Extracellular Vesicle Subproteome Differences among Filifactor alocis Clinical Isolates

Abstract

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod of the Firmicutes phylum, which has recently been implicated in oral infections. Extracellular vesicles (EVs) are crucial conveyors of microbial virulence in bacteria and archaea. Previously, in highly purified EVs from the F. alocis reference strain ATCC 35896 (CCUG 47790), 28 proteins were identified. The present study aimed to use label-free quantification proteomics in order to chart these EV proteins, in the reference strain, and in nine less-well-characterized clinical F. alocis isolates. In total, 25 of the EV proteins were identified and 24 were quantified. Sixteen of those were differentially expressed between the ten strains and the novel FtxA RTX toxin and one lipoprotein were among them. Consistent expression was observed among ribosomal proteins and proteins involved in L-arginine biosynthesis and type IV pilin, demonstrating a degree of EV protein expression preservation among strains. In terms of protein-protein interaction analysis, 21 functional associations were revealed between 19 EV proteins. Interestingly, FtxA did not display predicted interactions with any other EV protein. In conclusion, the present study charted 25 EV proteins in ten F. alocis strains. While most EV proteins were consistently identified among the strains, several of them were also differentially expressed, which justifies that there may be potential variations in the virulence potential among EVs of different F. alocis strains.

Keywords: Filifactor alocis; extracellular vesicles (EVs); oral infections; predicted EV subproteome; proteomics.

Figure 1

A schematic overview of the study design of the present work. The 28 EV proteins identified in the F. alocis reference strain ATCC 35896 and reported on previously [21] were assessed for predicted protein–protein interactions and expression levels in the reference strain and in nine additional, less-characterized F. alocis clinical isolates. A colony from F. alocis strain 624B-08U is shown at the top.

Figure 2

Predicted protein–protein interactions between EV proteins. Shown are networks established using STRING 10.5 based on the low confident score (0.15) of EV proteins (Supplementary Table S2). The interactions with high confident scores (0.9) are shown in circles. Proteins enriched for “signal” (UniProt: KW-0732) are indicated with red balls and the others with white. Colors of lines indicate different types of protein–protein interactions. Blue and purple lines indicate interaction determined from the curated database and experimental results, respectively. Green, red, and dark blue lines indicate predicted interactions determined from gene neighborhood, gene fusions, and gene co-occurrence, respectively. Yellow and black lines indicate interactions deduced from text mining and co-expression, respectively.

Figure 3

Heatmap of normalized abundance for EV proteins. The colors in the map display the mean value for log2-transformed normalized abundance value plus one for the individual proteins (represented by a single row) within each experimental group (represented by a single column). Expression values are shown as a color scale, with higher values represented by red and lower represented by blue. The EV proteins that were expressed at consistent levels (p > 0.05) among all strains are indicated with red text. Color-frame boxes and short explanations with the same color were given for proteins with consistent expressions as well as proteins with interaction confidence of more than 0.9 based on STRING analysis.