In vivo induced antigenic determinants of Actinobacillus actinomycetemcomitans

Abstract

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.

Use of In Vivo-Induced Antigen Technology for Identification of Escherichia coli O157:H7 Proteins Expressed during Human Infection

“…Also, consistent with the anaerobic gut environment, IVIAT identified glycolytic enzymes, hydrogenases involved in fermentation of carbon compounds, an alcohol dehydrogenase, and reductases (including two cryptic nitrate reductases) involved in energy generation from carbohydrates via anaerobic respiration and fermentation (42). These results were expected and consistent with those of other studies of diverse organisms using techniques such as transcriptional profiling (59), in vivo expression technology (15,33), recombinase in vivo expression technology (4), signature-tagged mutagenesis (14,37,38), selective capture of transcribed sequences (SCOTS) (7), and IVIAT of other organisms (5).…”

Identification of Group A Streptococcus Antigenic Determinants Upregulated In Vivo

“…This was accomplished by using IVIAT, which identifies genes that are expressed by a pathogen during an actual infection process. IVIAT has been previously applied to other gram-negative organisms, mycobacteria, and yeast (3,4,8,18,24), but this is the first report of the use of this technology in a gram-positive organism. The basis of IVIAT is that pooled sera from patients with disease adsorbed against in vitro-derived GAS antigens can be used to identify genes upregulated in vivo.…”

Proteomic and immunoproteomic analysis of Aggregatibacter actinomycetemcomitans JP2 clone strain HK1651

“…The technique has been used to screen the entire A. actinomycetemcomitans genome and revealed 116 in vivo expressed antigens [23]. Further investigations using this method have shown that patients have a significantly greater serum antibody titer against six antigens than healthy controls, which supports the in vivo expression of these antigens, and even suggests their usefulness as disease markers [24].…”