UVM Theses and Dissertations
Format:
Print
Author:
Yu, Chunxiao
Dept./Program:
Microbiology and Molecular Genetics
Year:
2009
Degree:
PhD
Abstract:
Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative, facultative anaerobic oral pathogen that is strongly associated with localized aggressive periodontitis (LAP) and some cases of chronic periodontitis. This oral pathogen is also associated with extra-oral diseases such as infectious endocarditis. Colonization of the extracellular matrix of the periodontium and extra-oral tissues is a critical step for the pathogenesis ofA. actinomycetemcomitans. EmaA (extracellular matrix protein adhesin A) mediates the adherence ofA. actinomycetemcomitans to extracellular matrix proteins and has been shown to be a virulence determinant of this pathogen. The molecular characteristics of EmaA were investigated in this thesis to understand the structure/function and pathogenic role of this adhesin.
The monomer of EmaA is a 202 kDa protein that belongs to the oligomeric coiled-coil autotransporter adhesin (Oca) family. Oligomerization of EmaA monomers form surface appendages containing a long stalk terminating with an ellipsoidal head domain. The collagen binding activity of EmaA was localized to the ellipsoidal head region, corresponding to aminoterminal amino acids 70-386, as determined using in-frame deletion mutants and transmission electron microscopy. Finer deletion mapping in the collagen binding domain indicated that multiple sequences in this region are required for collagen binding. Conserved pentameric repeats were identified in the sequence and were shown to playa structural role in the formation of the functional oligomeric binding domain.
The collagen binding domain is followed by a stalk domain with a length of at least 150 nm. Specific bending positions were found along the EmaA stalk domain in the 3D structures. A major bend at 29.4 nm from the distal end of the EmaA appendages was localized to the second neck sequence, which is a conserved structural motif in Oca family proteins. This sequence is proposed to stabilize the oligomer and transition between the different structural domains. In this study, the collagen binding domain and critical amino acids within this domain of EmaA were identified. The 3D structure of EmaA revealed unique characteristics of the adhesin and the collagen binding domain. This study initiates the investigation of the molecular mechanism of the interaction of this adhesin with collagen and the role of EmaA in pathogenesis.
The monomer of EmaA is a 202 kDa protein that belongs to the oligomeric coiled-coil autotransporter adhesin (Oca) family. Oligomerization of EmaA monomers form surface appendages containing a long stalk terminating with an ellipsoidal head domain. The collagen binding activity of EmaA was localized to the ellipsoidal head region, corresponding to aminoterminal amino acids 70-386, as determined using in-frame deletion mutants and transmission electron microscopy. Finer deletion mapping in the collagen binding domain indicated that multiple sequences in this region are required for collagen binding. Conserved pentameric repeats were identified in the sequence and were shown to playa structural role in the formation of the functional oligomeric binding domain.
The collagen binding domain is followed by a stalk domain with a length of at least 150 nm. Specific bending positions were found along the EmaA stalk domain in the 3D structures. A major bend at 29.4 nm from the distal end of the EmaA appendages was localized to the second neck sequence, which is a conserved structural motif in Oca family proteins. This sequence is proposed to stabilize the oligomer and transition between the different structural domains. In this study, the collagen binding domain and critical amino acids within this domain of EmaA were identified. The 3D structure of EmaA revealed unique characteristics of the adhesin and the collagen binding domain. This study initiates the investigation of the molecular mechanism of the interaction of this adhesin with collagen and the role of EmaA in pathogenesis.