Adesão, biofilme e invasão: investigando os mecanismos de virulência de Prevotella spp e Fusobacterium nucleatum

Abstract

The Prevotella genus and the species Fusobacterium nucleatum are strict anaerobic organisms associated with opportunistic infections in the vaginal, oral, and gastrointestinal cavities. During infection, virulence mechanisms such as adhesion to host tissues, invasion of cells and connective tissue, and evasion of the immune system are essential for bacterial establishment and host persistence. In the present study, we investigated the adhesion to human extracellular matrix proteins, biofilm formation, Matrigel invasion, and plasminogen activation of strains from the Prevotella species, including P. intermedia, P. melaninogenica, and P. nigrescens, as well as a strain of F. nucleatum. The bacterial adhesion capacity was quantified by the interaction of these bacteria with extracellular matrix proteins, including fibronectin, collagen type IV, collagen type I, laminin type 1, and Matrigel, previously immobilized on glass slides. P. intermedia and P. nigrescens demonstrated adhesion to fibronectin, type IV collagen, and Matrigel. F. nucleatum adhered to laminin, fibronectin, type IV collagen, and Matrigel. P. melaninogenica did not adhere to the substrates under the study conditions. To identify ligands in Prevotella and Fusobacterium, outer membrane protein extracts were purified from P. intermedia, P. nigrescens, and F. nucleatum and subjected to affinity chromatography using NHS-activated Sepharose columns containing immobilized laminin, fibronectin, and type IV and type I collagen. Eluted fractions containing potential ligands were sent for mass spectrometry analysis. In P. intermedia, six proteins were identified as potential laminin adhesins and 15 as potential type IV collagen adhesins. In P. nigrescens, five proteins were identified as potential laminin adhesins and three as potential type IV collagen adhesins. For F. nucleatum, 44 proteins were identified as potential fibronectin adhesins and 12 as potential type I collagen adhesins. Biofilm experiments were also conducted in the presence and absence of Matrigel. Biofilm formation was reduced in the presence of this substrate in P. intermedia and P. melaninogenica, while no significant difference was observed in the other species tested. To analyze the biofilm architecture, scanning electron microscopy was performed. It was observed that, in the presence of Matrigel, the biofilm surface of the analyzed species was altered. P. melaninogenica did not form biofilm on the glass surface used for SEM. A transwell invasion assay was performed for all Prevotella species. It was observed that only P. melaninogenica was capable of crossing the Matrigel layer. Matrigel degradation assays using SDS-PAGE showed that P. melaninogenica degrade matrix proteins and plasminogen. To understand the interaction between species and plasminogen, a plasminogen activation kinetic assay was conducted, in which only P. melaninogenica activated this molecule, likely utilizing this strategy to destroy tissues. Understanding the mechanisms involved in virulence may help develop new strategies to prevent periodontitis and biofilm formation in the gingival sulcus.