The diverse variety and numbers of bacteria in the endodontal space makes investigating the pathogenesis of endodontic infections a complicated project. In the spirit of true community cooperativity, each individual microbial species contributes to the development and success of infection. For example, some bacteria create a biofilm as a growth surface for other, more invasive, species to colonize, while others secrete enzymes to break down host tissue to permit colonization. Understanding disease progression thus requires examination of the whole microbial community and its metaproteome.
Using a shotgun proteomics technique, Provenzano et al. have analyzed the endodontic metaproteome, comparing the community exoproteome involved in abscess formation with that found alongside asymptomatic periodontal lesions.1
The researchers collected samples directly from the endodontic spaces of patients undergoing root canal surgery (n=12). Samples were taken when access to the root canal was first established (S1) and then after flushing the cavity (S2) with either sodium hypochlorite (NaOCl; n=6) or chlorhexidine (CHX; n=6) subsequent to mechanical debridement. The volumes collected in these cases were minimal, so samples were pooled for analysis. Samples of purulent material were collected by direct aspiration from two patients with acute apical abscesses. All samples were centrifuged immediately to remove cellular content and maximize recovery of secreted proteins in the supernatant.
Provenzano and co-workers confirmed the presence of bacteria using specific polymerase chain reaction (PCR) probes. They found that bacteria were present in both apical abscess samples and in 4/6 NaOCl and 5/6 CHX S1 samples.
Following overnight in-solution trypsin digestion, samples were analyzed by nanoflow liquid chromatography coupled with a linear ion trap quadrupole (LTQ) Velos Orbitrap mass spectrometer (Thermo Scientific) and by liquid chromatography–quadrupole time-of-flight (LC-QTOF). LTQ Velos Orbitrap data were generated from raw files using Proteome Discoverer v1.3 (Thermo Scientific) with Sequest search engine. Orbitrap and LC-QTOF data were searched against human and bacterial sequences in the UniProt protein database.
The researchers identified 308 bacterial proteins and 139 human proteins in the samples. Protein numbers decreased following CHX flushing (S1 = 74, S2 = 31) but rose when NaOCl was used (S1 = 14, S2 = 35). The latter could be due to the local effect of the chemical on host cells.
More proteins were identified in purulent exudate from the two abscess cases (n=173) than in aspirates taken from the asymptomatic apical periodontitis cases (n=88). The researchers consider that this is probably due to the increased number of cells (bacterial and host immune response) found in pus as compared to the low sample volumes occurring in the asymptomatic root canals. They also consider that cell lysis, either during sample collection or due to inflammation, boosts protein concentrations. Although measures were taken to maximize exoproteome analysis, cytoplasmic proteins were detected in all samples.
The bacterial proteins identified included those with roles in disease development such as adhesion, tissue invasion and proteolysis. For example, S1 samples contained glycosyltransferase 1, which is involved in adhesion and biofilm creation through production of polysaccharides. Proteins linked to antibiotic resistance, TetR and a beta-lactamase, were also found in S1 samples. Collagenase and proteases were identified in samples from the two apical abscess cases. Proteins involved with host defense responses were identified in the pool of human proteins recovered.
Provenzano et al. suggest that by examining and qualitatively defining the metaproteome of endodontitis, the role of bacteria in the development and pathogenesis of this condition has been further defined, although more work is needed to expand the database.
1. Provenzano, J.C., et al. (2013) “Metaproteome Analysis of Endodontic Infections in Association with Different Clinical Conditions,” Public Library of Science ONE, 8(10): e76108, doi:10.1371/journal.pone.0076108.
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