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Adhesins of Leptospira interrogans mediate the interaction to fibrinogen and inhibit fibrin clot formation in vitro.

Oliveira R, Domingos RF, Siqueira GH, Fernandes LG, Souza NM, Vieira ML, de Morais ZM, Vasconcellos SA, Nascimento AL - PLoS Negl Trop Dis (2013)

Bottom Line: The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade.We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins.The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.

ABSTRACT
We report in this work that Leptospira strains, virulent L. interrogans serovar Copenhageni, attenuated L. interrogans serovar Copenhageni and saprophytic L. biflexa serovar Patoc are capable of binding fibrinogen (Fg). The interaction of leptospires with Fg inhibits thrombin- induced fibrin clot formation that may affect the haemostatic equilibrium. Additionally, we show that plasminogen (PLG)/plasmin (PLA) generation on the surface of Leptospira causes degradation of human Fg. The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade. We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins. The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration. The calculated dissociation equilibrium constants (K D ) of these reactions ranged from 733.3 ± 276.8 to 128 ± 89.9 nM for rLIC12238 and Lsa33, respectively. The interaction of recombinant proteins with human Fg resulted in inhibition of fibrin clot by thrombin-catalyzed reaction, suggesting that these versatile proteins could mediate Fg interaction in Leptospira. Our data reveal for the first time the inhibition of fibrin clot by Leptospira spp. and presents adhesins that could mediate these interactions. Decreasing fibrin clot would cause an imbalance of the coagulation cascade that may facilitate bleeding and help bacteria dissemination.

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Binding characterization of recombinant proteins to human Fg.(A) Ten µg/mL of Fg was immobilized into 96-wells ELISA plates and 0 to 4,000 nM of each recombinant protein was added for interaction. The bindings were detected using antiserum raised in mice against each protein followed by HRP-conjugated anti-mouse IgG. Gelatin was employed as negative control for each assayed protein (not shown) and Lsa25, as a leptospiral recombinant protein negative control. Data represent the mean absorbance values ± the standard deviation of three replicates for each experimental group of three independent experiments. The insert shows the dose response curve obtained with 10 µg/mL immobilized Fg and 0 to 3,000 nM LigB7-12, employed as leptospiral Fg-binding protein. The binding was detected with monoclonal anti-polyhistidine HRP antibodies. (B) The dissociation constant (KD) was calculated based on ELISA data for the recombinant proteins that have reached the equilibrium concentrationMean absorbance values at 492nm (± the standard deviations of three independent experiments) were compared to those obtained with untreated Fg (0 mM).
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pntd-0002396-g005: Binding characterization of recombinant proteins to human Fg.(A) Ten µg/mL of Fg was immobilized into 96-wells ELISA plates and 0 to 4,000 nM of each recombinant protein was added for interaction. The bindings were detected using antiserum raised in mice against each protein followed by HRP-conjugated anti-mouse IgG. Gelatin was employed as negative control for each assayed protein (not shown) and Lsa25, as a leptospiral recombinant protein negative control. Data represent the mean absorbance values ± the standard deviation of three replicates for each experimental group of three independent experiments. The insert shows the dose response curve obtained with 10 µg/mL immobilized Fg and 0 to 3,000 nM LigB7-12, employed as leptospiral Fg-binding protein. The binding was detected with monoclonal anti-polyhistidine HRP antibodies. (B) The dissociation constant (KD) was calculated based on ELISA data for the recombinant proteins that have reached the equilibrium concentrationMean absorbance values at 492nm (± the standard deviations of three independent experiments) were compared to those obtained with untreated Fg (0 mM).

Mentions: The interactions between the recombinant proteins and Fg was assessed on a quantitative basis, as indicated in Fig. 5A. Dose-dependent and saturable binding was observed when increasing concentrations (0 to 4,000 nM) of recombinant proteins rLIC12238, Lsa33, OmpL1, rLIC11975 and rLIC11360, or (0 to 2,000) of Lsa30, were allowed to individually adhere to a fixed human Fg amount (10 µg/mL) for 2 h. Saturation was reached with all except Lsa30 protein, due to the impossibility to achieve this protein at higher concentrations. In the case of LigB7-12, dose-response curve was measured using monoclonal anti-His tag antibodies, and the results depicted in the insert of Fig. 5A shows that saturation was not reached in the protein concentration range employed. Based on the ELISA data, the calculated dissociation equilibrium constants (KD) for the recombinant proteins with Fg are depicted in Fig. 5B; the highest and the lowest KD values were for rLIC12238 (733.3±276.8 nM) and Lsa33 (128±89.9 nM), respectively.


Adhesins of Leptospira interrogans mediate the interaction to fibrinogen and inhibit fibrin clot formation in vitro.

Oliveira R, Domingos RF, Siqueira GH, Fernandes LG, Souza NM, Vieira ML, de Morais ZM, Vasconcellos SA, Nascimento AL - PLoS Negl Trop Dis (2013)

Binding characterization of recombinant proteins to human Fg.(A) Ten µg/mL of Fg was immobilized into 96-wells ELISA plates and 0 to 4,000 nM of each recombinant protein was added for interaction. The bindings were detected using antiserum raised in mice against each protein followed by HRP-conjugated anti-mouse IgG. Gelatin was employed as negative control for each assayed protein (not shown) and Lsa25, as a leptospiral recombinant protein negative control. Data represent the mean absorbance values ± the standard deviation of three replicates for each experimental group of three independent experiments. The insert shows the dose response curve obtained with 10 µg/mL immobilized Fg and 0 to 3,000 nM LigB7-12, employed as leptospiral Fg-binding protein. The binding was detected with monoclonal anti-polyhistidine HRP antibodies. (B) The dissociation constant (KD) was calculated based on ELISA data for the recombinant proteins that have reached the equilibrium concentrationMean absorbance values at 492nm (± the standard deviations of three independent experiments) were compared to those obtained with untreated Fg (0 mM).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757074&req=5

pntd-0002396-g005: Binding characterization of recombinant proteins to human Fg.(A) Ten µg/mL of Fg was immobilized into 96-wells ELISA plates and 0 to 4,000 nM of each recombinant protein was added for interaction. The bindings were detected using antiserum raised in mice against each protein followed by HRP-conjugated anti-mouse IgG. Gelatin was employed as negative control for each assayed protein (not shown) and Lsa25, as a leptospiral recombinant protein negative control. Data represent the mean absorbance values ± the standard deviation of three replicates for each experimental group of three independent experiments. The insert shows the dose response curve obtained with 10 µg/mL immobilized Fg and 0 to 3,000 nM LigB7-12, employed as leptospiral Fg-binding protein. The binding was detected with monoclonal anti-polyhistidine HRP antibodies. (B) The dissociation constant (KD) was calculated based on ELISA data for the recombinant proteins that have reached the equilibrium concentrationMean absorbance values at 492nm (± the standard deviations of three independent experiments) were compared to those obtained with untreated Fg (0 mM).
Mentions: The interactions between the recombinant proteins and Fg was assessed on a quantitative basis, as indicated in Fig. 5A. Dose-dependent and saturable binding was observed when increasing concentrations (0 to 4,000 nM) of recombinant proteins rLIC12238, Lsa33, OmpL1, rLIC11975 and rLIC11360, or (0 to 2,000) of Lsa30, were allowed to individually adhere to a fixed human Fg amount (10 µg/mL) for 2 h. Saturation was reached with all except Lsa30 protein, due to the impossibility to achieve this protein at higher concentrations. In the case of LigB7-12, dose-response curve was measured using monoclonal anti-His tag antibodies, and the results depicted in the insert of Fig. 5A shows that saturation was not reached in the protein concentration range employed. Based on the ELISA data, the calculated dissociation equilibrium constants (KD) for the recombinant proteins with Fg are depicted in Fig. 5B; the highest and the lowest KD values were for rLIC12238 (733.3±276.8 nM) and Lsa33 (128±89.9 nM), respectively.

Bottom Line: The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade.We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins.The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.

ABSTRACT
We report in this work that Leptospira strains, virulent L. interrogans serovar Copenhageni, attenuated L. interrogans serovar Copenhageni and saprophytic L. biflexa serovar Patoc are capable of binding fibrinogen (Fg). The interaction of leptospires with Fg inhibits thrombin- induced fibrin clot formation that may affect the haemostatic equilibrium. Additionally, we show that plasminogen (PLG)/plasmin (PLA) generation on the surface of Leptospira causes degradation of human Fg. The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade. We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins. The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration. The calculated dissociation equilibrium constants (K D ) of these reactions ranged from 733.3 ± 276.8 to 128 ± 89.9 nM for rLIC12238 and Lsa33, respectively. The interaction of recombinant proteins with human Fg resulted in inhibition of fibrin clot by thrombin-catalyzed reaction, suggesting that these versatile proteins could mediate Fg interaction in Leptospira. Our data reveal for the first time the inhibition of fibrin clot by Leptospira spp. and presents adhesins that could mediate these interactions. Decreasing fibrin clot would cause an imbalance of the coagulation cascade that may facilitate bleeding and help bacteria dissemination.

Show MeSH
Related in: MedlinePlus