UVM Theses and Dissertations
Format:
Print
Author:
Jiang, Xuan
Dept./Program:
Microbiology and Molecular Genetics
Year:
2012
Degree:
PhD
Abstract:
Aggregatibacter actinomycetemcomitans is a Gram-negative facultative anaerobic oral pathogen, which is associated with different forms of periodontitis and several systemic infections. Colonization of the extracellular matrix of host tissues is a contributing factor in the pathogenesis of A. actinomycetemcomitans. EmaA (extracellular matrix protein adhesin A) is a virulence determinant mediating the interaction between the extracellular matrix protein, collagen, and this bacterium. The mechanism for the secretion of EmaA has not been characterized. ln this study, the signal peptide and the secretion pathway of presecretory EmaA were investigated. The amino acids required for inner membrane protein targeting and translocation were investigated utilizing gene fusion products and analyzed in both Escherichia coli and A. actinomycetemcomitans. The signal peptide of the presecretory EmaA was determined to be composed of 56 amino acids.
The sequence contains the canonical domains of positively charged amino acids (N) followed by hydrophobic amino acids (H) adjacent to the signal peptidase cleavage site (C) and a conserved N-terminal extension juxtaposed to the NHC domains. The substitution of the long signal peptide with a typical signal peptide did not affect EmaA localization or function. In-frame deletion constructs were generated to determine if all of the amino acids composing the signal peptide were necessary for secretion. Analysis of these constructs indicated that amino acids 2 to 23 and 24 to 53 were required for protein secretion. Whereas, amino acids 36 to 56 were competent for reduced secretion but abolished the collagen binding activity. Interestingly, a strain expressing the signal peptide with amino acids 16 to 39 deleted was competent for secretion when grown at 37 °C, but demonstrated reduced secretion at elevated temperatures.
EmaA secretion was demonstrated to be reliant on a chaperone-dependent pathway. The secretion-dedicated chaperone SecB was shown to participate in the secretion of EmaA utilizing a secB deletion strain. This strain demonstrated a partial reduction in the amount and the collagen binding activity of EmaA. The partial reduction of EmaA was attributed to an increase in the synthesis of DnaK. Overexpression of a DnaK substitution mutant with diminished activity (A174T), in the [Greek delta]secB strain reduced further the amount of EmaA in the membrane. Expression of dnaK A174T in the wild type strain did not affect the amount of EmaA when grown under typical laboratory conditions. However, the amount of EmaA was reduced when this strain was grown at elevated temperatures. A chromosomal deletion strain of amino acids 16 to 39 of the signal peptide, transformed with either the wild type or dnaK A174T expressing plasmid, did not affect the amount of EmaA. In addition, the level of EmaA in a [Greek delta]secB/emaA⁻ double mutant strain expressing EmaA[Greek delta]16-39 was unaffected when grown at both temperatures.
In this study, the portions of the long signal peptide of EmaA have been demonstrated to be required for protein folding or assembly of this collagen adhesin. Furthermore, the data suggest that chaperones are required for the targeting of EmaA to the membrane and a specific region of the signal peptide is necessary for secretion under stress conditions. These novel findings lay the foundation for future investigations into the secretion of virulence factors in this and other pathogenic microorganisms.
The sequence contains the canonical domains of positively charged amino acids (N) followed by hydrophobic amino acids (H) adjacent to the signal peptidase cleavage site (C) and a conserved N-terminal extension juxtaposed to the NHC domains. The substitution of the long signal peptide with a typical signal peptide did not affect EmaA localization or function. In-frame deletion constructs were generated to determine if all of the amino acids composing the signal peptide were necessary for secretion. Analysis of these constructs indicated that amino acids 2 to 23 and 24 to 53 were required for protein secretion. Whereas, amino acids 36 to 56 were competent for reduced secretion but abolished the collagen binding activity. Interestingly, a strain expressing the signal peptide with amino acids 16 to 39 deleted was competent for secretion when grown at 37 °C, but demonstrated reduced secretion at elevated temperatures.
EmaA secretion was demonstrated to be reliant on a chaperone-dependent pathway. The secretion-dedicated chaperone SecB was shown to participate in the secretion of EmaA utilizing a secB deletion strain. This strain demonstrated a partial reduction in the amount and the collagen binding activity of EmaA. The partial reduction of EmaA was attributed to an increase in the synthesis of DnaK. Overexpression of a DnaK substitution mutant with diminished activity (A174T), in the [Greek delta]secB strain reduced further the amount of EmaA in the membrane. Expression of dnaK A174T in the wild type strain did not affect the amount of EmaA when grown under typical laboratory conditions. However, the amount of EmaA was reduced when this strain was grown at elevated temperatures. A chromosomal deletion strain of amino acids 16 to 39 of the signal peptide, transformed with either the wild type or dnaK A174T expressing plasmid, did not affect the amount of EmaA. In addition, the level of EmaA in a [Greek delta]secB/emaA⁻ double mutant strain expressing EmaA[Greek delta]16-39 was unaffected when grown at both temperatures.
In this study, the portions of the long signal peptide of EmaA have been demonstrated to be required for protein folding or assembly of this collagen adhesin. Furthermore, the data suggest that chaperones are required for the targeting of EmaA to the membrane and a specific region of the signal peptide is necessary for secretion under stress conditions. These novel findings lay the foundation for future investigations into the secretion of virulence factors in this and other pathogenic microorganisms.