Proteomics of protein secretion by Aggregatibacter actinomycetemcomitans.
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ABSTRACT:
SUBMITTER: Zijnge V
PROVIDER: S-EPMC3405016 | biostudies-literature | 2012
REPOSITORIES: biostudies-literature
Publications
Proteomics of protein secretion by Aggregatibacter actinomycetemcomitans.
Zijnge Vincent V Kieselbach Thomas T Oscarsson Jan J
PloS one 20120725 7
The extracellular proteome (secretome) of periodontitis-associated bacteria may constitute a major link between periodontitis and systemic diseases. To obtain an overview of the virulence potential of Aggregatibacter actinomycetemcomitans, an oral and systemic human pathogen implicated in aggressive periodontitis, we used a combined LC-MS/MS and bioinformatics approach to characterize the secretome and protein secretion pathways of the rough-colony serotype a strain D7S. LC-MS/MS revealed 179 pr ...[more]
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Project description:Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.
| S-EPMC4575117 | biostudies-literature
Project description: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.
2013-08-13 | PXD000411 | Pride
Project description:Periodontitis is an infectious disease that causes the inflammatory destruction of the tooth-supporting (periodontal) tissues, caused by polymicrobial biofilm communities growing on the tooth surface. Aggressive periodontitis is strongly associated with the presence of Aggregatibacter actinomycetemcomitans in the subgingival biofilms. Nevertheless, whether and how A. actinomycetemcomitans orchestrates molecular changes within the biofilm is unclear. The aim of this work was to decipher the interactions between A. actinomycetemcomitans and other bacterial species in a multi-species biofilm using proteomic analysis. An in vitro 10-species "subgingival" biofilm model, or its derivative that included additionally A. actinomycetemcomitans, were anaerobically cultivated on hydroxyapatite discs for 64 h. When present, A. actinomycetemcomitans formed dense intra-species clumps within the biofilm mass, and did not affect the numbers of the other species in the biofilm. Liquid chromatography-tandem mass spectrometry was used to identify the proteomic content of the biofilm lysate. A total of 3225 and 3352 proteins were identified in the biofilm, in presence or absence of A. actinomycetemcomitans, respectively. Label-free quantitative proteomics revealed that 483 out of the 728 quantified bacterial proteins (excluding those of A. actinomycetemcomitans) were accordingly regulated. Interestingly, all quantified proteins from Prevotella intermedia were up-regulated, and most quantified proteins from Campylobacter rectus, Streptococcus anginosus, and Porphyromonas gingivalis were down-regulated in presence of A. actinomycetemcomitans. Enrichment of Gene Ontology pathway analysis showed that the regulated groups of proteins were responsible primarily for changes in the metabolic rate, the ferric iron-binding, and the 5S RNA binding capacities, on the universal biofilm level. While the presence of A. actinomycetemcomitans did not affect the numeric composition or absolute protein numbers of the other biofilm species, it caused qualitative changes in their overall protein expression profile. These molecular shifts within the biofilm warrant further investigation on their potential impact on its virulence properties, and association with periodontal pathogenesis.
| S-EPMC4355292 | biostudies-literature
Project description:ObjectivesWe and others have previously shown that epigallocatechin gallate (EGCg) inhibits the activity of an important virulence factor, leukotoxin (LtxA), produced by the oral bacterium Aggregatibacter actinomycetemcomitans, suggesting the potential use of this molecule as an anti-virulence strategy to treat periodontal infections. Here, we sought to better understand the effects of EGCg on toxin secretion and A. actinomycetemcomitans pathogenicity in a co-culture model.MethodsWe used a quantitative immunoblot assay to determine the concentrations of LtxA in the bacterial supernatant and on the bacterial cell surface. Using a co-culture model, consisting of A. actinomycetemcomitans and THP-1 cells, we studied the impact of EGCg-mediated changes in LtxA secretion on the toxicity of A. actinomycetemcomitans.Key findingsEGCg increased production of LtxA and changed the localization of secreted LtxA from the supernatant to the surface of the bacterial cells. In the co-culture model, a single low dose of EGCg did not protect host THP-1 cells from A. actinomycetemcomitans-mediated cytotoxicity, but a multiple dosing strategy had improved effects.ConclusionsTogether, these results demonstrate that EGCg has important, but complicated, effects on toxin secretion and activity; new dosing strategies and comprehensive model systems may be required to properly develop these anti-virulence activities.
| S-EPMC7947220 | biostudies-literature
Project description:Gram-negative bacteria display either a flat or an irregular outer membrane. The periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans has an irregular outer membrane. We have identified a gene that is associated with the biogenesis of this morphology. The gene is part of a three-gene operon and codes for a 141-kDa protein designated morphogenesis protein C (MorC), which is conserved in several gram-negative bacteria including Haemophilus influenzae and Pasteurella multocida. Insertional inactivation of this gene resulted in the conversion of an irregularly shaped membrane to a flat membrane. Associated with this morphological change were the autoaggregation of the bacteria during planktonic growth and a concomitant increase in the surface hydrophobicity of the bacterium. The absence of MorC also resulted in the loss of the secretion of leukotoxin but not the ltxA transcription. Our findings suggest that MorC is critical for membrane morphology and leukotoxin secretion in A. actinomycetemcomitans.
| S-EPMC2519528 | biostudies-literature
Project description:The extracellular matrix protein adhesin A (EmaA) surface antennae-like structures of the periodontal pathogen Aggregatibacter actinomycetemcomitans are composed of three identical protein monomers. Recently, we have demonstrated that the protein is synthesized with an extended signal peptide of 56 amino acids necessary for membrane targeting and protein translocation. In this study, EmaA secretion was demonstrated to be reliant on a chaperone-dependent secretion pathway. Deletion of secB partially reduced but did not abolish the amount of EmaA in the membrane. This observation was attributed to an increase in the synthesis of DnaK in the ΔsecB strain. Overexpression of a DnaK substitution mutant (A174T), with diminished activity, in the ΔsecB strain further reduced the amount of EmaA in the membrane. Expression of dnaK A174T in the wild-type strain did not affect the amount of EmaA in the membrane when grown under optimal growth conditions at 37°C. However, EmaA was found to be reduced when this strain was grown at heat-shock temperature. A chromosomal deletion of amino acids 16-39 of the EmaA extended signal peptide, transformed with either the wild-type or dnaK A174T-expressing plasmid, did not affect the amount of EmaA in the membrane. In addition, the level of EmaA in a ΔsecB/emaA(-) double mutant strain expressing EmaAΔ16-39 was unchanged when grown at both temperatures. 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.
| S-EPMC3442948 | biostudies-literature
Project description:Gram-negative facultative Aggregatibacter actinomycetemcomitans is an oral pathogen associated with periodontitis. The genetic heterogeneity among A. actinomycetemcomitans strains has been long recognized. This study provides a comprehensive genomic analysis of A. actinomycetemcomitans and the closely related nonpathogenic Aggregatibacter aphrophilus. Whole genome sequencing by Illumina MiSeq platform was performed for 31 A. actinomycetemcomitans and 2 A. aphrophilus strains. Sequence similarity analysis shows a total of 3,220 unique genes across the 2 species, where 1,550 are core genes present in all genomes and 1,670 are variable genes (accessory genes) missing in at least 1 genome. Phylogenetic analysis based on 397 concatenated core genes distinguished A. aphrophilus and A. actinomycetemcomitans. The latter was in turn divided into 5 clades: clade b (serotype b), clade c (serotype c), clade e/f (serotypes e and f), clade a/d (serotypes a and d), and clade e' (serotype e strains). Accessory genes accounted for 14.1% to 23.2% of the A. actinomycetemcomitans genomes, with a majority belonging to the category of poorly characterized by Cluster of Orthologous Groups classification. These accessory genes were often organized into genomic islands (n = 387) with base composition biases, suggesting their acquisitions via horizontal gene transfer. There was a greater degree of similarity in gene content and genomic islands among strains within clades than between clades. Strains of clade e' isolated from human were found to be missing the genomic island that carries genes encoding cytolethal distending toxins. Taken together, the results suggest a pattern of sequential divergence, starting from the separation of A. aphrophilus and A. actinomycetemcomitans through gain and loss of genes and ending with the divergence of the latter species into distinct clades and serotypes. With differing constellations of genes, the A. actinomycetemcomitans clades may have evolved distinct adaptation strategies to the human oral cavity.
| S-EPMC4700661 | biostudies-literature
Project description:The extracellular matrix protein adhesin A (EmaA) of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a fibrillar collagen adhesin belonging to the family of trimeric autotransporters. The protein forms antenna-like structures on the bacterial surface required for collagen adhesion. The 202-kDa protein monomers are proposed to be targeted and translocated across the inner membrane by a long signal peptide composed of 56 amino acids. The predicted signal peptide was functionally active in Escherichia coli and A. actinomycetemcomitans using truncated PhoA and Aae chimeric proteins, respectively. Mutations in the signal peptide were generated and characterized for PhoA activity in E. coli. A. actinomycetemcomitans strains expressing EmaA with the identical mutant signal peptides were assessed for cellular localization, surface expression, and collagen binding activity. All of the mutants impaired some aspect of EmaA structure or function. A signal peptide mutant that promoted alkaline phosphatase secretion did not allow any cell surface presentation of EmaA. A second mutant allowed for cell surface exposure but abolished protein function. A third mutant allowed for the normal localization and function of EmaA at 37°C but impaired localization at elevated temperatures. Likewise, replacement of the long EmaA signal peptide with a typical signal peptide also impaired localization above 37°C. The data suggest that the residues of the EmaA signal peptide are required for protein folding or assembly of this collagen adhesin.
| S-EPMC3232865 | biostudies-literature
Project description:The cyclic-AMP receptor protein (CRP) acts as a global regulatory protein among bacteria. Here, the CRP regulon has been defined in Aggregatibacter actinomycetemcomitans using microarray analysis of A. actinomycetemcomitans strain JP2 wild type cells compared to an isogenic crp deletion mutant. Genes whose expression levels changed at least 2-fold with p≤0.05 were considered significant. Of the 300 genes identified as being CRP-regulated, 139 were CRP-activated, including leukotoxin, with the remaining being CRP-repressed. The 300 genes represent 14.2% of ORFs probed which is significantly higher than what has been reported for CRP regulons in other bacteria. If the CRP-regulated genes are put into 17 functional classes, all 17 categories had at least 1 CRP-regulated gene. Several functional categories, mainly transport and binding proteins and energy metabolism proteins, were disproportionately represented in the CRP-regulated subset of genes relative to their overall representation in the genome. This is similar to the patterns seen in other bacteria. Finally, quantitative RT-PCR was used to show that the leukotoxin RNA levels were repressed 16-fold in the CRP mutant indicating that CRP activates leukotoxin transcription. However, this regulation appears to be acting through another regulatory protein since the leukotoxin promoter, unlike ∼129 other promoters of CRP-regulated genes, does not have a match to the consensus CRP-binding site. Several candidate genes for this intermediary transcription factor have been identified in the CRP regulon.
| S-EPMC3120918 | biostudies-literature
Project description:The cell envelope of gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 27% of the predicted open reading frames in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. A total of 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, whereas others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity.
| S-EPMC4305030 | biostudies-literature