Structure Analysis of a Sugar-moiety Chimera of EmaA, a Collagen Adhesin of a Gram-negative Bacterial Pathogen

Abstract

The gram-negative non-motile bacterium, Aggregatibacter actinomycetemcomitans is an oropharyngeal colonizer. A. actinomycetemcomitans causes aggressive periodontitis and systemic infections, including endocarditis and pneumonia. Multiple virulence factors are involved in the infective process, and EmaA (extracellular matrix adhesin A) is one of the important virulence determinants in the early stage of infection. EmaA is a trimeric autotransporter adhesin, composed of three 202 kDa monomers, and forms antenna-like appendages extending more than 150 nm from the bacterial surface [1]. The functional Nterminus, encompasses approximately 30 nm from the distal end, and comprises three subdomains (SI, SII and SIII), with a linker region between subdomains SII and SIII [2]. EmaAs are hypothesized to be glycosylated by a novel post-translational mechanism. This mechanism shares the same enzyme that ligates the O-polysaccharide (O-PS) sugars to the lipopolysaccharide [3]. Seven different serotypes associated with A. actinomycetemcomitans have been identified based on different O-PS sugars. Accordingly, the sugars present in the EmaA of each serotype are different. The O-PS of serotype a strains is composed of repeated disaccharide units of talose, while the O-PS of serotype b strains consists of repeated rhamnose-fucose-acetylgalactosamine trisaccharides. Glycosylation, as well as the primary sequence, are important for the function and stability of the EmaA adhesin [4]. Therefore, 3D structures of EmaA (the serotype b sequence) modified with the sugars from a serotype a strain (talose), a sugar-moiety chimera, were reconstructed for better understanding the functional role of glycosylation in this adhesin.Electron tomography analysis was performed to resolve the 3D structure of a sugar-moiety chimera of EmaA. A plasmid carrying the full-length serotype b emaA sequence was transformed into a serotype a emaA -mutant strain (a-emaA -) to generate a new strain a-emaA -/b-emaA + . The sugar-moiety chimera EmaA (a-EmaA) expressed in this strain was compared to the wild type b-EmaA expressed in a serotype b strain. The collagen binding activity of the a-emaA -/b-emaA + strain was determined using an enzymelinked absorbent assay (ELISA) [3,4]. Electron microscopy was performed on deep negatively stained whole-mount bacterial preparations [5]. Bacteria were grown in broth and collected by centrifugation at ×3,000 g for 1 min at 4°C, A 5 µl aliquot of bacterial suspension was placed onto carbon-coated grids pretreated with low molecular weight polylysine (~3 kDa) and colloidal gold, and negatively stained with Nano W (Nanoprobes, NY). Grids were imaged at 100 kV with a Tecnai 12 electron microscope (FEI, OR) Tomographic tilt series were acquired within a ±64° angular range at 2° angular intervals. Tomographic reconstructions of the whole imaged area were calculated using IMOD. For each selected EmaA from the tomogram, a tilt series of subprojections was extracted and subvolumes were calculated with the long-axis of the adhesin ap...

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

“…Projections were initially aligned by cross correlation and further refined using fiducial markers located outside of the bacterial surface. EmaA functional domains were orientationally selected from the tomograms by marking two points along their long axis (11, 12, 17, 23) with the first point located at the tip of the adhesin. For each selected molecule, a tilt series of subprojections was extracted, subprojection angles were recalculated and subvolumes were reconstructed with the molecules’ long-axis oriented approximately parallel to the Y-axis using algorithms implemented in EMIRA (15).…”