LytR Inhibition, an Approach to Avoid Biofilm Formation in Streptococcus dysgalactiae subs. dysgalactiae

• Autor(a) principal: Ferreira, João Carlos Paquete
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/87722
• topic: biofilms; Streptococcus dysgalactiae subs. dysgalactiae; teichoic acids; LCP proteins; X-ray crystallography; inhibition; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

Biofilm is a sessile bacterial growth phenotype, characterized by the cells being enclosed in a self-produced matrix, and attached to a surface. This makes the biofilms less susceptible to antibiotics, making them hard to eliminate, leading to chronic infections. Streptococcus dysgalactiae subs. dysgalactiae (SDSD) is a Gram-positive bacterium with the ability to form biofilms and also, a known animal pathogen, responsible for causing bovine mastitis and fish streptococcosis. Both diseases represent huge losses for the diary and fish industries. It is, then, very important to develop drugs capable of inhibiting the production of biofilms of this bacterium. Teichoic acids are glycopolymers present in the cell wall of all Gram-positive bacteria. These molecules are very important in the early stages of biofilm development, as they participate in the attachment events. SDSD possesses a LytR protein, belonging to the LytR-CpsA-Psr (LCP) family of proteins, that is necessary for the attachment of the teichoic acids to the peptidoglycan, which makes this protein a good target for biofilm inhibition. In this thesis, the LCP domain of SDSD LytR, that contains the LCP domains was overexpressed, in E. coli BL21. Purification of the protein was performed with an IMAC and a SEC. The produced protein was used for crystallization experiments and for the biophysical characterization of the interaction of SDSD LytR with putative ligands/substrates/inhibitors, by thermal shift assay (TSA), circular dichroism (CD), urea gel electrophoresis and microscale thermophoresis (MST). In the course of this work, the three-dimensional structure of SDSD LytR was determined, for the first time, at a resolution of 2.80 Å. Improvement of the resolution was not successful, as well as, the co-crystallization experiments. The techniques used did not reveal binding of the protein towards the tested compounds. We suggest that it might be due to low affinity of the compounds towards SDSD LytR.