Biology subjects

Mintz, K.

Publications and source records attributed to Mintz, K..

2 recordsLinked to original sources

bioRxiv2022

Dual function of the O-antigen WaaL ligase of Aggregatibacter actinomycetemcomitans

Protein glycosylation is critical to the quaternary structure and collagen binding activity of the extracellular matrix protein adhesin A (EmaA) associated with Aggregatibacter actinomycetemcomitans. The glycosylation of this large, trimeric autotransporter adhesin is postulated to be mediated by WaaL, an enzyme with the canonical function to ligate the O-polysaccharide (O-PS) antigen with a terminal sugar of the lipid A-core oligosaccharide of lipopolysaccharide (LPS). In this study, we have determined that the Escherichia coli waaL ortholog (rflA) does not restore collagen binding of a waaL mutant strain of A. actinomycetemcomitans but does restore O-PS ligase activity following transformation of a plasmid expressing waaL. Therefore, a heterologous E. coli expression system was developed constituted of two independently replicating plasmids expressing either waaL or emaA of A. actinomycetemcomitans to directly demonstrate the necessity of ligase activity for EmaA collagen binding. Proper expression of the protein encoded by each plasmid was characterized, and the individually transformed strains did not promote collagen binding. However, co-expression of the two plasmids resulted in a strain with a significant increase in collagen binding activity and a change in the biochemical properties of the protein. These results provide additional data supporting the novel hypothesis that the WaaL ligase of A. actinomycetemcomitans shares a dual role as a ligase in LPS biosynthesis and is required for collagen binding activity of EmaA. ImportanceThe human oral pathogen A. actinomycetemcomitans is a causative agent of periodontal and several systemic diseases. The organism expresses an adhesin, EmaA, important for the colonization of this pathobiont via collagen binding and biofilm formation. EmaA is suggested to be modified with sugars and the modification is mediated using the same enzymes involved in lipopolysaccharide (LPS) biosynthesis. In this study, evidence is presented which suggests that the WaaL ligase, the enzyme that ligates the O-polysaccharide (O-PS) antigen with a terminal sugar of the lipid A-core oligosaccharide of LPS, is required for the collagen binding activity of EmaA. This finding represents a new paradigm for the posttranslational modification of this type of autotransporter protein.

microbiology↗

bioRxiv2022

Serotype specific sugars impact structure but not functions of the trimeric autotransporter adhesin EmaA of Aggregatibacter actinomycetemcomitans.

The human oral pathobiont Aggregatibacter actinomycetemcomitans expresses multiple virulence factors including the trimeric, extracellular matrix protein adhesin A (EmaA). The posttranslational modification of EmaA is proposed to be dependent on the sugars and enzymes associated with O-polysaccharide (O-PS) synthesis of the lipopolysaccharide (LPS). This modification is important for the structure and function of this adhesin. To determine if the composition of the sugars alters structure and/or function, the prototypic 202 kDa protein was expressed in a non-serotype b, emaA mutant strain. The transformed strain displayed EmaA adhesins similar in appearance to the prototypic adhesin as observed by 2D electron microscopy of whole-mount negatively stained bacterial preparations. Biochemical analysis indicated that the protein monomers were post-translationally modified. 3D electron tomographic reconstruction and structure analyses of the functional domain revealed three well-defined subdomains (SI, SII and SIII) with a linker region between SII and SIII. Structural changes were observed in all three subdomains and the linker region of the adhesins synthesized compared with the known structure. These changes however did not affect the ability of the strain to bind collagen or form biofilms. The data suggest that changes in the composition of the glycan moiety alter the 3D structure of the molecule without negatively affecting the function(s) associated with this adhesin. IMPORTANCEThe human oral pathogen A. actinomycetemcomitans is a causative agent of periodontal and several systemic diseases. EmaA is a trimeric autotransporter protein adhesin important for the colonization of this pathobiont in vivo. This adhesin is modified with sugars associated with the O-polysaccharide (O-PS) and the modification is mediated using the same enzymes involved in lipopolysaccharide (LPS) biosynthesis. The interaction with collagen is not mediated by the specific binding between the glycans and collagen but is attributed to changes in the final quaternary structure necessary to maintain an active adhesin. In this study, we have determined that the composition of the sugars utilized in the post-translational modification of this adhesin is exchangeable without compromising functional activities.

microbiology↗