Mapping transcriptional regulation of biofilm-related genes promoters in Pseudomonas aeruginosa

• Autor(a) principal: Pereira, Greicy Kelly Bonifacio
• Data de Publicação: 2022
• Tipo de documento: Tese
• Idioma: eng
• Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
• Texto Completo: https://www.teses.usp.br/teses/disponiveis/17/17136/tde-03022023-101053/
• topic: Biofilm; Biofilme; Fatores de transcrição; Gene regulation; Pseudomonas aeruginosa; Pseudomonas aeruginosa; Regulação gênica; Transcription factors

Pseudomonas aeruginosa is a gram-negative bacterium predominant in soil, vegetation, and water. Being an opportunistic pathogen that affects immunosuppressed people, P. aeruginosa has great clinical importance. It is quite resistant to antimicrobials and has several virulence factors that contribute to its high pathogenicity. An important factor of bacterial virulence is the formation of biofilms which are aggregates of microorganisms incorporated into an extracellular matrix that protects bacteria from hostile environments. Biofilm formation is a crucial and precisely regulated process at the transcriptional level. Most of this regulation is carried out directly by transcription factors that modulate the activity of promoters aimed at expressing virulence factors. In this sense, we aim to characterize the behaviour of different promoters of genes involved in biofilm regulation under different conditions and to search for possible new DNAbinding proteins associated with their promoter regions. For this, the upstream regions of the gacA, gacS, ladS, retS, rsmA, rsmZ and PA1611 genes were cloned separately into a miniTn7 vector and inserted into the PAO1 chromosome. We then evaluated the growth and activity of the upstream regions by lux expression under different carbon sources and iron deprivation. We saw no differences in the activity of promoters under growth with glucose and glycerol. However, under citrate, the retS upstream region showed lower activity, while the activity of the rsmA upstream region was induced. When conducting DNA-affinity chromatography pulldown for the upstream regions of retS and rsmA in LB media, we found pqsC and pqsH binding to the upstream region of retS and mvaT binding to the upstream region of rsmA. mvaT is a major regulator, exerting negative control in many genes described. In our findings, mvaT has a negative influence on regulating rsmA, since its inactivation leads to a higher expression of the upstream region of rsmA. pqsC and pqsH are involved in quorum sensing and biofilm formation. In our study, we hypothesized that both have a role in the activation of the upstream region of the retS in LB media and MOPS glucose since their mutation led to higher expression of the upstream region. On the other hand, the opposite seems to happen when the carbon source is acetate and succinate. The expression of retS over time was also evaluated by western blot when co-cultivated with Staphylococcus aureus and Candida albicans in artificial sputum media and we noticed that there was no change in the expression of this gene when compared to the axenically cultured PAO1.