Quantitative Proteomics Reveal Distinct Protein Regulations Caused by Aggregatibacter actinomycetemcomitans within Subgingival Biofilms

Abstract

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.

Streptococcus oralis maintains homeostasis in oral biofilms by antagonizing the cariogenic pathogen Streptococcus mutans

“…Among all tested species, omission of F. nucleatum from the biofilm composition resulted in a decrease of the total bacterial numbers. This may not be surprising as F. nucleatum is a “bridging” microorganism between early and late colonizing species, and has a scattered distribution throughout the biofilm, as shown in the present and earlier studies . Hence, absence of F. nucleatum may compromise the structural integrity of the biofilm and hinder overall microbial growth, as evidenced by the reduced total bacterial numbers in the present study.…”

“…Deciphering the interactions between species within biofilms is crucial for understanding the factors that ensure their stability, or those that drive changes that lead to dysbiosis. The Zürich in vitro biofilm models are useful tools to define such interactions of groups of species, individual species, or their virulence factors …”

The effect of metronidazole plus amoxicillin or metronidazole plus penicillin V on periodontal pathogens in an in vitro biofilm model

“…A . actinomycetemcomitans regulates protein expression of other species present in the biofilm (Bao, Bostanci, Selevsek, Thurnheer, & Belibasakis, ).…”

Metabolic interactions in microbial communities: untangling the Gordian knot

“…For instance, analysis of transcriptional patterns in coculture of a marine bacterium and a diatom, as well as ocean samples, pinpointed cross-feeding of 2,3-dihydroxypropane-1-sulfonate, a new link in marine microbial food web [41 ]. Metabolic applications of meta-proteomics are more commonly used for relatively simple systemsit was used to demonstrate metabolic adjustments made by three species comprising a model oral biofilm [43] or to show how the presence/absence of Aggregatibacter actinomycetemcomitans modulates metabolism of other bacteria in a 10-species biofilm [44]. Although not distinguishing between species, these results give a sense of the complexity and scale of metabolic adjustments that happen in 'real-world' communities.…”