Hemorrhagic Activity of HF3, a Snake Venom Metalloproteinase: Insights from the Proteomic Analysis of Mouse Skin and Blood Plasma

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and blood extravasation. The mechanism of action of SVMPs has been investigated using various methodologies however the precise molecular events associated with microvessel disruption remains not fully understood. To gain insight into the hemorrhagic process, we analyzed the global effects of HF3, an extremely hemorrhagic SVMP from <i>Bothrops jararaca</i>, in the mouse skin and plasma. We report that in the HF3-treated skin there was evidence of degradation of extracellular matrix (collagens and proteoglycans), cytosolic, cytoskeleton, and plasma proteins. Furthermore, the data suggest that direct and indirect effects promoted by HF3 contributed to tissue injury as the activation of collagenases was detected in the HF3-treated skin. In the plasma analysis after depletion of the 20 most abundant proteins, fibronectin appeared as degraded by HF3. In contrast, some plasma proteinase inhibitors showed higher abundance compared to control skin and plasma. This is the first study to assess the complex <i>in vivo</i> effects of HF3 using high-throughput proteomic approaches, and the results underscore a scenario characterized by the interplay between the hydrolysis of intracellular, extracellular, and plasma proteins and the increase of plasma inhibitors in the hemorrhagic process

Hemorrhagic Activity of HF3, a Snake Venom Metalloproteinase: Insights from the Proteomic Analysis of Mouse Skin and Blood Plasma

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and blood extravasation. The mechanism of action of SVMPs has been investigated using various methodologies however the precise molecular events associated with microvessel disruption remains not fully understood. To gain insight into the hemorrhagic process, we analyzed the global effects of HF3, an extremely hemorrhagic SVMP from <i>Bothrops jararaca</i>, in the mouse skin and plasma. We report that in the HF3-treated skin there was evidence of degradation of extracellular matrix (collagens and proteoglycans), cytosolic, cytoskeleton, and plasma proteins. Furthermore, the data suggest that direct and indirect effects promoted by HF3 contributed to tissue injury as the activation of collagenases was detected in the HF3-treated skin. In the plasma analysis after depletion of the 20 most abundant proteins, fibronectin appeared as degraded by HF3. In contrast, some plasma proteinase inhibitors showed higher abundance compared to control skin and plasma. This is the first study to assess the complex <i>in vivo</i> effects of HF3 using high-throughput proteomic approaches, and the results underscore a scenario characterized by the interplay between the hydrolysis of intracellular, extracellular, and plasma proteins and the increase of plasma inhibitors in the hemorrhagic process

Table4.XLSX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p

Table1.XLSX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p

Table3.XLSX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p

Image1.TIF

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p

Table2.XLSX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p

Table5.DOCX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p