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Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod that has been isolated from a variety of oral infections including periodontitis, peri-implantitis, and odontogenic abscesses. As a newly emerging pathogen, its type strain has been investigated for pathogenic properties, yet little is known about its virulence variations among strains. We previously screened the whole genome of nine clinical oral isolates and a reference strain of F. alocis, and they expressed a novel RTX toxin, FtxA. In the present study, we aimed to use label-free quantification proteomics to characterize the full proteome of those ten F. alocis strains. A total of 872 proteins were quantified, and 97 among them were differentially expressed in FtxA-positive strains compared with the negative strains. In addition, 44 of these differentially expressed proteins formed 66 pairs of associations based on their predicted functions, which included clusters of proteins with DNA repair/mediated transformation and catalytic activity-related function, indicating different biosynthetic activities among strains. FtxA displayed specific interactions with another six intracellular proteins, forming a functional cluster that could discriminate between FtxA-producing and non-producing strains. Among them were FtxB and FtxD, predicted to be encoded by the same operon as FtxA. While revealing the broader qualitative and quantitative proteomic landscape of F. alocis, this study also sheds light on the deeper functional inter-relationships of FtxA, thus placing this RTX family member into context as a major virulence factor of this species.

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod of the phylum Firmicutes, and is considered an emerging pathogen in various oral infections, including periodontitis. We here aimed to perform phylogenetic analysis of a genome-sequenced F. alocis type strain (ATCC 35896; CCUG 47790), as well as nine clinical oral strains that we have independently isolated and sequenced, for identification and deeper characterization of novel genomic elements of virulence in this species. We identified that 60% of the strains carried a gene encoding a hitherto unrecognized member of the large repeats-in-toxins (RTX) family, which we have designated as FtxA. The clinical infection origin of the ftxA-positive isolates largely varied. However, according to MLST, a clear monophylogeny was reveled for all ftxA-positive strains, along with a high co-occurrence of lactate dehydrogenase (ldh)-positivity. Cloning and expression of ftxA in E. coli, and purification of soluble FtxA yielded a protein of the predicted molecular size of approximately 250 kDa. Additional functional and proteomics analyses using both the recombinant protein and the ftxA-positive, and -negative isolates may reveal a possible role and mechanism(s) of FtxA in the virulence properties of F.alocis, and whether the gene might be a candidate diagnostic marker for more virulent strains.

The Gram-positive bacterium, Filifactor alocis is an oral pathogen, and approximately 50% of known strains encode a recently identified repeat-in-toxin (RTX) protein, FtxA. By assessing a longitudinal Ghanaian study population of adolescents (10-19 years of age; mean age 13.2 years), we recently discovered a possible correlation between deep periodontal pockets measured at the two-year follow-up, presence of the ftxA gene, and a high quantity of F. alocis . To further understand the contribution of F. alocis and FtxA in periodontal disease, we used qPCR in the present study to assess the carriage loads of F. alocis and the prevalence of its ftxA gene in subgingival plaque specimens, sampled at baseline from the Ghanaian cohort (n=500). Comparing these results with the recorded clinical attachment loss (CAL) longitudinal progression data from the two-year follow up, we concluded that carriers of ftxA -positive F. alocis typically exhibited higher loads of the bacterium. Moreover, high carriage loads of F. alocis and concomitant presence of the ftxA gene were two factors that were both associated with an enhanced prevalence of CAL progression. Interestingly, CAL progression appeared to be further promoted upon the simultaneous presence of F. alocis and the non-JP2 genotype of Aggregatibacter actinomycetemcomitans . Taken together, our present findings are consistent with the notion that F. alocis and its ftxA gene promotes CAL during periodontal disease.

The Gram-positive organism Filifactor alocis is implicated in multiple oral diseases including periodontitis, and approximately 50% of known strains encode and produce a recently identified repeat-in-toxin (RTX) protein, FtxA, partly homologous to the Aggregatibacter actinomycetemcomitans leukotoxin. By assessing a longitudinal Ghanaian study population of adolescents, we recently identified a possible correlation between F. alocis levels, ftxA gene carriage, and progression of clinical attachment loss (CAL). To extend knowledge on the possible significance of F. alocis and its FtxA in periodontal disease, we have in the present work analyzed saliva samples in an independent cohort of periodontitis (n=156), collected at two private periodontal specialist practices in Perth, Western Australia. The present results corroborate that high loads of F. alocis and the presence of its ftxA gene together are associated with parameters of periodontal tissue destruction and severity. Moreover, among the individuals carrying A. actinomycetemcomitans , a majority also exhibited an ftxA -positive F. alocis , supporting the notion of the synergistic behavior of these two species. This emphasizes that F. alocis and its ftxA are involved in the pathogenesis of periodontitis and may have ecological roles, with diagnostic and prognostic implications for the disease.

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod that has been isolated from a variety of oral infections including periodontitis, peri-implantitis, and odontogenic abscesses. As a newly emerging pathogen, its type strain has been investigated for pathogenic properties, yet little is known about its virulence variations among strains. We previously screened the whole genome of nine clinical oral isolates and a reference strain of F. alocis, and they expressed a novel RTX toxin, FtxA. In the present study, we aimed to use label-free quantification proteomics to characterize the full proteome of those ten F. alocis strains. A total of 872 proteins were quantified, and 97 among them were differentially expressed in FtxA-positive strains compared with the negative strains. In addition, 44 of these differentially expressed proteins formed 66 pairs of associations based on their predicted functions, which included clusters of proteins with DNA repair/mediated transformation and catalytic activity-related function, indicating different biosynthetic activities among strains. FtxA displayed specific interactions with another six intracellular proteins, forming a functional cluster that could discriminate between FtxA-producing and non-producing strains. Among them were FtxB and FtxD, predicted to be encoded by the same operon as FtxA. While revealing the broader qualitative and quantitative proteomic landscape of F. alocis, this study also sheds light on the deeper functional inter-relationships of FtxA, thus placing this RTX family member into context as a major virulence factor of this species.