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1.
mSystems ; 7(1): e0139321, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35076266

RESUMO

Elasnin is a new antibiofilm compound that was recently reported to have excellent activity against methicillin-resistant Staphylococcus aureus (MRSA) biofilms. In this study, we established that elasnin also has antibacterial activity against growing S. aureus planktonic cells. To explore elasnin's potential as an antibiotic, we applied adaptive laboratory evolution (ALE) and produced evolved strains with elevated elasnin tolerance. Interestingly, they were more sensitive toward daptomycin and lysostaphin. Whole-genome sequencing revealed that all of the evolved strains possessed a single point mutation in a putative phosphate transport regulator. Subsequently, they exhibited increased intracellular phosphate (Pi) and polyphosphate levels. Inhibition of the phosphate transport regulator gene changed the phenotype of the wild type to one resembling those observed in the evolved strains. Proteomics and transcriptomics analyses showed that elasnin treatment resulted in the downregulation of many proteins related to cell division and cell wall synthesis, which is important for the survival of growing exponential-phase cells. Other downregulated processes and factors were fatty acid metabolism, glycolysis, the two-component system, RNA degradation, and ribosomal proteins. Most importantly, transport proteins and proteins involved in oxidative phosphorylation and the phosphotransferase system were more upregulated in the evolved strain than in the ancestral strain, indicating that they are important for elasnin tolerance. Overall, this study showed that elasnin has antibacterial activity against growing S. aureus cells and revealed the altered processes due to elasnin treatment and those associated with its tolerance. IMPORTANCE Besides the excellent antibiofilm properties of elasnin, we discovered that it can also kill growing methicillin-resistant Staphylococcus aureus (MRSA) planktonic cells. We subjected MRSA cells to an in vitro evolution experiment, and the resulting evolved strains exhibited increased elasnin tolerance, reduced growth rate, loss of pigmentation, and an increased proportion of small-colony formation, and they became more sensitive toward daptomycin and lysostaphin. Through multiomics analysis, we uncovered the affected processes in growing S. aureus planktonic cells following elasnin treatment, including the downregulation of cell wall synthesis, cell division, and some genes/proteins for the two-component system. These findings suggest that elasnin suppressed processes important for the cells' survival and adaptation to environmental stresses, making it an ideal drug adjuvant candidate. Overall, our study provides new insights into the mechanism of elasnin in S. aureus planktonic cells and pointed out the potential application of elasnin in clinics.


Assuntos
Daptomicina , Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus Resistente à Meticilina/genética , Daptomicina/metabolismo , Staphylococcus aureus/genética , Lisostafina/genética , Proteômica , Transcriptoma , Antibacterianos/metabolismo
2.
Biotechnol Appl Biochem ; 68(5): 1058-1066, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32918836

RESUMO

Antibiotic resistance and the colonization of resistant bacteria such as Staphylococcus aureus on surfaces, often in the form of biofilms, prolong hospitalization periods and increase mortality, thus is a significant concern for healthcare providers. To prevent biofilm formation, the inadequate concentration of using nanoparticles as antibacterial coating agents is one of the major obstacles. This study aimed to design a hypervalency TiO2 nanocomposite as a reserved base to carry a high amount of active antibacterial agents such as lysostaphin via a biotin-streptavidin-biotin bridge. The utilization of the streptavidin-biotin system could increase the abundance of lysostaphin. Lysostaphin was expressed in Escherichia coli and purified. Both recombinant lysostaphin and titanium oxide nanocomposite were conjugated with biotin and linked to a streptavidin bridge. The kinetics and activity of the enzyme were examined after each step utilizing N-acetylhexaglycine as a substrate. Physical characteristics of nanoparticles containing lysostaphin were determined using AFM, SEM, FTIR, and zeta potential. The results showed changes in size, charge, and morphology of the nanoparticles following the lysostaphin attachment. Also, the stability and kinetics of the active biological enzymes on nanoparticles were reexamined following 8 months of storage. Exploiting this approach, various biotinylated antibacterial agents could be prepared and rapidly immobilized on a nanoparticle as an active net against related infectious agents.


Assuntos
Antibacterianos/farmacologia , Lisostafina/metabolismo , Nanopartículas/química , Infecções Estafilocócicas/tratamento farmacológico , Titânio/farmacologia , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Biotina/química , Biotina/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lisostafina/química , Lisostafina/genética , Tamanho da Partícula , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Infecções Estafilocócicas/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo , Propriedades de Superfície , Titânio/química
3.
Protein Expr Purif ; 177: 105753, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32950627

RESUMO

Staphylococcus aureus (S. aureus), which has developed multidrug resistance, leads to many healthcare-associated infections resulting in significant medical and economic losses. Therefore, the development of new efficient strategies to deal with these bacteria has been gaining importance. Lysostaphin is a peptidoglycan hydrolase that has considerable potential as a bacteriocin. However, there have been few reported optimization and scale-up studies of the lysostaphin bioproduction process. Our preliminary results have revealed that the composition of auto-induction media at 30 °C increases the produced lysostaphin around 10-fold in shake flasks. In this study, achieving higher yields for recombinant lysostaphin in E. coli at a laboratory scale has been the aim, through the use of auto-induction media. Optimized medium composition and fermentation parameters were transferred to a laboratory-scale bioreactor. The tested conditions improved protein yields up to 184 mg/L in a 3 L stirred bioreactor and the productivity was improved 2-fold in comparison to previously published reports. Furthermore, this study also showed that lysostaphin is an effective bacteriocin on both commercially available and isolated S. aureus strains. These results will contribute to future larger-scale production of lysostaphin via the proposed fermentation conditions.


Assuntos
Antibacterianos/biossíntese , Lisostafina/biossíntese , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Reatores Biológicos , Clonagem Molecular , Meios de Cultura/química , Meios de Cultura/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Glicerol/metabolismo , Glicerol/farmacologia , Lisostafina/genética , Lisostafina/isolamento & purificação , Lisostafina/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus/genética , Staphylococcus aureus/crescimento & desenvolvimento
4.
ACS Synth Biol ; 9(3): 475-485, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32105449

RESUMO

Engineered living materials have the potential for wide-ranging applications such as biosensing and treatment of diseases. Programmable cells provide the functional basis for living materials; however, their release into the environment raises numerous biosafety concerns. Current designs that limit the release of genetically engineered cells typically involve the fabrication of multilayer hybrid materials with submicrometer porous matrices. Nevertheless the stringent physical barriers limit the diffusion of macromolecules and therefore the repertoire of molecules available for actuation in response to communication signals between cells and their environment. Here, we engineer a novel living material entitled "Platform for Adhesin-mediated Trapping of Cells in Hydrogels" (PATCH). This technology is based on engineered E. coli that displays an adhesion protein derived from an Antarctic bacterium with a high affinity for glucose. The adhesin stably anchors E. coli in dextran-based hydrogels with large pore diameters (10-100 µm) and reduces the leakage of bacteria into the environment by up to 100-fold. As an application of PATCH, we engineered E. coli to secrete the bacteriocin lysostaphin which specifically kills Staphyloccocus aureus with low probability of raising antibiotic resistance. We demonstrated that living materials containing this lysostaphin-secreting E. coli inhibit the growth of S. aureus, including the strain resistant to methicillin (MRSA). Our tunable platform allows stable integration of programmable cells in dextran-based hydrogels without compromising free diffusion of macromolecules and could have potential applications in biotechnology and biomedicine.


Assuntos
Adesinas Bacterianas/metabolismo , Materiais Biocompatíveis/farmacologia , Escherichia coli/genética , Engenharia Genética/métodos , Lisostafina/farmacologia , Adesinas Bacterianas/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Materiais Biocompatíveis/metabolismo , Membrana Celular/metabolismo , Dextranos/química , Escherichia coli/metabolismo , Hidrogéis/química , Hidrogéis/metabolismo , Lisostafina/genética , Lisostafina/metabolismo , Marinomonas/genética , Teste de Materiais , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos
5.
Int J Nanomedicine ; 14: 9777-9792, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849468

RESUMO

PURPOSE: Staphylococcus aureus is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary. METHODS: In the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against S. aureus. Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against S. aureus in vitro. RESULTS: The optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of S. aureus for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations. CONCLUSION: This study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of S. aureus in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Lisostafina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Liberação Controlada de Fármacos , Sinergismo Farmacológico , Fibroblastos/efeitos dos fármacos , Lipossomos/química , Lipossomos/farmacologia , Lisostafina/genética , Lisostafina/farmacocinética , Camundongos , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Catelicidinas
6.
Mol Biotechnol ; 61(10): 774-782, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31446565

RESUMO

Lysostaphin is a staphylolytic protein of growing interest from biotechnological and pharmaceutical industry due to its potential use in preventing and combating staphylococcal infections. Here, we describe an optimized method for production of lysostaphin in an inductionless system utilizing constitutive promoter from staphylococcal toxin-antitoxin system PemIK-Sa1. We investigated the influence of ribosome-binding site sequence, Escherichia coli producer strain and growth media on yield and kinetics of recombinant protein production. Lysostaphin was purified in its native active form using one-step cation-exchange chromatography. The system provides a method for cost-efficient and scalable protein production, and can be applied to produce other biotechnologically significant proteins.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Lisostafina/isolamento & purificação , Regiões Promotoras Genéticas , Staphylococcus/genética , Cromatografia por Troca Iônica , Clonagem Molecular , Meios de Cultura , Escherichia coli/genética , Lisostafina/genética , Engenharia de Proteínas , Proteínas Recombinantes/metabolismo , Sistemas Toxina-Antitoxina
7.
Molecules ; 24(16)2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31395814

RESUMO

Antibacterial lysins are promising proteins that are active against both antibiotic-susceptible and antibiotic-resistant bacterial strains. However, a major limitation of antibacterial lysins is their fast elimination from systemic circulation. PEGylation increases the plasma half-life of lysins but renders them inactive. Here we report the construction of a fusion protein of lysostaphin, a potent anti-staphylococcal lysin, and an albumin-binding domain from streptococcal protein G. The resulting fusion protein was less active than the parent enzyme lysostaphin, but it still retained significant antibacterial activity even when bound to serum albumin. The terminal half-life of the fusion protein in rats was five-fold greater than that of lysostaphin (7.4 vs. 1.5 h), and the area under the curve increased more than 115 times. Most importantly, this increase in systemic circulation time compensated for the decrease in activity. The plasma from rats that received an injection of the fusion protein retained bactericidal activity for up to 7 h, while plasma from rats that received plain lysostaphin lacked any detectable activity after 4 h. To the best of our knowledge, this is the first report of an antibacterial lysin with both improved pharmacokinetic parameters and prolonged bactericidal activity in the systemic circulation.


Assuntos
Proteínas de Bactérias , Lisostafina , Proteínas Recombinantes de Fusão , Albumina Sérica/química , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacocinética , Proteínas de Bactérias/farmacologia , Feminino , Lisostafina/química , Lisostafina/genética , Lisostafina/farmacocinética , Lisostafina/farmacologia , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia
8.
Sci Rep ; 9(1): 5965, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979923

RESUMO

Staphylococcus simulans lysostaphin cleaves pentaglycine cross-bridges between stem peptides in the peptidoglycan of susceptible staphylococci, including S. aureus. This enzyme consists of an N-terminal catalytic domain and a cell wall binding domain (SH3b), which anchors the protein to peptidoglycan. Although structures of SH3bs from lysostaphin are available, the binding modes of peptidoglycan to these domains are still unclear. We have solved the crystal structure of the lysostaphin SH3b domain in complex with a pentaglycine peptide representing the peptidoglycan cross-bridge. The structure identifies a groove between ß1 and ß2 strands as the pentaglycine binding site. The structure suggests that pentaglycine specificity of the SH3b arises partially directly by steric exclusion of Cß atoms in the ligand and partially indirectly due to the selection of main chain conformations that are easily accessible for glycine, but not other amino acid residues. We have revealed further interactions of SH3b with the stem peptides with the support of bioinformatics tools. Based on the structural data we have attempted engineering of the domain specificity and have investigated the relevance of the introduced substitutions on the domain binding and specificity, also in the contexts of the mature lysostaphin and of its bacteriolytic activity.


Assuntos
Lisostafina/química , Peptidoglicano/química , Sequência de Aminoácidos , Biologia Computacional , Simulação por Computador , Escherichia coli , Lisostafina/genética , Lisostafina/metabolismo , Modelos Moleculares , Peptidoglicano/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas , Staphylococcus
9.
Lett Appl Microbiol ; 14(3): 72-6, 1992 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1367977

RESUMO

The lysostaphin structural gene was cloned in Bacillus subtilis DSM402 and in Lactobacillus casei 102S. The gene was expressed in both organisms and active lysostaphin was released into the medium. Lysostaphin produced by these organisms induced lysis of growing and heat inactivated staphylococci. Expression in a protective starter organism is a prerequisite to produce lysostaphin in situ in fermenting foods and hence, to reduce the hygienical risk of staphylococcal food poisoning.


Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Lacticaseibacillus casei/genética , Lisostafina/genética , Staphylococcus/efeitos dos fármacos , Bacillus subtilis/metabolismo , Clonagem Molecular , Vetores Genéticos , Lacticaseibacillus casei/metabolismo , Lisostafina/biossíntese , Lisostafina/farmacologia , Fotometria , Plasmídeos , Mapeamento por Restrição , Staphylococcus/metabolismo , Transformação Bacteriana
10.
Gene ; 102(1): 105-9, 1991 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-1677905

RESUMO

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.


Assuntos
N-Acetil-Muramil-L-Alanina Amidase/genética , Homologia de Sequência do Ácido Nucleico , Staphylococcus aureus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Lisostafina/genética , Dados de Sequência Molecular , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Fases de Leitura Aberta , Staphylococcus aureus/enzimologia
11.
Biochem Biophys Res Commun ; 160(3): 1106-9, 1989 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-2730641

RESUMO

Staphylococcus simulans biovar staphylolyticus, the lysostaphin-producing organism, secretes a staphylolytic endopeptidase (EC 3.4.99.17) that is encoded on plasmid pACK1. Susceptibility of pACK1-cured strains to lysis by endopeptidase established that resistance to this enzyme is not an inherent property of the organism but rather is encoded on this dispensable plasmid. Furthermore, the enzyme is not an autolysin that is essential for cell wall synthesis because strains lacking the endopeptidase gene grew normally.


Assuntos
Lisostafina/genética , Plasmídeos , Staphylococcus/enzimologia , Lisostafina/farmacologia , Mutação , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética
12.
Mol Gen Genet ; 209(3): 563-9, 1987 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-2828883

RESUMO

The gene encoding lysostaphin of Staphylococcus staphylolyticus was cloned in Escherichia coli and its DNA sequence was determined. The complete coding region comprises 1440 base pairs corresponding to a precursor of 480 amino acids (molecular weight 51 669). It was shown by NH2-terminal amino acid sequence analysis of the purified extracellular lysostaphin from S. staphylolyticus that the mature lysostaphin consists of 246 amino acid residues (molecular weight 26926). Polyacrylamide gel electrophoresis revealed a similar molecular weight for the most active form. By computer analysis the secondary protein structure was predicted. It revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 aa), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 aa) and the hydrophobic mature lysostaphin. The lysostaphin precursor protein appears to be organized as a preprolysostaphin.


Assuntos
Escherichia coli/genética , Genes Bacterianos , Genes , Lisostafina/genética , Staphylococcus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon , Enzimas de Restrição do DNA , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico
13.
Proc Natl Acad Sci U S A ; 84(5): 1127-31, 1987 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-3547405

RESUMO

A 1.5-kilobase-pair fragment of DNA that contains the lysostaphin gene from Staphylococcus simulans and its flanking sequences has been cloned and completely sequenced. The gene encodes a preproenzyme of Mr 42,000. The NH2-terminal sequence of the preproenzyme is composed of a signal peptide followed by seven tandem repeats of a 13-amino acid sequence. Conversion of prolysostaphin to the mature enzyme occurs extracellularly in cultures of S. simulans and involves removal of the NH2-terminal portion of the proenzyme that contains the tandem repeats. The high degree of homology of the repeats suggests that they have arisen by duplication of a 39-base-pair sequence of DNA. In S. simulans, the lysostaphin gene is present on a large beta-lactamase plasmid.


Assuntos
Genes Bacterianos , Genes , Lisostafina/genética , Staphylococcus/genética , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Lisostafina/farmacologia
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