Project Details
Projekt
Characterization of sialic acid specific O-acetyltransferases: from neuroinvasive bacteria to human hosts
Subject Area
Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
Term
from 2014 to 2018
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 262794208
Pathogens causing bacterial sepsis and meningitis have frequently evolved the ability to decorate their cell surfaces with sialic acid (Sia), a sugar molecule that is used for cellular recognition events in the host organism. Escherichia coli K1 and serogroup B, C, W135 and Y meningococci are covered by Sia-containing capsular polysaccharides (CPS) that represent important virulence factors. Sia-specific O-acetyltransferases (OATs) can further modify the CPS, a process that modulates the specific characteristics of the capsular antigen. During the first granting period, we demonstrated that the function of Sia O-acetylation evolved on the basis of structurally diverse protein folds and succeeded in crystallization of an OAT with left-handed beta-helix fold. Now, we aim at the biochemical and structural characterization of OATs with putative alpha/beta-hydrolase fold. To dissect the determinants of Sia-specificity, a comparative analysis will be performed based on the capsule-modifying OATs of serogroup C and A meningococci, which are encapsulated by Sia- and mannose-phosphate-polymers, respectively.Preliminary studies on Escherichia coli K1 revealed the unexpected finding that O-acetylation positive but not negative strains show dynamic, growth phase-dependent variations in capsule expression. To unravel the underlying molecular mechanism, we propose studies on the cellular localization of OATs and their putative interactions with the capsule biosynthesis and export machinery. The relevance of Sia O-acetylation on host-pathogen interactions will be studied in the context of serum resistance. Questions to be addressed are: (i) does O-acetylation impacts interactions with factor H, a Sia-binding complement-regulator, and (ii) does the observed dynamic capsule reduction demask the bacterial surface protein OmpA, which would facilitate OmpA-mediated binding of complement regulator C4BP, a process known to mediate serum resistance.Regarding host-pathogen interactions of bacteria carrying an O-acetylated Sia-capsule, it might be of particular relevance that the host organism uses O-acetylation of sialo-glycans as a molecular switch to regulate Sia-mediated recognition processes, especially in the immune system. So far, the genetic basis of eukaryotic Sia-modifying OATs is not known. In preliminary work, we used our knowledge gained on the bacterial counterparts to identify a human candidate enzyme and performed initial cellular experiments to prove an involvement in Sia O-acetylation. By generating a homology-based structural model, we identified a putative catalytic triad, a feature that was also found for the meningococcal enzyme OatC. In the frame of this grant application, we propose a first biochemical characterization of the human enzyme with particular focus on the determination of Sia-specific OAT activity in vitro.
DFG Programme
Research Grants
