IN-SILICO ANALYSIS OF THE GIGANT EBH PROTEIN FROM Staphylococcus aureus AND ITS ROLE IN THE INTERACTION WITH Pseudomonas aeruginosa

SOLAR VENERO ESMERALDA C.; GALEANO MARIANA BELEN; SOSA EZEQUIEL; SOLA CLAUDIA DEL VALLE; TRIBELLI PAULA M.

Congreso; XVI CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE; 2021

SAMIGE

Staphylococcus aureus y Pseudomonas aeruginosa are two opportunistic pathogenic bacteria that can cause co-infections. The interaction between these species has been described primarily as antagonistic with P. aeruginosa overcoming S. aureus. However, the co-existence in-vivo appears to be higher. Our main goal was to identify S. aureus mutations that could possibly modify the dynamics of its interaction with P.aeruginosa. In a primary screening of the S. aureus USA300 Nebraska library co-cultured with P. aeruginosa PAO1 we found a clone carrying a mutation in ebh gene that presented a decrease in its survival in comparison with S. aureus USA300 wild type, 1-fold and 5 folds under aerobic and microaerobic conditions respectively. Ebh is a 1.1-MDa giant protein present on the surface of bacteria belonging to the genus Staphylococcus. Ebh is present in species that colonize and cause infections in humans, such as S. aureus, S. epidermidis and S. haemolyticus but it is rarely found in species not associated with human infections. Ebh sequence harbors a YSIRK-G/S motif, which targets proteins to the cell wall, at the N-terminal. Additionally, it presents 6 FIVAR domains, 45 FIVAR-GA domains and 8 DUF1542 (domain of unknown function) domains. These sugar and albumin binding repeat domains are thought to mediate in the binding to extracellular matrix proteins. In several strains, such as S. aureus USA300, S. aureus COL, and S. aureus NCTC8325, Ebh is encoded in a single 31200 nucleotides-long open reading frame (ORF). However, in strains such as S. aureus N315 and S. aeureus Newman, Ebh it is encoded in two different ORFs, ebhA and ebhB. We analyzed the presence of ebh in 64 genomes corresponding to S. auereus isolates obtained from pulmonary infections in patients with Cystic Fibrosis (CF), whose sequences are deposited in public databases. We also included the analysis of 4 isolates obtained from nasal and wound samples from patients in the province of Córdoba, Argentina. We found homologues to ebh in 66 of the 68 isolates analyzed. Analysis of these sequences allowed us to observe that in several of the strains there are variable STOP codons that could generate proteins of different sizes, ranging from 10624 to 2367 amino acids. Most of the isolates belong to clonal complexes 5 (CC5) and 8 (CC8) but there´s no apparent correlation between premature STOP codon generating mutations and CC grouping. The analysis of the domains and motifs present in the different Ebh versions present in the different isolates showed the conservation of the N-terminal of the protein, which contains the signal peptide. This suggests that the truncated proteins could still be exported to the cell wall. However, their role in the interaction with Pseudomonas remains to be elucidated.