Role of the Filifactor alocis Hypothetical Protein FA519 in Oxidative Stress Resistance

Filifactor alocis is an emerging member of the periodontal community and is now proposed to be a diagnostic indicator of periodontal disease. However, due to the lack of genetic tools available to study this organism, not much is known about its virulence attributes.

Filifactor alocis: Recent Insights and Advances

Filifactor alocis, a fastidious Gram-positive obligate anaerobic bacterium, is a newly appreciated member of the periodontal community that is now proposed to be a diagnostic indicator of periodontal disease. Its pathogenic characteristics are highlighted by its ability to survive in the oxidative stress–rich environment of the periodontal pocket and to significantly alter the microbial community dynamics by forming biofilms and interacting with several oral bacteria. Here, we describe the current understanding of F. alocis virulence attributes, such as its comparative resistance to oxidative stress, production of unique proteases and collagenases that can cause structural damage to host cells, and dysregulation of the immune system, which enable this bacterium to colonize, survive, and outcompete other traditional pathogens in the inflammatory environment of the periodontal pocket. Furthermore, we explore the recent advancements and future directions for F. alocis research, including the potential mechanisms for oxidative stress resistance and our evolving understanding of the interactions and mechanisms of bacterial survival inside neutrophils. We also discuss the current genetic tools and challenges involved in manipulating the F. alocis genome for the functional characterization of the putative virulence genes. Collectively, this information will expedite F. alocis research and should lead to the identification of prime targets for the development of novel therapeutics to aid in the control and prevention of periodontal disease.

Filifactor alocis and Tumor Necrosis Factor-Alpha Stimulate Synthesis of Visfatin by Human Macrophages

There is little known about the effect of the periodontopathogen Filifactor alocis on macrophages as key cells of the innate immune defense in the periodontium. Therefore, the aim of the present study was to investigate the effect of F. alocis and additionally of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) on visfatin and other pro-inflammatory and proteolytic molecules associated with periodontitis in human macrophages. The presence of macrophage markers CD14, CD86, CD68, and CD163 was examined in gingival biopsies from healthy individuals and periodontitis patients. Human macrophages were incubated with F. alocis and TNFα for up to 2 d. The effects of both stimulants on macrophages were determined by real-time PCR, ELISA, immunocytochemistry, and immunofluorescence. F. alocis was able to significantly stimulate the synthesis of visfatin by human macrophages using TLR2 and MAPK pathways. In addition to visfatin, F. alocis was also able to increase the synthesis of cyclooxygenase 2, TNFα, and matrix metalloproteinase 1. Like F. alocis, TNFα was also able to stimulate the production of these proinflammatory and proteolytic molecules. Our results highlight the pathogenetic role of F. alocis in periodontal diseases and also underline the involvement of visfatin in the aetiopathogenesis of periodontitis.

Phylogenetic Analysis of Filifactor alocis Strains Isolated from Several Oral Infections Identified a Novel RTX Toxin, FtxA

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.