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Immune response induced by mono, dual and multispecies biofilms formed by Sporothrix schenckii and/or Staphylococcus spp.

Grant number: 17/13334-5
Support Opportunities:Scholarships in Brazil - Post-Doctoral
Effective date (Start): February 01, 2018
Effective date (End): February 28, 2022
Field of knowledge:Biological Sciences - Immunology - Cellular Immunology
Principal Investigator:Iracilda Zeppone Carlos
Grantee:Thais de Cassia Negrini
Host Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
Associated scholarship(s):18/18258-8 - "Disruption of mannan-mediated bacterial-fungal cross-kingdom interaction as an approach for controlling mixed-species biofilm formation", BE.EP.PD

Abstract

Sporotrichosis is a fungal disease caused by species of the Sporothrix schenckii complex, which can affect animals and humans. The most frequently clinical manifestation is the lymphocutaneos form which begins with a nodular or ulcerated lesion at the site of traumatic inoculation of the fungus on the skin. Despite the protective mechanisms of this tissue to aggressive agents, when the skin is damaged, wounds can develop increasing the risk of infections. In this context, the microbial species most frequently found in skin lesions are Staphylococcus aureus and Staphylococcus epidermidis, which are commensal under normal conditions, but which may delay the re-epithelization of wounds and alter the pathogenicity of fungal infections. In view of the above and knowing that sporotrichosis is a cutaneous mycosis that can start with an ulcerated lesion; that epithelial cells play an important role in the immune response against aggressive agents; that Staphylococcus spp. are considered the main colonizers of the skin and potentially present in infectious processes and that the inter-relationships between bacteria and fungi can alter the pathogenicity of the fungal infection, the aim of this study is to investigate whether epithelial cells in contact with skin microbiota associated with S. schenckii will construct a defense response different from that found only in the presence of the fungus. For this, microbial biofilms monospecies (S. aureus; S. epidermidis; S. schenckii), dual species (S. aureus + S. epidermidis; S. aureus + S. schenckii; S. epidermidis + S. schenckii) and multispecies (S. aureus + S. epidermidis + S. schenckii) will be cultured on nitrocellulose membranes until are obtained 105 UFC /ml. In parallel, 104 HaCat epithelial cells will be inserted into each well of the 24-well plate containing DMEM medium and exposed to the membranes containing the different biofilms. At the end of each co-cultivation period, counting of microorganisms and viable cells will be performed. In addition, the DMEM medium and the epithelial cells present in each well of the 24-well plate will be collected for quantification of the production of IL-33, IL-6, TGF-², h-BD3, histatin-5 and LL-37 by ELISA and analysis of TLR-2 and TLR-4 expression by flow cytometry, respectively. Three different experiments will be performed for each analysis with sample duplicate.

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