The Extended Signal Peptide of the Trimeric Autotransporter EmaA of Aggregatibacter actinomycetemcomitans Modulates Secretion

Abstract

The extracellular matrix protein adhesin A (EmaA) of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a fibrillar collagen adhesin belonging to the family of trimeric autotransporters. The protein forms antenna-like structures on the bacterial surface required for collagen adhesion. The 202-kDa protein monomers are proposed to be targeted and translocated across the inner membrane by a long signal peptide composed of 56 amino acids. The predicted signal peptide was functionally active in Escherichia coli and A. actinomycetemcomitans using truncated PhoA and Aae chimeric proteins, respectively. Mutations in the signal peptide were generated and characterized for PhoA activity in E. coli. A. actinomycetemcomitans strains expressing EmaA with the identical mutant signal peptides were assessed for cellular localization, surface expression, and collagen binding activity. All of the mutants impaired some aspect of EmaA structure or function. A signal peptide mutant that promoted alkaline phosphatase secretion did not allow any cell surface presentation of EmaA. A second mutant allowed for cell surface exposure but abolished protein function. A third mutant allowed for the normal localization and function of EmaA at 37°C but impaired localization at elevated temperatures. Likewise, replacement of the long EmaA signal peptide with a typical signal peptide also impaired localization above 37°C. The data suggest that the residues of the EmaA signal peptide are required for protein folding or assembly of this collagen adhesin.

O-Polysaccharide Glycosylation Is Required for Stability and Function of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans

“…The EmaA structures were visualized using transmission electron microscopy (TEM) of whole-cell mount preparations as described earlier (11,25). Briefly, bacteria were grown to early logarithmic phase (OD 495 , 0.2 to 0.3), pelleted at 960 ϫ g, resuspended in phosphate-buffered saline (PBS; 10 mM NaH 2 PO 4 , 150 mM NaCl; pH 7.4), applied to grids, and negatively stained using Nano-W (Nanoprobes, Yephank, NY).…”

“…The EmaA monomers are synthesized as a preprotein by the ribosome in the cytoplasm, and the pre-EmaA protein contains a 56-amino-acid signal peptide that is removed after translocation across the inner membrane (11). Following inner membrane translocation, the C termini of three EmaA monomer proteins are proposed to integrate into the outer membrane to form a pore for the translocation of the remaining N-terminal portions of the proteins across the outer membrane, as suggested for other trimeric autotransporters (8).…”

Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans

“…EmaA, similar to other members of the trimeric auto transporter adhesin family, is secreted via the type V c secretion system. The protein is exported to the periplasm via the sec-dependent machinery with the guidance of the signal sequence (Jiang et al, 2011). The carboxyl termini of three monomers of the protein form an integral β-barrel pore in the outer membrane and then the passenger domain is transported through this pore into the extracellular space (Dautin and Bernstein, 2007; Henderson et al, 2004; Roggenkamp et al, 2003).…”

New findings on the function and potential applications of the trimeric autotransporter adhesin

“…In turn, the Omp complex facilitates the formation and insertion of bbarrels into the outer membrane (Voulhoux et al 2003). The signal peptide is also reported to contribute to the TAAs translocation process in addition to leading the proteins across the inner membrane (Jiang et al 2011). Removement or replacement of the signal peptide of EspP, an autotransporter of Escherichia coli that owns a long signal peptide, with a generic signal peptide impaired the translocation of the passenger domain across the OM, highlighting the importance of these specific signal peptides in maintaining the passenger domain in a suitable state for translocation across the OM by preventing its misfolding (Szabady et al 2005).…”