Acetylation of Surface Carbohydrates in Bacterial Pathogens Requires Coordinated Action of a Two-Domain Membrane-Bound Acyltransferase

Abstract

Membrane bound acyltransferase-3 (AT3) domain-containing proteins are implicated in a wide range of carbohydrate O-acyl modifications, but their mechanism of action is largely unknown. O-antigen acetylation by AT3 domain-containing acetyltransferases of Salmonella spp. can generate a specific immune response upon infection and can influence bacteriophage interactions. This study integrates in situ and in vitro functional analyses of two of these proteins, OafA and OafB (formerly F2GtrC), which display an “AT3-SGNH fused” domain architecture, where an integral membrane AT3 domain is fused to an extracytoplasmic SGNH domain. An in silico-inspired mutagenesis approach of the AT3 domain identified seven residues which are fundamental for the mechanism of action of OafA, with a particularly conserved motif in TMH1 indicating a potential acyl donor interaction site. Genetic and in vitro evidence demonstrate that the SGNH domain is both necessary and sufficient for lipopolysaccharide acetylation. The structure of the periplasmic SGNH domain of OafB identified features not previously reported for SGNH proteins. In particular, the periplasmic portion of the interdomain linking region is structured. Significantly, this region constrains acceptor substrate specificity, apparently by limiting access to the active site. Coevolution analysis of the two domains suggests possible interdomain interactions. Combining these data, we propose a refined model of the AT3-SGNH proteins, with structurally constrained orientations of the two domains. These findings enhance our understanding of how cells can transfer acyl groups from the cytoplasm to specific extracellular carbohydrates. IMPORTANCE Acyltransferase-3 (AT3) domain-containing membrane proteins are involved in O-acetylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, resistance to antimicrobials, and biosynthesis of antibiotics. Their mechanism of action, however, is poorly characterized. We analyzed two acetyltransferases as models for this important family of membrane proteins, which modify carbohydrates on the surface of the pathogen Salmonella enterica, affecting immunogenicity, virulence, and bacteriophage resistance. We show that when these AT3 domains are fused to a periplasmic partner domain, both domains are required for substrate acetylation. The data show conserved elements in the AT3 domain and unique structural features of the periplasmic domain. Our data provide a working model to probe the mechanism and function of the diverse and important members of the widespread AT3 protein family, which are required for biologically significant modifications of cell-surface carbohydrates.

An acquired acyltransferase promotes Klebsiella pneumoniae ST258 respiratory infection

“…Deletion of atf3 does not alter the physical properties of KP35 Neither the primary sequence nor the predicted protein structure of the KP35 atf3 gene product indicates its function. However, atf3 is colocated with multiple genes involved in capsular production (ugd, gnd; components of the cps cluster in K. pneumoniae) (Pan et al, 2015) and O-antigen synthesis (wbgU, tagGH) (Bruchmann et al, 2021;Caboni et al, 2015) (Figure 2A). To study the role of atf3 in pathogenesis, a null mutant was constructed using a recently adapted CRISPR-Cas9 system, Lambda Red Recombineering genes to improve efficiency, and Zeocin selection due to the presence of multiple antimicrobial resistance elements in the KP35 genome (McConville et al, 2020).…”

“…There are numerous bacterial acyltransferases with diverse functions (Rö ttig and Steinb€ uchel, 2013) and >60 independent proteins identified as targets for acylation (Hentchel and Escalante-Semerena, 2015). Members of the acyltransferase superfamily 3, of which atf3 of KP35 belongs, are present in prokaryotes and eukaryotes, with enrichment of matching orthologous sequences across Gammaproteobacteria, a class of bacteria to which K. pneumoniae belongs (Pearson et al, 2020). A notable member of this family includes OatA, an enzyme that O-acetylates the peptidoglycan of Staphylococcus aureus, leaving it resistant to lysozyme degradation (Bera et al, 2006;Herbert et al, 2007).…”

Diverse functions for acyltransferase-3 proteins in the modification of bacterial cell surfaces

“…Although having known O-acetylesterase activity, the SGNH domain is essential for the acyltransferase activity of AT3-SGNH fused carbohydrate acyltransferases [4,11,29,30] (Fig. 1).…”

Rewiring host–microbe interactions and barrier function during gastrointestinal inflammation

“…Elevated levels of LPS can be detected in the plasma of humans showing features of metabolic syndrome, including obesity, insulin resistance, dyslipidemia, and chronic low-grade inflammation [ 166 ], which is exacerbated upon high fat intake [ 167 ]. Still, as LPS is a complex molecule found in different acetylation forms affecting molecular structure [ 168 ] and thereby binding capacity, not all types of LPS are metabolically detrimental to the host. Comparing the metabolic effects of Escherichia coli -derived and Rhodobacter sphaeroides -derived LPS, it was recently demonstrated that only E. coli LPS mediated intestinal barrier disruption, dysglycemia, and low-grade inflammation of adipose tissue [ 169 ].…”