Individual Variability in the Venom Proteome of Juvenile <i>Bothrops jararaca</i> Specimens

Snake venom proteomes/peptidomes are highly complex and subject to ontogenetic changes. Individual variation in the venom proteome of juvenile snakes is poorly known. We report the proteomic analysis of venoms from 21 juvenile specimens of <i>Bothrops jararaca</i> of different geographical origins and correlate it with the evaluation of important venom features. Individual venoms showed similar caseinolytic activities; however, their amidolytic activities were significantly different. Rather intriguingly, plasma coagulant activity showed remarkable variability among the venoms but not the prothrombin-activating activity. LC–MS analysis showed significant differences between venoms; however, an interesting finding was the ubiquitous presence of the tripeptide ZKW, an endogenous inhibitor of metalloproteinases. Electrophoretic profiles of proteins submitted to reduction showed significant variability in total proteins, glycoproteins, and in the subproteomes of proteinases. Moreover, identification of differential bands revealed variation in most <i>B. jararaca</i> toxin classes. Profiles of venoms analyzed under nonreducing conditions showed less individual variability and identification of proteins in a conserved band revealed the presence of metalloproteinases and l-amino acid oxidase as common components of these venoms. Taken together, our findings suggest that individual venom proteome variability in <i>B. jararaca</i> exists from a very early animal age and is not a result of ontogenetic and diet changes

Dynamic Rearrangement in Snake Venom Gland Proteome: Insights into <i>Bothrops jararaca</i> Intraspecific Venom Variation

We carried out an analysis of the venom gland proteome of <i>Bothrops jararaca</i> taking into account two distinct phases of its ontogenetic development (i.e., newborn and adult) and the marked sexual dimorphism recently reported on its venom proteome. Proteomic data analysis showed a dynamic rearrangement in the proteome landscape of <i>B. jararaca</i> venom gland upon development and gender-related changes. Differentially expressed proteins covered a number of biological pathways related to protein synthesis, including proteins associated with transcription and translation, which were found to be significantly higher expressed in the newborn venom gland. Our results suggest that the variation in the expression levels of cellular proteins might give rise to an even higher variation in the levels of the expressed toxins. Upon aging, the venom gland proteome repertoire related to the protein synthesis together with ecological traits would have an impact on the toxin repertoire, which, in the case of <i>B. jararaca</i> species, would enable the species to deal with different prey types during its lifespan. Proteomic data are available via ProteomeXchange with identifier PXD004186

Exploring Potential Virulence Regulators in <i>Paracoccidioides brasiliensis</i> Isolates of Varying Virulence through Quantitative Proteomics

Few virulence factors have been identified for <i>Paracoccidioides brasiliensis</i>, the agent of paracoccidioidomycosis. In this study, we quantitatively evaluated the protein composition of <i>P. brasiliensis</i> in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC–MS/MS) analysis, we identified and quantified 316 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 295 proteins were overexpressed in the virulent fungus. Three proteins were found to be up-regulated in both media, suggesting the potential roles of these proteins in virulence regulation in <i>P. brasiliensis</i>. Moreover, genes up-regulated in virulent Pb18 showed an increase in its expression after the recovery of virulence of attenuated Pb18. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and provided a foundation for achieving a molecular understanding of how <i>Paracoccidioides</i> modulates the host–pathogen interaction to its advantage

Exploring Potential Virulence Regulators in <i>Paracoccidioides brasiliensis</i> Isolates of Varying Virulence through Quantitative Proteomics

Few virulence factors have been identified for <i>Paracoccidioides brasiliensis</i>, the agent of paracoccidioidomycosis. In this study, we quantitatively evaluated the protein composition of <i>P. brasiliensis</i> in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC–MS/MS) analysis, we identified and quantified 316 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 295 proteins were overexpressed in the virulent fungus. Three proteins were found to be up-regulated in both media, suggesting the potential roles of these proteins in virulence regulation in <i>P. brasiliensis</i>. Moreover, genes up-regulated in virulent Pb18 showed an increase in its expression after the recovery of virulence of attenuated Pb18. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and provided a foundation for achieving a molecular understanding of how <i>Paracoccidioides</i> modulates the host–pathogen interaction to its advantage

AgB8 protein species identified in sQS_f and bQS_f by 2-DGE followed by MALDI-TOF/TOF.

<p>AgB8 protein species identified in sQS_f and bQS_f by 2-DGE followed by MALDI-TOF/TOF.</p

AgB8 protein species identified in sQS_f and bQS_f by LC-MS/MS.

<p>AgB8 protein species identified in sQS_f and bQS_f by LC-MS/MS.</p

Composition of native AgB: sQS_f apolipoproteins identification by 2-DGE plus MALDI-TOF/TOF and sLd_f lipid moiety analysis.

<p><b>A)</b> Analysis of sQS_f by 2-DGE (Figure is representative of analytical triplicates), using a 3–10 lineal gradient of pH in the first dimension, and a 15% polyacrylamide gel for SDS-PAGE in the second dimension. Gels were stained with colloidal coomassie. The presence of host and parasite components was studied by analysing all spots by MS (MALDI-TOF/TOF). AgB was found in spots regularly spaced at around 8, 16 and 24 kDa (bold circles and arrows). The table illustrates which AgB8 subunits were identified in spots corresponding to the monomeric, dimeric and trimeric forms of AgB. MW: molecular weight (KDa). <b>B)</b> Analysis of sLd_f by HPTLC (Figure is representative of analytical triplicates). Standards and samples (about 10 μg) were applied onto HPTLC plates and resolving using double development solvent system for characterisation of both neutral and polar lipid classes. Lipid bands were visualised using iodine vapour. Std: standard containing polar and neutral lipids; PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; CLP: cardiolipin and PE: phsophatidylethanolamine; CHO: cholesterol; FA: free fatty acids; TAG: triacylglycerols; FAMEs: fatty acid methyl esters; SE: sterol esters.</p

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

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

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