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1.
Appl Environ Microbiol ; 79(13): 4015-23, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23603688

RESUMO

The oral bacterium Streptococcus mutans, strain JH1140, produces the antibiotic mutacin 1140. Mutacin 1140 belongs to a group of antibiotics called lanthipeptides. More specifically, mutacin 1140 is related to the epidermin type A(I) lanthipeptides. Mutagenesis experiments of this group of lanthipeptides have been primarily restricted to the posttranslationally modified meso-lanthionine and 3-methyllanthionine residues. Site-directed mutagenesis of the core peptide of mutacin 1140 was performed using the suicide vector pVA891. Substitutions of the N-terminal residue, the charged residue in the hinge region, and residues in ring A and intertwined rings C and D were investigated. A truncation and insertion of residues in ring A and intertwined rings C and D were also performed to determine whether or not they would alter the antimicrobial activity of the producing strain. Bioassays revealed that five of 14 mutants studied had improved antimicrobial activity against the indicator strain Micrococcus luteus ATCC 10240. MICs against Streptococcus mutans UA159, Streptococcus pneumoniae ATCC 27336, Staphylococcus aureus ATCC 25923, Clostridium difficile UK1, and Micrococcus luteus ATCC 10240 were determined for three mutacin 1140 variants that had the most significant increases in bioactivity in the M. luteus bioassay. This mutagenesis study of the epidermin group of lanthipeptides shows that antimicrobial activity can be significantly improved.


Assuntos
Bacteriocinas/genética , Biotecnologia/métodos , Mutagênese Sítio-Dirigida/métodos , Peptídeos/genética , Streptococcus mutans/genética , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Clostridioides difficile/efeitos dos fármacos , Genes Transgênicos Suicidas/genética , Vetores Genéticos/genética , Micrococcus luteus/efeitos dos fármacos , Estrutura Molecular , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Staphylococcus aureus/efeitos dos fármacos , Streptococcus/efeitos dos fármacos , Streptococcus mutans/metabolismo
2.
Biochemistry ; 48(35): 8312-21, 2009 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-19673482

RESUMO

Bacterial strain Burkholderia contaminans MS14 was isolated from soil that suppressed brown patch disease of lawn grass. An antifungal compound was purified from the liquid culture of this bacterium. In this study, complete covalent structures of two purified closely related antifungal compounds were determined by the experiments of TOCSY, NOESY, ROESY, 13C HSQC 2D NMR, and ESI-MS and GC. The analysis of monoisotopic masses of the purified preparation indicated the presence of two related compounds with masses determined to be 1199.543 and 1215.518 Da; the difference corresponds to the mass of an oxygen atom. GC analysis identified a xylose sugar attached to the antifungal compound. NMR experiments revealed that the compound is cyclic and composed of eight amino acids, two of which are beta-hydroxy derivatives of Tyr and Asn, and one being a novel amino acid. The novel amino acid serves as the scaffold for the attachment of the xylose and a short acyl chain. The spectrum and concentration of antifungal activity were determined using a microtiter plate assay. The antifungal compound demonstrated potent antifungal activities against a broad panel of fungal plant and animal pathogens, as well as two Pythium spp. Microscopic observations showed that the antifungal compound disrupts normal membrane morphology. The cells fill with large inclusion bodies and the membrane becomes irregularly shaped and swollen following the exposure to subinhibitory concentrations of the antifungal compound. Our data support the identification of a novel fungicide and the compound has been named occidiofungin, meaning fungal killer.


Assuntos
Antifúngicos/farmacologia , Burkholderia/química , Glicopeptídeos/farmacologia , Antifúngicos/química , Fungos/efeitos dos fármacos , Glicopeptídeos/química , Testes de Sensibilidade Microbiana , Peso Molecular , Doenças das Plantas/microbiologia , RNA Ribossômico 16S/efeitos dos fármacos , RNA Ribossômico 16S/farmacologia , Análise de Sequência de DNA
3.
Appl Environ Microbiol ; 75(5): 1381-7, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19114522

RESUMO

The peptide antibiotic nisin A belongs to the group of antibiotics called lantibiotics. They are classified as lantibiotics because they contain the structural group lanthionine. Lanthionines are composed of a single sulfur atom that is linked to the beta-carbons of two alanine moieties. These sulfur atoms are vulnerable to environmental oxidation. A mild oxidation reaction was performed on nisin A to determine the relative effects it would have on bioactivity. High-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry data revealed the addition of seven, eight, and nine oxygens. These additions correspond to the five lanthionines, two methionines, and two histidines that would be susceptible to oxidation. Subsequent bioassays revealed that the oxidized form of nisin A had a complete loss of bactericidal activity. In a competition study, the oxidized nisin did not appear to have an antagonistic affect on the bioactivity of nisin A, since the addition of an equal molar concentration of the oxidized variant did not have an influence on the bactericidal activity of the native antibiotic. Electron microscopy data revealed that the oxidized forms were still capable of assembling into large circular complexes, demonstrating that oxidation does not disrupt the lateral assembly mechanism of the antibiotic. Affinity thin-layer chromatography and fluorescence microscopy experiments suggested that the loss of activity is due to the inability of the oxidized form of nisin to bind to the cell wall precursor lipid II. Given the loss of bioactivity following oxidation, oxidation should be an important factor to consider in future production, purification, pharmacokinetic, and pharmacodynamic studies.


Assuntos
Alanina/análogos & derivados , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Nisina/metabolismo , Nisina/farmacologia , Sulfetos/metabolismo , Alanina/metabolismo , Substâncias Macromoleculares , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Micrococcus luteus/efeitos dos fármacos , Microscopia Eletrônica , Modelos Moleculares , Oxirredução , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
4.
Can J Microbiol ; 55(11): 1261-6, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19940934

RESUMO

The peptide antibiotic mutacin 1140 belongs to the group of antibiotics called lantibiotics. They are ribosomally synthesized and undergo extensive enzymatic modifications before being excreted into the culture medium. By using reverse-phase-high-performance liquid chromatography (RP-HPLC) and a semiquantitative bacteriocin bioassay to track mutacin 1140, an efficient ammonium sulfate (AS) precipitation method has been developed for removing mutacin 1140 from a complex medium containing 5% yeast extract. This method minimizes the amounts of fermentation by-products and media components that make downstream purification processes more difficult and economically infeasible. The method may be adaptable for the initial purification step of other lantibiotics. A threefold decrease in the precipitation of the medium components found in yeast extract, at pH 8.0 vs. pH 2.0, may have broad utility for the isolation of secondary metabolites produced in this complex medium. The average yield of mutacin 1140 from the fermentation medium was determined as 66%.


Assuntos
Antibacterianos/isolamento & purificação , Bacteriocinas/isolamento & purificação , Fermentação , Microbiologia Industrial/métodos , Peptídeos/isolamento & purificação , Leveduras/química , Sequência de Aminoácidos , Antibacterianos/química , Bacteriocinas/química , Cromatografia Líquida de Alta Pressão , Misturas Complexas/química , Meios de Cultura/química , Meios de Cultura/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/química , Alinhamento de Sequência
5.
Recent Pat Antiinfect Drug Discov ; 6(2): 175-85, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21517737

RESUMO

Lantibiotics are an interesting group of antimicrobial peptides. There are a number of reviews that describe the potential application of lantibiotics for controlling foodborne illnesses and their potential to treat Gram positive infections caused by organisms like Staphylococcus aureus. In this review, commercial potential for the producing organism for promoting health and their potential for protein chemistry applications are discussed. Lantibiotics are ribozomally synthesized as a prepropeptide, comprised of a leader sequence that directs the propeptide to the enzymes that perform a number of post-translational modifications. This system affords several possibilities for the synthesis of a library of peptides that may have therapeutic use. We also take a look at a couple of lantibiotic producing organisms that have potential applications in the field of probiotics, given their ability to displace pathogenic microorganisms. The use of application along with the discussion of recent patents are discussed in this review article.


Assuntos
Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Bacteriocinas/uso terapêutico , Probióticos , Animais , Bactérias/genética , Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Bacteriocinas/biossíntese , Bacteriocinas/genética , Descoberta de Drogas , Humanos , Patentes como Assunto , Engenharia de Proteínas
6.
Biochemistry ; 47(10): 3308-14, 2008 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-18266322

RESUMO

Mutacin 1140 and nisin A are peptide antibiotics that belong to the lantibiotic family. N-Terminal rings A and B of nisin A and mutacin 1140 (lipid II-binding domain) share many structural and sequence similarities. Nisin A binds lipid II and thus disrupts cell wall synthesis and also forms transmembrane pores. Very little is known about mutacin 1140 in this regard. We performed fluorescence-based studies using a bacteria-mimetic membrane system. The results indicated that lipid II monomers are arranged differently in the mutacin 1140 complex than in the nisin A complex. These differences in complex formation may be attributed to the fact that nisin A uses lipid II to form a distinct pore complex, while mutacin 1140 does not form pores in this membrane system. Further experiments demonstrated that the mutacin 1140-lipid II and nisin A-lipid II complexes are very stable and capable of withstanding competition from each other. Transmembrane electrical potential experiments using a Streptococcus rattus strain, which is sensitive to mutacin 1140, demonstrated that mutacin 1140 does not form pores in this strain even at a concentration 8 times higher than the minimum inhibitory concentration (MIC). Circular complexes of mutacin 1140 and nisin A were observed by electron microscopy, providing direct evidence for a lateral assembly mechanism for these antibiotics. Mutacin 1140 did exhibit a membrane disruptive function in another commonly used artificial bacterial membrane system, and its disruptive activity was enhanced by increasing amounts of anionic phospholipids.


Assuntos
Antibacterianos/química , Bacteriocinas/química , Peptídeos/química , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Sequência de Aminoácidos , Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Nisina/química , Nisina/metabolismo , Peptídeos/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
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