The role of RppH pyrophosphatase in c-di-GMP-mediated signaling pathways in Pseudomonas aeruginosa

• Grant number: 24/10212-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: August 01, 2025
• End date: April 30, 2029
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Regina Lúcia Baldini
• Grantee: Erik Gabriel Silva Rios
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

Pseudomonas aeruginosa is a ubiquitous microorganism, used as a model for the study of regulatory and infectious processes in bacteria. The regulation of these processes depends on the concentration of the second messenger cyclic bis-(3',5')-di-guanosine monophosphate (c-di-GMP), which fluctuation is dependent on the synthesis activities of diguanylate cyclases and degradation from phosphodiesterase. Intracellular levels of c-di-GMP are directly associated with the planktonic and biofilm lifestyles, that correlate to acute and chronic infections, respectively. Modulation of gene expression is also essential for controlling these phenotypes, as can be observed when studying the Gac/Rsm system, in which the RsmA and RsmF proteins bind to target transcripts in the cell and prevent or activate their translation, playing a central role in responses. In previous experiments with the diguanylate cyclase PA14_04420 overexpressing strain, various phenotypes associated with a high concentration of c-di-GMP were observed, indicating its enzymatic activity (unpublished data). PA14_04420 is encoded in an operon with the gene for a hypothetical pyrophosphatase, RppH, that cleaves triphosphates into monophosphates at the 5' end of RNAs. The deletion mutant of this gene also had an influence on various phenotypes associated with fluctuating levels of c-di-GMP in the cell, such as swimming and swarming motility, and higher resistance to tobramycin, an antibiotic that acts on the ribosome, inhibiting translation. Therefore, this project aims to investigate the roles of RppH, focusing on its relationship with c-di-GMP-dependent phenotypes, as well as its participation in the control of gene expression, initially targeting the Gac/Rsm system. To this end, phenotypic analyses of the mutant, overexpression and wild-type RppH strains will be carried out with regard to the influence of this enzyme on motility, biofilm formation, antibiotic tolerance and resistance, exopolysaccharide production and quantification of pyocyanin and pyoverdine production. Also, for the initial characterization of the influence of RppH at a global level, a transcriptome assay at the sub-inhibitory concentration of tobramycin will be carried out todetermine new targets of regulation. Finally, to determine the direct interaction between RppH and these transcripts, a ChIP-qPCR (Chromatin Immunoprecipitation followed by quantitative PCR) assay would be used. From these tests, it is expected that it will be possible to elucidate the role of RppH in these pathways, resulting in a better understanding of the regulatory processes as a basis for the development of new therapies in the future.