares of family members and co-workers. A veterinarian obtained swabs from the nares, throat and perineum of 10 pigs. Swabs were cultured following a national protocol to detect MRSA that included the use of an enrichment broth. Animal and human strains were characterized by PFGE, spa-typing, MLST analysis, SSCmec, AGR typing, and the detection for PVL, LukM, and TSST toxin genes. Three family members, three co-workers, and 8 of the 10 pigs were MRSA positive. With the exception of the initial case (the mother) all persons were solely colonized, with no signs of clinical infections. After digestion with SmaI, none of the strains showed any bands using PFGE. All isolates belonged to spa type t108 and ST398. CONCLUSION: 1. This report clearly shows clonal spread and transmission between humans and pigs in the Netherlands. 2. MLST sequence type 398 might be of international importance as pig-MRSA, since this type was shown earlier to be present in epidemiologically unrelated French pigs and pig-farmers. 3. Research is needed to evaluate whether this is a local problem or a new source of MRSA, that puts the until now successful Search and Destroy policy of the Netherlands at risk.
Assuntos
Criação de Animais Domésticos , Infecções Comunitárias Adquiridas/transmissão , Resistência a Meticilina , Infecções Estafilocócicas/transmissão , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacos , Suínos/microbiologia , Adulto , Animais , Animais Domésticos , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Portador Sadio/microbiologia , Infecções Comunitárias Adquiridas/microbiologia , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Recém-Nascido , Masculino , Mastite/microbiologia , Resistência a Meticilina/genética , Cavidade Nasal/microbiologia , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Doenças dos Suínos/microbiologia , Doenças dos Suínos/transmissão , ZoonosesRESUMO
BACKGROUND: Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution. METHODOLOGY/PRINCIPAL FINDINGS: A new MLVA scheme for S. aureus was validated using 1681 S. aureus isolates collected from Dutch patients and 100 isolates from pigs. MLVA using 8 tandem repeat loci was performed in 2 multiplex PCRs and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer. The assessed number of repeats was used to create MLVA profiles consisting of strings of 8 integers that were used for categorical clustering. MLVA yielded 511 types that clustered into 11 distinct MLVA complexes which appeared to coincide with MLST clonal complexes. MLVA was at least as discriminatory as PFGE and twice as discriminatory as spa-sequence typing. There was considerable congruence between MLVA, spa-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. CONCLUSIONS: The MLVA described in this study is a high throughput, relatively low cost genotyping method for S. aureus that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology.
Assuntos
Staphylococcus aureus Resistente à Meticilina/genética , Sequências de Repetição em Tandem , Eletroforese em Gel de Campo Pulsado , Corantes Fluorescentes , Reação em Cadeia da PolimeraseAssuntos
Resistência a Meticilina , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Casas de Saúde/estatística & dados numéricos , Infecções Estafilocócicas/epidemiologia , Idoso de 80 Anos ou mais , Humanos , Países Baixos/epidemiologia , Prevalência , Infecções Estafilocócicas/microbiologiaRESUMO
An outbreak of community-acquired methicillin-resistant Staphylococcus aureus occurred among members and close contacts of a soccer team. Typing of the isolates showed the outbreak was caused by the well-known European ST80-IV strain. To our knowledge, this is the first report of an outbreak of this strain among members of a sports team.
Assuntos
Surtos de Doenças , Resistência a Meticilina , Futebol , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Adolescente , Adulto , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Testes de Sensibilidade Microbiana , Países Baixos/epidemiologia , Infecções Estafilocócicas/transmissão , Staphylococcus aureus/classificação , Staphylococcus aureus/isolamento & purificaçãoRESUMO
This paper describes the design and synthesis of gramicidin S (GS) analogues 10a-c containing arylated sugar amino acids (SAAs) as a replacement of one of the two (D)Phe-Pro beta-turn regions. The cyclic, amphiphilic peptides adopt a beta-sheet conformation featuring an unusual reverse turn induced by the SAAs. The altered turn region induces a slight distortion of the antiparallel beta-sheet, as compared to GS; the overall geometry however closely resembles that of the nonarylated GS analogue 1. GS analogues 10a-c proved to be as active as the parent GS itself as antibacterial agents and are equally efficient in lysing red blood cells. These properties are in sharp contrast to the diminished biological activity displayed by 1. We conclude that the presence of aromaticity in the turn regions of GS derivatives is required for biological activity, whereas the native conformation of the beta-hairpin is not. Our findings may guide future research toward efficient and nonhemolytic GS analogues for combating bacterial infections.
Assuntos
Aminoácidos/química , Amino Açúcares/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Produtos Biológicos/farmacologia , Eritrócitos/efeitos dos fármacos , Gramicidina/farmacologia , Hemólise/efeitos dos fármacos , Acrilatos/química , Sequência de Aminoácidos , Antibacterianos/síntese química , Produtos Biológicos/síntese química , Gramicidina/síntese química , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fenilalanina/química , Prolina/química , Estrutura Secundária de ProteínaRESUMO
Multienzyme multiplex PCR-amplified fragment length polymorphism (ME-AFLP) typing is a reliable and simple method for typing of bacterial species. In this study we analyzed two well-documented strain collections of Staphylococcus aureus and compared ME-AFLP typing results with results of various other typing methods. The discriminatory power of ME-AFLP was found comparable to pulsed-field gel electrophoresis, and typing results were highly concordant. ME-AFLP typing presents a suitable method for prescreening of large strain collections. Furthermore, the obtained typing patterns were found to cluster according to the staphylococcal cassette chromosome mec types of the strains.
Assuntos
Eletroforese em Gel de Campo Pulsado/métodos , Resistência a Meticilina , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacos , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Cromossomos Bacterianos , Surtos de Doenças , Humanos , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genéticaRESUMO
The design and synthesis of analogues of the cyclic beta-sheet gramicidin S (GS), having additional functionalities in their turn regions, is reported. The monomeric GS analogues were transformed into dimers and their activities towards biological membranes, through antimicriobial and hemolytic assays, were evaluated. Finally, conductivity measurements have been performed to elucidate ion channel forming properties.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Gramicidina/síntese química , Gramicidina/farmacologia , Antibacterianos/química , Dimerização , Gramicidina/química , Hemólise , Canais Iônicos/química , Testes de Sensibilidade Microbiana , Conformação Molecular , Sensibilidade e Especificidade , Relação Estrutura-AtividadeRESUMO
A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.
Assuntos
Alelos , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , Sequência de Bases , Enterococcus faecium/classificação , Animais , Antibacterianos/farmacologia , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Variação Genética , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Dados de Sequência Molecular , Recombinação Genética , Análise de Sequência de DNA , Vancomicina/farmacologia , Resistência a VancomicinaRESUMO
The synthesis of novel gramicidin S analogues having additional functionalities in the turn region by employing a biomimetic approach is described. The preservation of beta-sheet character in all analogues was established by NMR and the biological activity was evaluated.
Assuntos
Gramicidina/análogos & derivados , Gramicidina/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Gramicidina/síntese química , Testes de Sensibilidade Microbiana , Mimetismo Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de ProteínaRESUMO
The epidemiology of vancomycin-resistant Entero- coccus faecium (VREF) in Europe is characterized by a large community reservoir. In contrast, nosocomial outbreaks and infections (without a community reservoir) characterize VREF in the United States. Previous studies demonstrated host-specific genogroups and a distinct genetic lineage of VREF associated with hospital outbreaks, characterized by the variant esp-gene and a specific allele-type of the purK housekeeping gene (purK1). We investigated the genetic relatedness of vanA VREF (n=108) and vancomycin-susceptible E. faecium (VSEF) (n=92) from different epidemiologic sources by genotyping, susceptibility testing for ampicillin, sequencing of purK1, and testing for presence of esp. Clusters of VSEF fit well into previously described VREF genogroups, and strong associations were found between VSEF and VREF isolates with resistance to ampicillin, presence of esp, and purK1. Genotypes characterized by presence of esp, purK1, and ampicillin resistance were most frequent among outbreak-associated isolates and almost absent among community surveillance isolates. Vancomycin-resistance was not specifically linked to genogroups. VREF and VSEF from different epidemiologic sources are genetically related; evidence exists for nosocomial selection of a subtype of E. faecium, which has acquired vancomycin-resistance through horizontal transfer.
Assuntos
Resistência a Ampicilina/genética , Enterococcus faecium/efeitos dos fármacos , Genes MDR/genética , Resistência a Vancomicina/genética , Surtos de Doenças , Enterococcus faecium/genética , Genótipo , Humanos , Reação em Cadeia da PolimeraseAssuntos
Antibacterianos/química , Antibacterianos/farmacologia , Gramicidina/análogos & derivados , Gramicidina/farmacologia , Aminoácidos/química , Bactérias/efeitos dos fármacos , Carboidratos/química , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Estrutura Molecular , Estrutura Secundária de ProteínaRESUMO
A practical gram-scale and high-yielding synthesis of the antimicrobial peptide gramicidin S is presented. An Fmoc-based solid-phase peptide synthesis protocol is employed for the generation of the linear decapeptide precursor, which is cyclized in solution to afford the target compound. The versatility of our method is demonstrated by the construction of eight gramicidin S analogues (15a-h) having nonproteinogenic sugar amino acid residues (4-7) incorporated in the turn regions.