ABSTRACT: Background. Periodontal diseases are bacterial-induced inflammatory diseases of the tooth-supporting tissues. Periodontitis is epidemiologically associated with systemic diseases such as rheumatoid arthritis and cardiovascular diseases. Secreted bacterial products and virulence factors are considered to link periodontitis and systemic diseases. As one of the most studied organisms in periodontal diseases, Aggregatibacter actinomycetemcomitans serves as model organism to study the secretion of bacterial products and virulence factors into host tissues. We applied bioinformatics and proteomics to define the virulence potential of the secretome of the A. actinomycetemcomitans strain D7S. Results. The genome of A. actinomycetemcomitans (EBI: ADCF01000001) shows the presence of the Sec and Tat secretion systems, and their activity was confirmed by the detection of specific substrates in the fractions of secreted proteins. Two preparations of secreted proteins were analyzed and 103 proteins were identified that were present in both preparations. Among the proteins that have a known pathogenic potential in in vitro studies are the outer membrane proteins Omp100, Omp64, Omp39, Omp16/18 and OmpA, and the leukotoxin, CdT. In addition, the secretome of A. actinomycetemcomitans revealed candidates for novel virulence factors. Most strikingly, these proteins included the lipoprotein LppC and a homologue to the factor H-binding protein (fHbp) of Neisseria spp. Furthermore, in our in silico approach VacJ, OapA, OapB and an uncharacterized protein were identified. Conclusions.The secretome of A. actinomycetemcomitans gives an overview of the pathogenic potential of this bacterium including established virulence factors and putative novel ones such as LppC and fHbp. Methods Bacterial strain and growth conditions A. actinomycetemcomitans serotype a rough-colony strain D7S was isolated from a patient suffering aggressive periodontal disease [1] and used in this study. The strain was cultured on blood agar plates (5% defibrinated horse blood, 5 mg hemin/l, 10 mg Vitamin K/l, Columbia agar base) incubated in air supplemented with 5% CO2, at 37 C for 3 d as previously described [2]. For biofilm growth, 2 - 108 bacterial cells were inoculated in 2 ml tryptic soy broth (Difco) in 24-well cell culture plates (Nunc), which were incubated in static culture in air supplemented with 5% CO2, at 37C for 42 h. Preparation of the D7S secretome Following biofilm cultivation, 2 ml of the growth medium of a single well was carefully collected and then centrifuged for 10 min at 10.000 x g to pellet down remaining bacterial cells. Supernatants were then filtered through 0.45 um and subsequently 0.22 um membranes prior to being desalted and concentrated into 120 ul H2O with Pall 10K membrane filters according to the manufacturer's instructions (Pall Corporation). SDS-PAGE and preparation of in-gel digests For mass-spectrometry, proteins were separated by SDS-PAGE in 12% polyacrylamide gels (length 12 cm, thickness 1.5 mm) [3] containing 0.78% N,N'-methylenbisacrylamide and 2M urea. Subsequent to electrophoresis the gels were fixated using 10% acetic acid, 30% ethanol and stained using hot Coomassie blue [4]. Preparations of peptides for analysis by mass spectrometry were generated essentially as described earlier [5]. Mass-spectrometry and MS/MS data processing LC-MS/MS analysis of peptides was performed using an HCT-Ultra ETD II ion trap mass spectrometer from Bruker (Bremen, Germany) linked to an Easy-nLC system from Proxeon (Odense, Denmark). Spectra were acquired using the enhanced scanning mode covering a mass range from m/z 400 to m/z 1300. The LC separation of peptides was performed using a 5 ?m C18 column (375 um OD/75 um IDx10 cm) from NanoSeparations (Nieuwkoop, The Netherlands) and a 60 min gradient ranging from 1 to 50 percent of acetonitrile. The solvents used were 0.1% formic acid from Baker (Tamro AB, Hisings Backa, Sweden) in 18 MOhm water (Millipore, Solna, Sweden) and 0.1% formic acid in acetoinitrile (LiChrosolv) from Merck (Darmstadt, Germany), and the flow rate was 300 nl min-1. In this study, each of the two independent preparations of secreted proteins of A. actinomycetemcomitans (strain D7S) used, were analyzed two times. The LC-MS/MS datasets were processed using Bruker DataAnalysis 4.0 SP4 using default settings for deconvolution and compound detection. Database searches using the peaklist files of the processed mass spectra were performed in the bacterial section of the NCBInr database using ProteinSacpe 2.1 (Bruker) and in-house licenses of Mascot 2.3.01 (www.matrixscience.com) and of Phenyx 2.6 (www.genbio.com). The search parameters allowed for one missed cleavage site and a mass error of 0.3 Da for both the MS and MS/MS mode. In addition, variable modifications including methionine oxidation, N-terminal acetylation, and derivation of cysteine by propionamide were considered. Non-redundant protein lists were obtained using the ProteinExtractor of ProteinScape 2.1 and settings for spectra acceptance as follows: Mascot score greater than 100 and at least one peptide with a peptide ion score greater than55. Peptides with a Mascot ion score less than 30 were ignored. As for Phenyx scores, the minimum threshold for protein acceptance was 18 and at least one peptide with a score of 10 as required. Peptides with a Phenyx score less than 7 were not considered. As ProteinScape considers different modified forms of the same peptide as individual peptides, all protein identifications on the final result list were manually inspected to ensure that each protein identification was based on at least two peptides with different sequences.
Thomas Kieselbach 
