Membrane Morphology and Leukotoxin Secretion Are Associated with a Novel Membrane Protein of Aggregatibacter actinomycetemcomitans (original) (raw)
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Microbiology (Reading, England), 2016
Fimbrial subunit synthesis, secretion and assembly on the surface of the periodontal pathogen Aggregatibacter actinomycetemcomitans is essential for biofilm formation. A recent quantitative proteomics study employing an afimbriated strain and a developed mutant isogenic for the inner membrane protein morphogenesis protein C (MorC) revealed that the abundance of the proteins of the fimbrial secretion apparatus in the membrane is dependent on MorC. To further investigate the relationship between MorC and fimbriation, we identified and complemented the defect in fimbriae production in the afimbriated laboratory strain. The transformed strain expressing a plasmid containing genes encoding the wild-type fimbrial subunit and the prepilin peptidase displayed all of the hallmarks of a fimbriated bacterium including the distinct star-like colony morphology, robust biofilm formation, biofilm architecture composed of discrete microcolonies, and presence of fimbriae. When the identical plasmid ...
Microbiology, 2016
Fimbrial subunit synthesis, secretion and assembly on the surface of the periodontal pathogen Aggregatibacter actinomycetemcomitans are essential for biofilm formation. A recent quantitative proteomics study employing an afimbriated strain and a developed mutant isogenic for the inner-membrane protein morphogenesis protein C (MorC) revealed that the abundance of the proteins of the fimbrial secretion apparatus in the membrane is dependent on MorC. To investigate further the relationship between MorC and fimbriation, we identified and complemented the defect in fimbriae production in the afimbriated laboratory strain. The transformed strain expressing a plasmid containing genes encoding the WT fimbrial subunit and the prepilin peptidase displayed all of the hallmarks of a fimbriated bacterium including the distinct star-like colony morphology, robust biofilm formation, biofilm architecture composed of discrete microcolonies and the presence of fimbriae. When the identical plasmid was transformed into a morC mutant strain, the bacterium did not display any of the phenotypes of fimbriated strains. Extension of these studies to a naturally fimbriated clinical strain showed that the resulting morC mutant maintained the characteristic colony morphology of fimbriated strains. There was, however, a reduction in the secretion of fimbrial subunits, and fewer fimbriae were observed on the surface of the mutant strain. Furthermore, the morC mutant of the fimbriated strain displayed a significantly altered biofilm microcolony architecture, while maintaining a similar biofilm mass to the parent strain. These results suggest that MorC influences fimbrial secretion and microcolony formation in A. actinomycetemcomitans.
Proteomics, 2015
Aggregatibacter actinomycetemcomitans is an important pathogen in the etiology of human periodontal and systemic diseases. Inactivation of the gene coding for the inner membrane protein, morphogenesis protein C (MorC), results is pleotropic effects pertaining to the membrane structure and function of this bacterium. The role of this protein in membrane biogenesis is unknown. To begin to understand the role of this conserved protein, stable isotope dimethyl labeling in conjunction with mass spectrometry was used to quantitatively analyze differences in the membrane proteomes of the isogenic mutant and wild-type strain. A total of 613 proteins were quantified and 601 of these proteins were found to be equal in abundance between the two strains. The remaining 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain compared with the wild-type strain. The 12 proteins were ascribed functions associated with protein quality control systems, oxidative stress responses, and protein secretion. The potential relationship between these proteins and the phenotypes of the morC mutant strain is discussed.
Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans
Molecular Microbiology, 2003
Actinobacillus actinomycetemcomitans ( Aa ) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa . Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti-Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knockout mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10-fold more than the wild-type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)-8, IL-6 and tumour necrosis factor (TNF) a a a a in epithelial cells, and induced IL-1 b b b b and TNF a a a a production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa .
Journal of Periodontal Research, 2000
The leukotoxin of Actinobacillus actinomycetemcomitans is considered to be an important virulence factor implicated in the pathogenesis of periodontal disease (1). It lyses human polymorphonuclear leukocytes (PMNL) and monocytes (2, 3) and can protect the bacterium from being phagocytized (4). Leukotoxin is therefore assumed to enable A. actinomycetemcomitans to evade the main defense line of the periodontal pocket. The leukotoxin is a 116-kD protein that belongs to the RTX family of pore-forming toxins (5). Although it shares considerable molecular homology (35±70%) with other RTX toxins (6, 7), the leukotoxin from A. actinomycetemcomitans exhibits the smallest spectrum of target cells compared to other human cell-aecting RTX toxins (7). Furthermore, A. actinomycetemcomitans leukotoxin is the only RTX toxin that remains bound to the bacterial surface. The mechanism behind the binding is unknown. A hydrophobic domain at the carboxyl terminus of the molecule was suggested to mediate association of the toxin to the outer Divisions of
Identification of six major outer membrane proteins from Actinobacillus actinomycetemcomitans
Gene, 2002
We have identified six major sarcosyl-insoluble outer membrane proteins (Omp) of Actinobacillus actinomycetemcomitans Y4, and designated them as Omp100, Omp64, Omp39, Omp29, Omp18 and Omp16 according to the molecular mass. A similar N-terminal sequence was found in the first 15 amino acid residues of Omp16 and Omp18. The N-terminal sequence of Omp29 matched perfectly with the sequence previously identified. We cloned and determined the DNA sequences of three complete genes encoding Omp100, Omp64 and Omp18/16, and one incomplete gene encoding Omp39. Each Omp revealed homologies with some bacterial virulence factors responsible for adhesion, invasion, serum resistance, or protein antigenicity. Serum from patients with periodontitis suspected to be related to A. actinomycetemcomintans infection strongly reacted with Omp100, Omp29 and Omp16 as did serum from mice immunized with A. actinomycetemcomitans Y4 whole bacteria. These findings suggest that Omps of A. actinomycetemcomitans can be associated with periodontal disease. q
Infection and immunity, 1998
The major outer membrane protein (OMP) of Actinobacillus actinomycetemcomitans is an OmpA homolog that demonstrates electrophoretic heat modifiability. The gene encoding this protein was isolated from a genomic library of A. actinomycetemcomitans NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis. Expression of the cloned gene in Escherichia coli and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa. The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins. We have named the major OMP of A. actinomycetemcomitans Omp34, and its corresponding gene has been named omp34.