RESUMEN
Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.
Asunto(s)
Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Staphylococcus aureus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Metionina/metabolismo , Modelos Moleculares , Péptido Hidrolasas/genética , Péptidos/química , Péptidos/metabolismo , Especificidad por SustratoRESUMEN
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
OBJECTIVE: Staphylococcus aureus (S. aureus) infection is known to contribute to the severity and recalcitrance of chronic rhinosinusitis (CRS), and its secreted products have been shown to alter the airway barrier. Extracellular proteases secreted by S. aureus are thought to be important in epithelial infection and immune evasion; however, their effect on airway mucosal barrier function is not known. METHODS: To investigate the impact of extracellular proteases on airway epithelial integrity, the purified S. aureus proteases V8 protease, Staphopain A, Staphopain B, Exfoliative toxin A, and serine protease-like A-F were applied to human nasal epithelial cell air-liquid interface (HNEC-ALI) cultures. Transepithelial electrical resistance (TEER), permeability (Papp) measurements, and immuno-localization of the tight junction proteins claudin-1 and ZO-1 were used to assess barrier integrity. Effects of the proteases on inflammation and cell viability were measured using interleukin-6 (IL-6) ELISA and a lactate dehydrogenase assay. RESULTS: Application of V8 protease to HNEC-ALI cultures caused a significant concentration and time-dependent decrease in TEER (22.67%, P < 0.0001), a reciprocal Papp increase (20.14-fold, P < 0.05), and a discontinuous ZO-1 immuno-localization compared to control. IL-6 production was significantly reduced in V8 protease-treated cells (153.5 pg/mL, P = 0.0069) compared to control (548.3 pg/mL), whereas no difference in cell viability was observed. CONCLUSION: S. aureus V8 protease causes dysfunction of mucosal barrier structure and function indicative of a leaky barrier. A reduction in IL-6 levels suggests that the mucosal immunity is impaired by this protease and thus has the potential to contribute to CRS recalcitrance. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E8-E15, 2018.
Asunto(s)
Interleucina-6/metabolismo , Mucosa Nasal/metabolismo , Serina Endopeptidasas/inmunología , Staphylococcus aureus/inmunología , Supervivencia Celular , Células Cultivadas , Colorimetría/métodos , Impedancia Eléctrica , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunidad Mucosa , Técnicas In Vitro , Inflamación/inmunología , Uniones EstrechasRESUMEN
Staphylococcus aureus is a dangerous human pathogen characterized by alarmingly increasing antibiotic resistance. Accumulating evidence suggests the role of Spl proteases in staphylococcal virulence. Spl proteases have restricted, non-overlapping substrate specificity, suggesting that they may constitute a first example of a proteolytic system in bacteria. SplA, SplB, and SplD were previously characterized in terms of substrate specificity and structural determinants thereof. Here we analyze the substrate specificity of SplE documenting its unique P1 preference among Spl proteases and, in fact, among all chymotrypsin-like (family S1) proteases characterized to date. This is interesting since our understanding of the general aspects of proteolysis is based on seminal studies of S1 family members. To better understand the molecular determinants of the unusual specificity of SplE, the crystal structure of the protein is determined here. Conclusions from structural analysis are evaluated by successful grafting of SplE specificity on the scaffold of SplB protease.
Asunto(s)
Proteínas Bacterianas/química , Péptidos/química , Serina Proteasas/química , Staphylococcus aureus/química , Factores de Virulencia/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutación , Biblioteca de Péptidos , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Serina Proteasas/genética , Serina Proteasas/metabolismo , Staphylococcus aureus/enzimología , Staphylococcus aureus/patogenicidad , Especificidad por Sustrato , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Staphylococcus aureus is a dangerous human pathogen characterized by growing antibiotic resistance. Virulence of S. aureus relies on a variety of secreted and cell surface associated virulence factors among which certain proteolytic enzymes play an important role. Amid staphylococcal extracellular proteases, those encoded by the spl operon remain poorly characterized, both in terms of enzymology and their physiological role. Initial data demonstrated that Spl proteases exhibit restricted substrate specificity. This study describes development of convenient protein FRET substrates for SplB protease and characterization of the substrate preference of the protease at the P1' position. Kinetic data on hydrolysis of a panel of substrates substituted at the said position is provided.
Asunto(s)
Proteínas Bacterianas/metabolismo , Péptido Hidrolasas/metabolismo , Serina Proteasas/metabolismo , Infecciones Estafilocócicas/enzimología , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Humanos , Hidrólisis , Cinética , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/patología , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Especificidad por SustratoRESUMEN
Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1') with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.