In vivo induced antigenic determinants of Actinobacillus actinomycetemcomitans.
Actinobacillus actinomycetemcomitans is a Gram-negative capnophilic rod and the etiological agent of localized aggressive periodontitis. The genome-wide survey of A. actinomycetemcomitans using in vivo induced antigen technology (IVIAT) has previously resulted in the discovery of antigenic determinants expressed specifically in diseased patients. The present study evaluated the potential of these antigens as putative disease markers, and investigating their contribution to the pathogenesis of the microorganism. Sera from patients had a significantly greater antibody titer than sera from healthy controls against six antigens, which supports the in vivo expression of these antigens, and suggests their usefulness as disease markers. A. actinomycetemcomitans invasion of epithelium-derived HeLa cells resulted in the induction of all three genes tested, as evidenced by real-time PCR. Isogenic mutants of these three genes were constructed and the adhesion and intracellular survival of the mutants was assayed in a competition assay with the wild-type strain. A significant defect in the intracellular survival of two of these mutant strains (orf1402 and orf859) was found. This defect could not be attributed to an adhesion defect. In contrast, a mutation in vapA, a homologue of a novel putative transcriptional regulator, out-competed the wild-type strain in the same assay. The virulent phenotype was restored for a mutant strain in orf859 upon complementation. This data provided new insight into the pathogenic personality of A. actinomycetemcomitans in vivo and supported the use of HeLa cells as a valid in vitro host-pathogen interactions model for that microorganism. IVIAT is applicable to most pathogens and will undoubtedly lead to the discovery of novel therapies, antibiotics and diagnostic tools.
Cao
Sam Linsen
SL
Center for Molecular Microbiology and Department of Oral Biology, University of Florida College of Dentistry, P.O. Box 100424, 1600 SW Archer Road, Gainesville, FL 32610, USA.
Progulske-Fox
Ann
A
Hillman
Jeffrey D
JD
Handfield
Martin
M
D000189:Actinobacillus Infections / Q000175:diagnosis; D000222:Adaptation, Physiological; D016976:Aggregatibacter actinomycetemcomitans / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000472:pathogenicity*; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D015415:Biomarkers; D003593:Cytoplasm / Q000382:microbiology; D000939:Epitopes; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics / Q000502:physiology
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D015415:Biomarkers; D000939:Epitopes; D037521:Virulence Factors