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Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection.

Atzingen MV, Barbosa AS, De Brito T, Vasconcellos SA, de Morais ZM, Lima DM, Abreu PA, Nascimento AL - BMC Microbiol. (2008)

Bottom Line: Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction.The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1.Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil. m.atzingen@butantan.gov.br

ABSTRACT

Background: It has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of L. interrogans allowed identification of a repertoire of putative leptospiral surface proteins.

Results: Here, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis.

Conclusion: Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis.

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Related in: MedlinePlus

Binding of Lsa21 recombinant protein to ECM components. Wells were coated with 1 μg of laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, and the control proteins BSA and fetuin. Recombinant Lsa21 and Loa22 proteins attachment to those ECM macromolecules was assessed by an ELISA-based assay. One microgram of recombinant protein was added per well. Optical densities were taken at 492 nm. Data represent the mean ± standard error of three independent experiments. For statistical analysis, the attachment of Lsa21 to ECM macromolecules was compared to its binding to BSA by the Student's two tailed t- test (*, P < 0.01).
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Figure 5: Binding of Lsa21 recombinant protein to ECM components. Wells were coated with 1 μg of laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, and the control proteins BSA and fetuin. Recombinant Lsa21 and Loa22 proteins attachment to those ECM macromolecules was assessed by an ELISA-based assay. One microgram of recombinant protein was added per well. Optical densities were taken at 492 nm. Data represent the mean ± standard error of three independent experiments. For statistical analysis, the attachment of Lsa21 to ECM macromolecules was compared to its binding to BSA by the Student's two tailed t- test (*, P < 0.01).

Mentions: As Lsa21 protein is suggested to be surface-exposed and might play a role in virulence, we queried whether it could mediate host colonization by adhering to extracellular matrix proteins. Therefore, we examined its interaction with laminin, collagen type I, collagen type IV, cellular fibronectin, and plasma fibronectin. BSA and fetuin were included as negative controls and binding assays were performed by an ELISA-based assay [9]. As shown in Fig. 5, Lsa21 protein bound to all immobilized ECM macromolecules tested, but no interaction was detected with BSA or fetuin. As a negative control, we have included another recombinant protein, rLIC10191, known as Loa22 [31], and recently described as a virulence factor in Leptospira species [32]. A stronger interaction was observed with laminin, collagen IV and plasma fibronectin. This interaction was also assessed on a quantitative basis as illustrated in Fig. 6A. A dose-dependent binding was observed when increasing concentrations of the recombinant protein (0 to 2,000 nM) were allowed to adhere to a fixed laminin, collagen IV or fibronectin concentration (1 μg).


Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection.

Atzingen MV, Barbosa AS, De Brito T, Vasconcellos SA, de Morais ZM, Lima DM, Abreu PA, Nascimento AL - BMC Microbiol. (2008)

Binding of Lsa21 recombinant protein to ECM components. Wells were coated with 1 μg of laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, and the control proteins BSA and fetuin. Recombinant Lsa21 and Loa22 proteins attachment to those ECM macromolecules was assessed by an ELISA-based assay. One microgram of recombinant protein was added per well. Optical densities were taken at 492 nm. Data represent the mean ± standard error of three independent experiments. For statistical analysis, the attachment of Lsa21 to ECM macromolecules was compared to its binding to BSA by the Student's two tailed t- test (*, P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2386478&req=5

Figure 5: Binding of Lsa21 recombinant protein to ECM components. Wells were coated with 1 μg of laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, and the control proteins BSA and fetuin. Recombinant Lsa21 and Loa22 proteins attachment to those ECM macromolecules was assessed by an ELISA-based assay. One microgram of recombinant protein was added per well. Optical densities were taken at 492 nm. Data represent the mean ± standard error of three independent experiments. For statistical analysis, the attachment of Lsa21 to ECM macromolecules was compared to its binding to BSA by the Student's two tailed t- test (*, P < 0.01).
Mentions: As Lsa21 protein is suggested to be surface-exposed and might play a role in virulence, we queried whether it could mediate host colonization by adhering to extracellular matrix proteins. Therefore, we examined its interaction with laminin, collagen type I, collagen type IV, cellular fibronectin, and plasma fibronectin. BSA and fetuin were included as negative controls and binding assays were performed by an ELISA-based assay [9]. As shown in Fig. 5, Lsa21 protein bound to all immobilized ECM macromolecules tested, but no interaction was detected with BSA or fetuin. As a negative control, we have included another recombinant protein, rLIC10191, known as Loa22 [31], and recently described as a virulence factor in Leptospira species [32]. A stronger interaction was observed with laminin, collagen IV and plasma fibronectin. This interaction was also assessed on a quantitative basis as illustrated in Fig. 6A. A dose-dependent binding was observed when increasing concentrations of the recombinant protein (0 to 2,000 nM) were allowed to adhere to a fixed laminin, collagen IV or fibronectin concentration (1 μg).

Bottom Line: Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction.The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1.Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil. m.atzingen@butantan.gov.br

ABSTRACT

Background: It has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of L. interrogans allowed identification of a repertoire of putative leptospiral surface proteins.

Results: Here, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis.

Conclusion: Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis.

Show MeSH
Related in: MedlinePlus