Filifactor alocis is a Gram-positive anaerobic rod frequently detected in periodontal lesions, and which has gained increased interest recently due to the discovery of Filifactor alocis toxin A (FtxA), a repeats-in-toxin (RTX) protein. Approximately 50% of isolated F. alocis strains carry the ftxA gene. FtxA is yet only the second recognized RTX protein known to be produced by oral bacterial species after the Aggregatibacter actinomycetemcomitans leukotoxin (LtxA). The co-occurrence of F. alocis and A. actinomycetemcomitans in the oral cavity, at diseased periodontal sites suggest possible synergistic interactions relevant to pathogenesis. However, the virulence properties of FtxA, its contribution to periodontal disease, and its potential diagnostic relevance have remained largely unexplored.
This PhD thesis aimed to investigate 1) the clinical prevalence and diagnostic potential of F. alocis and ftxA in periodontal disease, 2) to examine possible synergistic interactions between F. alocis and A. actinomycetemcomitans, 3) to characterize the virulence-related properties of FtxA, and 4) to evaluate the effects of FtxA on host immune responses.
Across the three clinical studies (papers I-III), high levels of F. alocis and the presence of ftxA were associated with increased periodontal severity and disease progression.
In the longitudinal Ghanaian adolescent cohort (paper I), carriage of ftxApositive F. alocis was associated with higher bacterial loads and enhanced clinical attachment loss progression. Disease progression appeared to be further promoted when F. alocis co-occurred with non-JP2 genotype A. actinomycetemcomitans, whereas the JP2 genotype showed strong virulence irrespective of F. alocis.
In a site-specific analysis from the same Ghanaian adolescent cohort (paper II), higher loads of both species were associated with deeper periodontal pockets and greater progression, with the highest levels observed at sites positive for both JP2 genotype A. actinomycetemcomitans and ftxA-positive F. alocis.
In an independent Australian periodontitis cohort (paper III), high F. alocis loads and ftxA carriage were linked to more severe disease, including higher frequencies in Grade C and Stage IV periodontitis, supporting the clinical relevance of these findings across populations.
Mechanistic studies using THP-1 cells (paper IV) showed that a ftxA-positive F. alocis strain, its extracellular vesicles, and recombinant FtxA were non-cytotoxic, although according to RNA-Seq and array analysis induced a transcriptional and cytokine profile consistent with immunosuppression. Key inflammatory pathways, including cytokine and chemokine signaling, were downregulated, cytokine secretion was reduced, and apoptosis- and necroptosis-related pathways were suppressed. In contrast, a ftxA-negative strain and its vesicles elicited a more pro-inflammatory response. These findings suggest that FtxA may contribute to periodontal pathogenesis by dampening host immune responses and altering hostcell survival and metabolism.
Together, the findings of this PhD thesis work support a role for F. alocis and its RTX protein FtxA in periodontal disease progression and severity. FtxA thus emerges as only the second known bacterial virulence factor, after LtxA, described to have this capability. Detection of high F. alocis levels and ftxA may have value as molecular risk markers, particularly in combination with A. actinomycetemcomitans. Moreover, FtxA may represent a previously unexplored candidate virulence factor and possible future therapeutic target in periodontitis. Overall, this work expands current understanding of how an emerging oral pathogen - F. alocis - and its novel RTX protein FtxA could contribute to periodontal dysbiosis and tissue destruction, potentially offering new avenues for diagnosis and treatment.