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Analysis of the effects of the Streptococcus sanguinis genes spxB, swan and nox in NET production by human neutrophils

Grant number: 23/16788-8
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): February 01, 2024
Effective date (End): January 31, 2025
Field of knowledge:Health Sciences - Dentistry
Principal Investigator:Renata de Oliveira Mattos Graner
Grantee:Emanoeli Daniel Bessa
Host Institution: Faculdade de Odontologia de Piracicaba (FOP). Universidade Estadual de Campinas (UNICAMP). Piracicaba , SP, Brazil

Abstract

Streptococcus sanguinis is an abundant commensal species in the oral cavity, capable of initiating the formation of dental biofilms. Bacteria of this species are commonly involved in cases of infectious endocarditis (IE) in susceptible individuals and are also detected in atherosclerotic plaques. Streptococcus sanguinis expresses three genes, spxB, swan, and nox, potentially involved in cytotoxicity and/or modulation of NETosis in neutrophils (PMN), the primary leukocytes of innate immunity in periodontal tissues and the bloodstream. These genes were previously reported as required for the production of H2O2, DNA degradation, and the response to oxidative stress, respectively. Data from our group indicate that these genes encode multifunctional proteins capable of interacting with serum proteins and/or extracellular matrix (ECM) proteins. The objective of this project is to investigate the influence of spxB, swan, and nox on NET production by human PMN exposed to S. sanguinis in the presence or absence of serum components. For this, isogenic mutant strains of each of these genes will be compared with the parental strain S. sanguinis SK36 regarding their ability to bind to ECM/plasma glycoproteins (fibronectin, fibrinogen, plasminogen, collagen type I) in fluorimetric assays. The ability of strains to induce NETosis in PMN isolated from human peripheral blood in the presence of 20% human serum (HS), 20% heat-inactivated human serum (HIHS), or PBS will be evaluated by scanning electron microscopy (SEM). Furthermore, the frequencies of NETosis induction by all strains will be quantified after their incubation in human blood for different times, followed by light microscopy analyses of PMN identified in blood smears stained with May-Grunwald-Giemsa. Similar analyses will be performed with PMN previously isolated and challenged with the studied strains in the presence of 20% HS, HIHS, or PBS. The results of this project are expected to reveal mechanisms through which S. sanguinis can induce NETosis by PMN, a key process not only for the elimination of microorganisms by PMN but also involved in the pathogenesis of cardiovascular diseases.

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