IN SILICO STUDY OF THE DOMAINS PRESENT IN PROTEINS INVOLVED IN THE REGULATION OF THE SECOND C-DI-GMP MESSENGER IN Bordetella bronchiseptica

BELHART, K.; MUGNI, S.; FERNANDEZ, J.; SISTI, F.

Congreso; SAIB SAMIGE 2021; 2021

SAIB y SAMIGE

Bordetella bronchiseptica causes respiratory infections in a variety of mammalian hosts. We have already described c-di-GMP(cdG) ´s role in motility, biofilm formation, and virulence. cdG intracellular concentration is regulated by diguanylate cyclases(DGC) and phosphodiesterases (PDE). DGC contain a GGDEF domain, responsible for the activity. PDE can have either EALor HD-GyP domains. In both cases, activity domains frequently are adjacent to one or more accessory domains. Most roles orligands of the accessory domains are unknown. In this work, we present a detailed in silico analysis of the 19 proteins involvedin B. bronchiseptica cdG network. We found 10 proteins with GGDEF domain, 4 with EAL domain, and 5 with both domains(dual proteins). Phobius program predicted 12 membranes and 7 cytosolic proteins. We used CLUSTALW to compare proteinssequences with known active PDE or DGC and found 9 proteins with probable DGC activity. Among EAL proteins weconfirmed that 4 of them (PdeA, BB3128, BB2110, and BB3116) presented all conserved amino acids important for activityand substrate binding. Accessory domains were identified using BLASTP and PROSITE software. Only half of the proteinsanalyzed presented known domains (CACHE, DosC, PAS, CSS, REC. MHyT, and CBS). We characterized in detail thedomains REC, CACHE, and HK present in proteins PdeA, BdcA, and BB2109 respectively. We modeled the 3D structures ofthe domains with the PHYRE2.0 program. REC domains are present in two-component system response regulators. PdeA hasbeen described as an orphan response regulator. We found similarities to REC domain from RocR, a response regulator of theRocASR system in Pseudomonas aeruginosa. All amino acids important for RocR function are conserved in PdeA, includingthe phosphorylation site. Accordingly, we proposed that PdeA activity may be regulated by phosphorylation by a still unknownhistidine kinase. BdcA is a membrane DGC that interact with LapD and regulates biofilm formation in B. bronchiseptica.Phobius predicted that CACHE domain is present in periplasmic space, probably sensing extracellular signals. AlthoughCACHE domains are widespread in bacteria, they present high variability and can sense different ligands. Comparison ofBdcA CACHE to CACHE domains with known ligands did not give us a strong idea of a putative ligand for BdcA. BB2109is a membrane, dual protein involved in motility regulation in B. bronchiseptica. Our analysis indicates that EAL and GGDEFdomains have degenerated, hence PDE or DGC activity are not expected. PHYRE2.0 analysis yielded structural similarity toWalK protein from Lactobacillus plantarum. The histidine that is phosphorylated in WalK is also present in BB2109,suggesting a role for this amino acid in BB2109 function. In silico description of these proteins involved in the regulation ofcdG in B. bronchiseptica is important to design new strategies and experiments to understand the role of this second messengerin the Bordetella pathogenesis.