RESUMEN
Staphylococcal SplB protease belongs to the chymotrypsin family. Chymotrypsin zymogen is activated by proteolytic processing at the N terminus, resulting in significant structural rearrangement at the active site. Here, we demonstrate that the molecular mechanism of SplB protease activation differs significantly and we characterize the novel mechanism in detail. Using peptide and protein substrates we show that the native signal peptide, or any N-terminal extension, has an inhibitory effect on SplB. Only precise N-terminal processing releases the full proteolytic activity of the wild type analogously to chymotrypsin. However, comparison of the crystal structures of mature SplB and a zymogen mimic show no rearrangement at the active site whatsoever. Instead, only the formation of a unique hydrogen bond network, distant form the active site, by the new N-terminal glutamic acid of mature SplB is observed. The importance of this network and influence of particular hydrogen bond interactions at the N terminus on the catalytic process is demonstrated by evaluating the kinetics of a series of mutants. The results allow us to propose a consistent model where changes in the overall protein dynamics rather than structural rearrangement of the active site are involved in the activation process.
Asunto(s)
Serina Proteasas/química , Serina Proteasas/metabolismo , Staphylococcus aureus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Quimotripsina/química , Quimotripsina/genética , Quimotripsina/metabolismo , Cristalografía por Rayos X , Precursores Enzimáticos/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Señales de Clasificación de Proteína/fisiología , Estructura Terciaria de Proteína , Serina Proteasas/genética , Staphylococcus aureus/genética , Relación Estructura-ActividadRESUMEN
Secretory leukocyte proteinase inhibitor (SLPI) is a well-established inhibitor of serine proteases such as human neutrophil elastase (HNE) and a NF-κB regulatory agent in immune cells. In this paper, we report that SLPI plays a previously uncharacterized role in regulating activation of plasmacytoid dendritic cells (pDCs). As the main source of IFN type I (IFNI), pDCs are crucial contributors to inflammatory and likely wound-healing responses associated with psoriasis. The mechanisms responsible for activation of pDCs in psoriatic skin are therefore of substantial interest. We demonstrate that in lesional skin of psoriasis patients, SLPI together with its enzymatic target HNE and DNA, is a component of neutrophil extracellular traps (NETs). Whereas SLPI(+) neutrophils and NETs were found to colocalize with pDCs in psoriatic skin, a mixture of SLPI with neutrophil DNA and HNE induced a marked production of IFNI by pDCs. IFNI synthesis by stimulated pDCs was dependent on intracellular DNA receptor TLR9. Thus, SLPI may contribute to psoriasis by enabling pDCs to sense extracellular DNA and produce IFNI.
Asunto(s)
ADN/inmunología , Células Dendríticas/inmunología , Psoriasis/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias/inmunología , Adulto , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Interferón Tipo I/inmunología , Elastasa de Leucocito/inmunología , MasculinoRESUMEN
Staphylococcus aureus strain CH-91, isolated from a broiler chicken with atopic dermatitis, has a highly proteolytic phenotype that is correlated with the disease. We describe the isolation and biochemical and molecular characterization of the AI-type lantibiotic BacCH91 from S. aureus CH-91 culture medium. The bacteriocin was purified using a three-stage procedure comprising precipitation with ammonium sulfate, extraction with organic solvents, and reversed-phase HPLC. The BacCH91 peptide is thermostable and highly resistant to cleavage by both prokaryotic and eukaryotic peptidases. The MIC for the Gram-positive bacteria ranged from 2.5 nM for Microococcus luteus through 1.3-6.0 µM for staphylococcal strains up to more than 100 µM for Lactococcus lactis. BacCH91 was ineffective against the Gram-negative strains tested at the maximal concentration (100 µM). The amino acid sequence of BacCH91 is similar to that of epidermin and gallidermin. The encoding gene (bacCH91) occurred in two allelic variants distinguishable in the restriction fragment length polymorphism assay. Variant I, identified in S. aureus CH-91, dominated in S. aureus strains of poultry origin, although strains with variant II were also identified in this group. S. aureus strains of human origin were characterized exclusively by variant II.
Asunto(s)
Bacteriocinas/farmacología , Staphylococcus aureus/metabolismo , Animales , Bacteriocinas/genética , Bacteriocinas/aislamiento & purificación , Bacteriocinas/metabolismo , Fraccionamiento Químico , Pollos , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Lactococcus lactis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Micrococcus luteus/efectos de los fármacos , Datos de Secuencia Molecular , Aves de Corral , Enfermedades de las Aves de Corral/microbiología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Staphylococcus aureus is a dangerous human pathogen whose antibiotic resistance is steadily increasing and no efficient vaccine is as yet available. This serious threat drives extensive studies on staphylococcal physiology and pathogenicity pathways, especially virulence factors. Spl (serine protease-like) proteins encoded by an operon containing up to six genes are a good example of poorly characterized secreted proteins probably involved in virulence. In the present study, we describe an efficient heterologous expression system for SplA and detailed biochemical and structural characterization of the recombinant SplA protease. The enzyme shares a significant sequence homology to V8 protease and epidermolytic toxins which are well documented staphylococcal virulence factors. SplA has a very narrow substrate specificity apparently imposed by the precise recognition of three amino acid residues positioned N-terminal to the hydrolysed peptide bond. To explain determinants of this extended specificity we resolve the crystal structure of SplA and define the consensus model of substrate binding. Furthermore we demonstrate that artificial N-terminal elongation of mature SplA mimicking a naturally present signal peptide abolishes enzymatic activity. The probable physiological role of the process is discussed. Of interest, even though precise N-terminal trimming is a common regulatory mechanism among S1 family enzymes, the crystal structure of SplA reveals novel significantly different mechanistic details.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Animales , Aniones , Biocatálisis , Quimotripsina/química , Cristalografía por Rayos X , Histidina , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Especificidad por SustratoRESUMEN
We have reported a bacterial infection in a dog with progressive dysplasia of the hips. Orthopedic surgery was performed. Seven weeks prior to the surgery, the patient was bitten by another dog. The postimplantation wound exuded for four days after the surgery. Microbiological analysis performed by standard identification techniques showed the presence of Staphylococcus intermedius, but an additional molecular analysis indicated S. pseudintermedius. This was followed by an evaluation of antibiotic susceptibility of the strain which showed cefoxitin, ciprofloxacin, clindamycin, trimethoprim/sulfamethoxazole, doksycycline, erythromycin, and gentamicin resistance. Minimal inhibitory concentration (MIC) values for selected antibiotics were reported. Resistance for cefoxitin indicates that methicillin resistant S. pseudintermedius (MRSP) strains were present in individual macroorganisms, but they can expand and persist the colonization of other hosts.
Asunto(s)
Artroplastia de Reemplazo/efectos adversos , Enfermedades de los Perros/microbiología , Complicaciones Posoperatorias/veterinaria , Infecciones Estafilocócicas/veterinaria , Staphylococcus/aislamiento & purificación , Animales , Antibacterianos/farmacología , Artroplastia de Reemplazo/veterinaria , Enfermedades de los Perros/cirugía , Perros , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Complicaciones Posoperatorias/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus/efectos de los fármacos , Staphylococcus/genéticaRESUMEN
CD44 is a type I transmembrane glycoprotein interacting with a number of extracellular components, including hyaluronic acid (HA). CD44-HA axis is involved in a variety of processes, including adhesion, migration, differentiation, trafficking, and others. CD44 is overexpressed in several cancers where binding of HA induces signal transduction leading to activation of antiapoptotic proteins and factors linked to drug resistance. As such, CD44 has been implicated in cancer growth, progression, and metastasis. It has been convincingly demonstrated that blocking CD44-HA interaction decreases cancer cell survival and metastasis. In this study, using in vitro selection, we have developed DNA aptamers recognizing a HA-binding domain of CD44 with high affinity and specificity. The aptamers bind to CD44 with nanomolar affinities and efficiently inhibit the growth of leukemic cancer cells characterized by high expression of CD44. The selectivity is demonstrated by an irrelevant effect on cells characterized by low CD44 levels. The obtained aptamers broaden the existing landscape of potential approaches to the development of antitumor strategies based on inhibition of the CD44 axis.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Receptores de Hialuranos/genética , Ácido Hialurónico/genética , Neoplasias/terapia , Aptámeros de Nucleótidos/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/patología , Dominios Proteicos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Staphylococcus aureus expresses several proteases, which are thought to contribute to the virulence of this bacterium. Here we focus on aureolysin, the major thermolysin-like metalloprotease. Despite the importance of aureolysin in the physiology and pathogenesis of S. aureus, relatively little information was so far available concerning the aur gene diversity and mobility within and between the major subdivisions of the S. aureus population. Therefore, an epidemiologically and genetically diverse collection of S. aureus strains was used to determine the range of aureolysin (aur) gene polymorphism. RESULTS: Sequence analyses support the conclusion that the aur gene occurs in two distinct types of related sequences. The aur gene was much more polymorphic but, at the same time, showed higher purifying selection than genes utilized for multilocus sequence typing (MLST). Gene trees constructed from aur and concatenated MLST genes revealed several putative assortative recombination events (i.e. entire aur gene exchanges) between divergent lineages of S. aureus. Evidence for intragenic recombination events (i.e. exchanges of internal aur segments) across aur genes was also found. The biochemical properties and substrate specificity of the two types of aureolysin purified to homogeneity were studied, revealing minor differences in their affinity to low molecular weight synthetic substrates. CONCLUSION: Although numerous nucleotide differences were identified between the aur alleles studied, our findings showed that a strong purifying selection is acting on the aur gene. Moreover, our study distinguishes between homologous exchanges of the entire aur gene (assortative recombination) between divergent S. aureus lineages and recombination events within aur genes.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Metaloendopeptidasas/genética , Polimorfismo Genético , Recombinación Genética , Staphylococcus aureus/genética , Alelos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Técnicas de Tipificación Bacteriana , Evolución Molecular , Metaloendopeptidasas/química , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Staphylococcus aureus/clasificaciónRESUMEN
Thermolysins constitute a family of secreted bacterial metalloproteases expressed, among others, by several pathogens. Strains of Staphylococcus pseudintermedius isolated from diseased dogs and judged as protease-positive, by skim milk agar plate culture, were investigated for protease content. No proteolytic activity was detected when the bacteria were grown in regular liquid media. Unexpectedly, supplementation of the medium with calcium ions resulted in expression of a metalloprotease and profound changes in the profile of extracellular proteins. On the basis of homology to other staphylococcal metalloproteases, the nucleotide sequence of the gene encoding this protease (Pst) and its flanking regions was determined. The full-length pst codes for a protein with an open reading frame of 505 amino acids. The internal region contains the HEXXH catalytic domain that is conserved in members of the thermolysin family. Regardless of the presence of calcium in the medium, the expression of the protease gene was of the same intensity. This suggests that regulation of the metalloprotease production by calcium ions is at a post-transcriptional level. Isolates of S. pseudintermedius exhibit a proteolytic phenotype due to the metalloprotease expression, however only in presence of calcium ions, which most probably stabilize the structure of the protease.
Asunto(s)
Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Staphylococcus/enzimología , Staphylococcus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Calcio/metabolismo , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Perros , Evolución Molecular , Expresión Génica , Genes Bacterianos , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Polimorfismo Genético , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Staphylococcus/aislamiento & purificación , Staphylococcus/patogenicidad , Especificidad por SustratoRESUMEN
Staphylococcus aureus is the major cause of nosocomial infections world-wide, with increasing prevalence of community-acquired diseases. The recent dramatic increase in multi-antibiotic resistance, including resistance to the last-resort drug, vancomycin, together with the lack of an effective vaccine highlight the need for better understanding of S.aureus pathogenicity. Comparative analysis of available bacterial genomes allows for the identification of previously uncharacterized S.aureus genes with potential roles in pathogenicity. A good example is a cluster of six serine protease-like (spl) genes encompassed in one operon, which encode for putative proteases with similarity to staphylococcal glutamylendopeptidase (V8 protease). Here, we describe an efficient expression system for the production of recombinant SplB and SplC proteases in Escherichia coli, together with structural and functional characterization of the purified enzymes. A unique mechanism of cytoplasm protection against activity of misdirected SplB was uncovered. Apparently, the co-translated signal peptide maintains protease latency until it is cleaved by the signal peptidase during protein secretion. Furthermore, the crystal structure of the SplC protease revealed a fold resembling that of the V8 protease and epidermolytic toxins. Arrangement of the active site cleft and substrate-binding pocket of SplC explains the mechanism of enzyme latency and suggests that some Spl proteases possess restricted substrate specificity similar to that of the V8 protease and epidermolytic toxins.
Asunto(s)
Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Activación Enzimática , Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Alineación de Secuencia , Serina Endopeptidasas/químicaRESUMEN
Eosinophils constitute an important component of helminth immunity and are not only associated with various allergies but are also linked to autoinflammatory disorders, including the skin disease psoriasis. Here we demonstrate the functional relationship between eosinophils and plasmacytoid dendritic cells (pDCs) as related to skin diseases. We previously showed that pDCs colocalize with neutrophil extracellular traps (NETs) in psoriatic skin. Here we demonstrate that eosinophils are found in psoriatic skin near neutrophils and NETs, suggesting that pDC responses can be regulated by eosinophils. Eosinophils inhibited pDC function in vitro through a mechanism that did not involve cell contact but depended on soluble factors. In pDCs stimulated by specific NET components, eosinophil-conditioned media attenuated the production of interferon α (IFNα) but did not affect the maturation of pDCs as evidenced by the unaltered expression of the costimulatory molecules CD80 and CD86. As pDCs and IFNα play a key role in autoimmune skin inflammation, these data suggest that eosinophils may influence autoinflammatory responses through their impact on the production of IFNα by pDCs.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Trampas Extracelulares/inmunología , Interferón-alfa/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Adulto , Degranulación de la Célula/inmunología , Trampas Extracelulares/genética , Trampas Extracelulares/metabolismo , Femenino , Expresión Génica , Humanos , Interferón-alfa/genética , Masculino , Psoriasis/diagnóstico , Psoriasis/inmunología , Psoriasis/metabolismo , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
The aim of this study was to reveal potential markers associated with drug dependence, using the proteomic approach. Gels containing samples derived from morphine-treated and control animals were compared and analyzed. Inspection of protein profiles, following TCA/acetone precipitation and the use of nano-scale liquid chromatography coupled to tandem mass spectrometry, allowed for identification of eleven potential dependence markers, mainly cytoplasmic and mitochondrial enzymes, e.g. proteins that belong to GTPase and GST superfamilies, ATPase, asparaginase or proteasome subunit p27 families.
Asunto(s)
Química Encefálica/fisiología , Dependencia de Morfina/metabolismo , Proteoma/metabolismo , Animales , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Masculino , Espectrometría de Masas , Nanotecnología , Ratas , Ratas WistarRESUMEN
Staphopain A is a staphylococcal cysteine protease. Genes encoding staphopain A and its specific inhibitor, staphostatin A, are localized in an operon. Staphopain A is an important staphylococcal virulence factor. It is difficult to perform studies on its interaction with other proteins due to problems in obtaining a sufficient amount of the enzyme from natural sources. Therefore efforts were made to produce a recombinant staphopain A. Sequences encoding the mature form of staphopain A and staphostatin A were PCR-amplified from Staphylococcus aureus genomic DNA and cloned into different compatible expression vectors. Production of staphopain A was observed only when the enzyme was co-expressed together with its specific inhibitor, staphostatin A. Loss of the function mutations introduced within the active site of staphopain A causes the expression of the inactive enzyme. Mutations within the reactive centre of staphostatin A result in abrogation of production of both the co-expressed proteins. These results support the thesis that the toxicity of recombinant staphopain A to the host is due to its proteolytic activity. The coexpressed proteins are located in the insoluble fraction. Ni2+-nitrilotriacetate immobilized metal-affinity chromatography allows for an efficient and easy purification of staphopain A. Our optimized refolding parameters allow restoration of the native conformation of the enzyme, with yields over 10-fold higher when compared with isolation from natural sources.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Escherichia coli/genética , Expresión Génica/genética , Proteínas Recombinantes/biosíntesis , Staphylococcus aureus/enzimología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cromatografía de Afinidad , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/aislamiento & purificación , Inhibidores de Cisteína Proteinasa/biosíntesis , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/genética , Mutación/genética , Renaturación de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/genéticaRESUMEN
Staphylococcus aureus is a commensal inhabitant of skin and mucous membranes in nose vestibule but also an important opportunistic pathogen of humans and livestock. The extracellular proteome as a whole constitutes its major virulence determinant; however, the involvement of particular proteins is still relatively poorly understood. In this study, we compared the extracellular proteomes of poultry-derived S. aureus strains exhibiting a virulent (VIR) and non-virulent (NVIR) phenotype in a chicken embryo experimental infection model with the aim to identify proteomic signatures associated with the particular phenotypes. Despite significant heterogeneity within the analyzed proteomes, we identified alpha-haemolysin and bifunctional autolysin as indicators of virulence, whereas glutamylendopeptidase production was characteristic for non-virulent strains. Staphopain C (StpC) was identified in both the VIR and NVIR proteomes and the latter fact contradicted previous findings suggesting its involvement in virulence. By supplementing NVIR, StpC-negative strains with StpC, and comparing the virulence of parental and supplemented strains, we demonstrated that staphopain C alone does not affect staphylococcal virulence in a chicken embryo model.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Proteoma/análisis , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/química , Factores de Virulencia/análisis , Animales , Embrión de Pollo , Modelos Animales de Enfermedad , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/patogenicidadRESUMEN
Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the reactive loop was significantly similar to carp and mammalian alpha1-antiproteinases. Both sequences were used to construct primers and obtain a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the similarity to carp alpha1-antiproteinase and mammalian alpha1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily and is similar to alpha1-antiproteinase.
Asunto(s)
Oncorhynchus mykiss/metabolismo , Semen/enzimología , Inhibidores de Tripsina , alfa 1-Antitripsina , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN Complementario/análisis , Glicosilación , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia , Inhibidores de Tripsina/química , Inhibidores de Tripsina/genética , Inhibidores de Tripsina/aislamiento & purificación , Inhibidores de Tripsina/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/aislamiento & purificación , alfa 1-Antitripsina/metabolismoRESUMEN
The increasing antibiotic resistance of pathogenic bacteria calls for the development of alternative antimicrobial strategies. Possible approaches include the development of novel, broad-spectrum antibiotics as well as specific targeting of individual bacterial virulence factors. It is impossible to decide currently which strategy will prove more successful in the future since they both promise different advantages, but also introduce diverse problems. Considering both approaches, our laboratory's research focuses on the evaluation of hemocidins, broad-spectrum antibacterial peptides derived from hemoglobin and myoglobin, and staphostatins, specific inhibitors of staphopains -- Staphylococcus aureus secreted proteases that are virulence factors regarded as possible targets for therapy. The article summarizes recent advances in both fields of study and presents perspectives for further development and possible applications.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Péptidos/síntesis química , Antibacterianos/química , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/química , Inhibidores Enzimáticos/química , Marcación de Gen , Hemoglobinas/química , Pruebas de Sensibilidad Microbiana , Mioglobina/química , Péptidos/farmacología , Staphylococcus aureus/químicaRESUMEN
X-ray crystallography provides important insights into structure-function relationship in biomolecules. However, protein crystals are usually hard to obtain which hinders our understanding of multiple important processes. Crystallization requires large amount of protein sample, whereas recombinant proteins are often unstable or insoluble. Green fluorescent protein (GFP) fusion is one of the approaches to increase protein synthesis, solubility and stability, facilitating crystallization. In this study we analyze the influence of the linker length, composition and the position of GFP relative to the fusion partner on the fusion protein production and stability. To this end, multiple constructs of enzymatically impaired variant of PemKSa toxin from Staphylococcus aureus CH91 fused to GFP were generated. Fusion protein production in Escherichia coli was evaluated. The proteins were purified and their stability tested. PemKSa-α14aa-GFP fusion provided best production and stability. Obtained results demonstrate the importance of optimization of fusion protein construct, including linker selection and the order of fusion partners, in obtaining high quantities of stable protein for crystallization.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , Escherichia coli/metabolismo , Hidrólisis , Datos de Secuencia Molecular , Concentración Osmolar , Solubilidad , Staphylococcus aureus/metabolismo , TemperaturaRESUMEN
Genetic methods based on PCR-restriction fragment length polymorphism (RFLP) are widely used for microbial species determination. In this study, we present the application of saoC gene as an effective tool for species determination and within-species diversity analysis for Staphylococcus genus. The unique sequence diversity of saoC allows us to apply four restriction enzymes to obtain RFLP patterns, which appear highly distinctive even among closely related species as well as atypical isolates of environmental origin. Such patterns were successfully obtained for 26 species belonging to Staphylococcus genus. What is more, tracing polymorphisms detected by different restriction enzymes allowed for basic phylogeny analysis for Staphylococcus aureus, which is potentially applicable for other staphylococcal species.
Asunto(s)
Proteínas Bacterianas/genética , Variación Genética , Tipificación Molecular/métodos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Staphylococcus/clasificación , Staphylococcus/genética , Genotipo , HumanosRESUMEN
Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups.
Asunto(s)
Bacteriocinas/farmacología , Péptidos/farmacología , Staphylococcus/metabolismo , Factores de Virulencia/farmacología , Secuencia de Aminoácidos , Animales , Bacteriocinas/química , Bacteriocinas/aislamiento & purificación , Secuencia de Bases , Línea Celular , Supervivencia Celular/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Péptidos/química , Péptidos/aislamiento & purificación , Estabilidad Proteica , Alineación de Secuencia , Staphylococcus/genética , Staphylococcus/patogenicidad , Factores de Virulencia/química , Factores de Virulencia/aislamiento & purificaciónRESUMEN
Neutrophil extracellular traps (NETs), web-like DNA structures, provide efficient means of eliminating invading microorganisms but can also present a potential threat to its host because it is a likely source of autoantigens or by promoting bystander tissue damage. Therefore, it is important to identify mechanisms that inhibit NET formation. Neutrophil elastase (NE)-dependent chromatin decondensation is a key event in the release of NETs release. We hypothesized that inhibitors of NE, secretory leukocyte protease inhibitor (SLPI) and α(1)-proteinase inhibitor (α(1)-PI), has a role in restricting NET generation. Here, we demonstrate that exogenous human SLPI, but not α(1)-PI markedly inhibited NET formation in human neutrophils. The ability of exogenous SLPI to attenuate NET formation correlated with an inhibition of a core histone, histone 4 (H4), cleavage, and partial dependence on SLPI-inhibitory activity against NE. Moreover, neutrophils from SLPI(-/-) mice were more efficient at generating NETs than were neutrophils from wild-type mice in vitro, and in experimental psoriasis in vivo. Finally, endogenous SLPI colocalized with NE in the nucleus of human neutrophils in vitro, as well as in vivo in inflamed skin of patients with psoriasis. Together, these findings support a controlling role for SLPI in NET generation, which is of potential relevance to infectious and autoinflammatory diseases.
Asunto(s)
Neutrófilos/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/fisiología , Adulto , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Psoriasis/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias/genéticaRESUMEN
Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Here, we present the 1.4 A crystal structure of staphostatin B and show that the fold can be described as a fully closed, highly sheared eight-stranded beta-barrel. Thus, staphostatin B is related to beta-barrel domains that are involved in the inhibition or regulation of proteases of various catalytic types and to the superfamily of lipocalins/cytosolic fatty acid binding proteins. Unexpectedly for a cysteine protease inhibitor, staphostatin B is not significantly similar to cystatins.