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1.
BMC Res Notes ; 9: 389, 2016 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-27488131

RESUMO

BACKGROUND: Staphylococcus aureus is a versatile pathogen expressing a number of virulence-associated adhesive molecules. In a previous study, we generated in a secretion-competent Escherichia coli strain a library of random FLAG-tag positive (FTP) polypeptides of S. aureus. To identify adhesive proteins and gain additional knowledge on putative virulence factors of S. aureus, we here screened the FTP library against human serum proteins. FINDINGS: Staphylococcus aureus NCTC 8325-4, origin of the FTP library, adhered to immobilized plasminogen in vitro. In an enzyme-linked immunoassay a C-terminal part of penicillin binding protein 3 (PBP3), included in the FTP library, bound to immobilized plasminogen. We expressed and purified full-length PBP3 and its C-terminal fragments as recombinant proteins. In a time-resolved fluorometry-based assay the PBP3 polypeptides bound to immobilized plasminogen. The polypeptides enhanced formation of plasmin from plasminogen as analyzed by cleavage of a chromogenic plasmin substrate. CONCLUSIONS: The present findings, although preliminary, demonstrate reliably that S. aureus NCTC 8325-4 adheres to immobilized plasminogen in vitro and that the adhesion may be mediated by a C-terminal fragment of the PBP3 protein. The full length PBP3 and the penicillin binding C-terminal domain of PBP3 expressed as recombinant proteins bound plasminogen and activated plasminogen to plasmin. These phenomena were inhibited by the lysine analogue ε-aminocaproic acid suggesting that the binding is mediated by lysine residues. A detailed molecular description of surface molecules enhancing the virulence of S. aureus will aid in understanding of its pathogenicity and help in design of antibacterial drugs in the future.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Plasminogênio/metabolismo , Staphylococcus aureus/metabolismo , Fibrinolisina/metabolismo , Humanos , Proteínas Imobilizadas/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Solubilidade
2.
Microbiology (Reading) ; 158(Pt 7): 1713-1722, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22516222

RESUMO

Lactobacilli belong to the normal gastrointestinal and genital tract microbiota of human and animal hosts. Adhesion is important for bacterial colonization; however, only a few Lactobacillus adhesins have been identified so far. We studied extracted surface proteins from an adhesive Lactobacillus crispatus strain, ST1, which efficiently colonizes the chicken alimentary tract, for their binding to tissue sections of the chicken crop, and identified a novel high-molecular-mass repetitive surface protein that shows specific binding to stratified squamous epithelium. The adhesin binds to both crop epithelium and epithelial cells from human vagina, and was named Lactobacillus epithelium adhesin (LEA). Expression of LEA is strain-specific among L. crispatus strains and corresponds directly to in vitro bacterial adhesion ability. The partial sequence of the lea gene predicts that the LEA protein carries an N-terminal YSIRK signal sequence and a C-terminal LPxTG anchoring motif, as well as a highly repetitive region harbouring 82 aa long repeats with non-identical sequences that show similarity to Lactobacillus Rib/alpha-like repeats. LEA-mediated epithelial adherence may improve bacterial colonization in the chicken crop and the human vagina, which are the natural environments for L. crispatus.


Assuntos
Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Células Epiteliais/microbiologia , Lactobacillus/genética , Lactobacillus/patogenicidade , Animais , Células Cultivadas , Galinhas , Fezes , Feminino , Trato Gastrointestinal/microbiologia , Humanos , Sequências Repetitivas de Aminoácidos , Análise de Sequência de Proteína , Vagina/microbiologia
3.
J Bacteriol ; 194(10): 2509-19, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22389474

RESUMO

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Glucose-6-Fosfato Isomerase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Lactobacillus/efeitos dos fármacos , Lactobacillus/enzimologia , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose-6-Fosfato Isomerase/genética , Glutamato-Amônia Ligase/genética , Humanos , Concentração de Íons de Hidrogênio , Lactobacillus/citologia , Lactobacillus/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ligação Proteica , Catelicidinas
4.
BMC Microbiol ; 11: 117, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21615970

RESUMO

BACKGROUND: Bacterial adhesive proteins, called adhesins, are frequently the decisive factor in initiation of a bacterial infection. Characterization of such molecules is crucial for the understanding of bacterial pathogenesis, design of vaccines and development of antibacterial drugs. Because adhesins are frequently difficult to express, their characterization has often been hampered. Alternative expression methods developed for the analysis of adhesins, e.g. surface display techniques, suffer from various drawbacks and reports on high-level extracellular secretion of heterologous proteins in Gram-negative bacteria are scarce. These expression techniques are currently a field of active research. The purpose of the current study was to construct a convenient, new technique for identification of unknown bacterial adhesive polypeptides directly from the growth medium of the Escherichia coli host and to identify novel proteinaceous adhesins of the model organism Staphylococcus aureus. RESULTS: Randomly fragmented chromosomal DNA of S. aureus was cloned into a unique restriction site of our expression vector, which facilitates secretion of foreign FLAG-tagged polypeptides into the growth medium of E. coli ΔfliCΔfliD, to generate a library of 1663 clones expressing FLAG-tagged polypeptides. Sequence and bioinformatics analyses showed that in our example, the library covered approximately 32% of the S. aureus proteome. Polypeptides from the growth medium of the library clones were screened for binding to a selection of S. aureus target molecules and adhesive fragments of known staphylococcal adhesins (e.g coagulase and fibronectin-binding protein A) as well as polypeptides of novel function (e.g. a universal stress protein and phosphoribosylamino-imidazole carboxylase ATPase subunit) were detected. The results were further validated using purified His-tagged recombinant proteins of the corresponding fragments in enzyme-linked immunoassay and surface plasmon resonance analysis. CONCLUSIONS: A new technique for identification of unknown bacterial adhesive polypeptides was constructed. Application of the method on S. aureus allowed us to identify three known adhesins and in addition, five new polypeptides binding to human plasma and extracellular matrix proteins. The method, here used on S. aureus, is convenient due to the use of soluble proteins from the growth medium and can in principle be applied to any bacterial species of interest.


Assuntos
Adesinas Bacterianas/metabolismo , Escherichia coli/genética , Biblioteca Gênica , Staphylococcus aureus/metabolismo , Adesinas Bacterianas/genética , Clonagem Molecular , Meios de Cultura/química , DNA Bacteriano/química , DNA Bacteriano/genética , Vetores Genéticos , Genética Microbiana/métodos , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Staphylococcus aureus/genética , Estados Unidos , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
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