Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display
Pathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still re...
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Frontiers Media S.A.
2022-11-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1051698/full |
| _version_ | 1811186978698297344 |
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| author | Fabiana Lauretti-Ferreira Fabiana Lauretti-Ferreira André Azevedo Reis Teixeira Ricardo José Giordano Josefa Bezerra da Silva Patricia Antonia Estima Abreu Angela Silva Barbosa Milena Apetito Akamatsu Paulo Lee Ho Paulo Lee Ho |
| author_facet | Fabiana Lauretti-Ferreira Fabiana Lauretti-Ferreira André Azevedo Reis Teixeira Ricardo José Giordano Josefa Bezerra da Silva Patricia Antonia Estima Abreu Angela Silva Barbosa Milena Apetito Akamatsu Paulo Lee Ho Paulo Lee Ho |
| author_sort | Fabiana Lauretti-Ferreira |
| collection | DOAJ |
| description | Pathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still remain unknown. Toxins and adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines with cross-protection and long-lasting effect against leptospirosis. For this aim, we used the shotgun phage display technique to unravel new proteins with adhesive properties. A shotgun library was constructed using fragmented genomic DNA from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and pG8SAET phagemid vector. Selection of phages bearing new possible cell-binding antigens was performed against VERO cells, using BRASIL biopanning methodology. Analysis of selected clones revealed the hypothetical protein LIC10778, a potentially exposed virulence factor that belongs to the virulence-modifying (VM) protein family (PF07598), composed of 13 members in the leptospiral strain Fiocruz L1-130. Prediction of LIC10778 tertiary structure indicates that the protein contains a cellular-binding domain (N-terminal portion) and an unknown domain of no assigned activity (C-terminal portion). The predicted N-terminal domain shared structural similarities with the cell-binding and internalization domain of toxins like Ricin and Abrin, as well as to the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin in Mycoplasma pneumoniae. Interestingly, recombinant portions of the N-terminal region of LIC10778 protein showed binding to laminin, collagens I and IV, vitronectin, and plasma and cell fibronectins using overlay blotting technique, especially regarding the binding site identified by phage display. These data validate our preliminary phage display biopanning and support the predicted three-dimensional models of LIC10778 protein and other members of PF07598 protein family, confirming the identification of the N-terminal cell-binding domains that are similar to ricin-like toxins. Moreover, fluorescent fused proteins also confirmed that N-terminal region of LIC10778 is capable of binding to VERO and A549 cell lines, further highlighting its virulence role during host-pathogen interaction in leptospirosis probably mediated by its C-terminal domain. Indeed, recent results in the literature confirmed this assumption by demonstrating the cytotoxicity of a closely related PF07598 member. |
| first_indexed | 2024-04-11T13:54:22Z |
| format | Article |
| id | doaj.art-e744df8bc5274e59a0f89a2981708a94 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-11T13:54:22Z |
| publishDate | 2022-11-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-e744df8bc5274e59a0f89a2981708a942022-12-22T04:20:24ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-11-011310.3389/fmicb.2022.10516981051698Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage displayFabiana Lauretti-Ferreira0Fabiana Lauretti-Ferreira1André Azevedo Reis Teixeira2Ricardo José Giordano3Josefa Bezerra da Silva4Patricia Antonia Estima Abreu5Angela Silva Barbosa6Milena Apetito Akamatsu7Paulo Lee Ho8Paulo Lee Ho9Bioindustrial Division, Butantan Institute, São Paulo, BrazilDepartment of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, BrazilDepartment of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, BrazilDepartment of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, BrazilLaboratory of Bacteriology, Butantan Institute, São Paulo, BrazilLaboratory of Bacteriology, Butantan Institute, São Paulo, BrazilLaboratory of Bacteriology, Butantan Institute, São Paulo, BrazilBioindustrial Division, Butantan Institute, São Paulo, BrazilBioindustrial Division, Butantan Institute, São Paulo, BrazilDepartment of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, BrazilPathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still remain unknown. Toxins and adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines with cross-protection and long-lasting effect against leptospirosis. For this aim, we used the shotgun phage display technique to unravel new proteins with adhesive properties. A shotgun library was constructed using fragmented genomic DNA from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and pG8SAET phagemid vector. Selection of phages bearing new possible cell-binding antigens was performed against VERO cells, using BRASIL biopanning methodology. Analysis of selected clones revealed the hypothetical protein LIC10778, a potentially exposed virulence factor that belongs to the virulence-modifying (VM) protein family (PF07598), composed of 13 members in the leptospiral strain Fiocruz L1-130. Prediction of LIC10778 tertiary structure indicates that the protein contains a cellular-binding domain (N-terminal portion) and an unknown domain of no assigned activity (C-terminal portion). The predicted N-terminal domain shared structural similarities with the cell-binding and internalization domain of toxins like Ricin and Abrin, as well as to the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin in Mycoplasma pneumoniae. Interestingly, recombinant portions of the N-terminal region of LIC10778 protein showed binding to laminin, collagens I and IV, vitronectin, and plasma and cell fibronectins using overlay blotting technique, especially regarding the binding site identified by phage display. These data validate our preliminary phage display biopanning and support the predicted three-dimensional models of LIC10778 protein and other members of PF07598 protein family, confirming the identification of the N-terminal cell-binding domains that are similar to ricin-like toxins. Moreover, fluorescent fused proteins also confirmed that N-terminal region of LIC10778 is capable of binding to VERO and A549 cell lines, further highlighting its virulence role during host-pathogen interaction in leptospirosis probably mediated by its C-terminal domain. Indeed, recent results in the literature confirmed this assumption by demonstrating the cytotoxicity of a closely related PF07598 member.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1051698/fullLeptospiravirulencephage displaybinding domainsprotein structure prediction |
| spellingShingle | Fabiana Lauretti-Ferreira Fabiana Lauretti-Ferreira André Azevedo Reis Teixeira Ricardo José Giordano Josefa Bezerra da Silva Patricia Antonia Estima Abreu Angela Silva Barbosa Milena Apetito Akamatsu Paulo Lee Ho Paulo Lee Ho Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display Frontiers in Microbiology Leptospira virulence phage display binding domains protein structure prediction |
| title | Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display |
| title_full | Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display |
| title_fullStr | Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display |
| title_full_unstemmed | Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display |
| title_short | Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display |
| title_sort | characterization of a virulence modifying protein of leptospira interrogans identified by shotgun phage display |
| topic | Leptospira virulence phage display binding domains protein structure prediction |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1051698/full |
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