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Filifactor alocis is an emerging oral pathogen, and approximately 50% of known F.alocis strains encode and express a Repeats-in-Toxin (RTX) protein, FtxA. FtxAappears to be associated with both progress and severity of periodontal disease.Mechanisms are not yet known but could be linked to increased loads of F. alocisin ftxA-positive strains. Here, we investigated mechanistic correlations based onFtxA-activity, as present in F. alocis cells and extracellular vesicles and as arecombinant protein, exploiting THP-1 macrophage-like cells. For this, we usedthe ftxA-expressing strain, ATCC 35896 (ftxA+), and F. alocis 148B-17U (ftxA−),which naturally lacks the ftxA gene. Using RNA sequencing analysis (RNA-Seq) andcytokine array analysis, we have pinpointed a role of FtxA in shifting host responsetoward immunosuppression, also inhibiting apoptosis and immune cellrecruitment, and with a potential role in downregulating mitochondrial andoxidative phosphorylation pathways. Such role(s) could provide a plausibleexplanation why FtxA is associated with progress and severity of periodontaldisease, and further studies on FtxA-host cell interactions might reveal novelpotential therapeutic targets.

The oral pathogen Filifactor alocis encodes a repeats-in-toxin (RTX) protein, FtxA, that isencoded by the ftxA gene; it is present in approximately 50% of known isolated strains from various infected oral sites, including periodontitis, peri-implantitis, and root canal infections. It has been determined from PCR assessment of periodontally diseased cohorts in Ghana and Australia. Based on current knowledge, ftxA appears to be associated with both the progress and severity of periodontitis. This finding could potentially be linked to enhanced levels of ftxA-positive F. alocis, relative to ftxA-negative strain, and/or, in addition, a synergy between ftxA-positive strains and other periodontal pathogens. The exact mechanism remains unclear but may depend on an FtxA-mediated shifting of the host cell response toward immunosuppression. The main objective of the present work was to evaluate the prevalence and loads of F. alocis and the presence of ftxA in subgingival plaque in patients recruited for periodontal treatment in Sweden. This observational study included all samples that were received from external clinics over one full year (n =71 patients). Our findings revealed that F. alocis was carried by 49 (69%) of the individuals, with the prevalence of ftxA amounting to 42.9% (n = 21). In 32 of the 71 samples, F. alocis could be quantitatively assessed. In this sub-population of F. alocis-positive patients, high loads of the bacterium were not related to age, and high loads were more frequently observed upon carriage of ftxA. The presence of, and co-colonization with, F. alocis with four additional periodontal pathogens was also evaluated. F. alocis was notable in that it cocolonized with all of the other species. Moreover, it was detected alongside two and even three of the other species within the same sample.

Aggregatibacter aphrophilus is the only known oral bacterium with a functional type VI secretion system (T6SS) that acts against Aggregatibacter actinomycetemcomitans. Bacteria use the T6SS to deliver toxic effectors into bacterial or eukaryotic cells during interbacterial competition or host colonization. To date, the T6SS of A. aphrophilus has not been demonstrated to participate in antieukaryotic activity, nor have the cytotoxic effectors involved been identified. Here, we identified 2 T6SS effectors in A. aphrophilus, a glycosyl hydrolase (Glh) and a phospholipase D (Tle5), which, upon inactivation of their respective genes, together resulted in abolished T6SS activity against A. actinomycetemcomitans in multispecies biofilms. Next, we probed the role of the 2 T6SS effectors in host–cell interactions using gingival keratinocytes. Interestingly, although neither of the effectors appeared to contribute to the acute inflammatory response directly, the co-presence of both species reduced the inflammatory effect, likely due to the T6SS-dependent elimination of A. actinomycetemcomitans, hence decreasing the bacterial abundance. This reduction was not observed using A. aphrophilus mutants lacking the effectors or the T6SS “tube” core protein, hemolysin co-regulated protein (Hcp). Here, we show that the T6SS effectors in A. aphrophilus have distinct functions in eukaryotic versus bacterial cell interactions. Hence, these T6SS effectors may represent novel mechanisms of interaction between bacteria and the oral–mucosal barrier, offering potential therapeutic targets for managing periodontal pathogens.

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.

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.

Microbial ecosystems experience spatial and nutrient restrictions, leading to the coevolution of cooperation and competition among cohabiting species. To increase their fitness for survival, bacteria exploit machinery to antagonizing rival species upon close contact. As such, the bacterial type VI secretion system (T6SS) nanomachinery, typically expressed by pathobionts, can transport proteins directly into eukaryotic or prokaryotic cells, consequently killing cohabiting competitors. Here we demonstrate first time that oral symbiont Aggregatibacter aphrophilus possesses a T6SS and can eliminate its close relative oral pathobiont Aggregatibacter actinomycetemcomitans using its T6SS. These findings bring newer the anti-bacterial prospects of symbionts against cohabiting pathobionts while introducing presence of an active T6SS in the oral cavity.

Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.

This paper aims to investigate the presence of Aggregatibacter actinomycetemcomitans and to assess potential indicators of the risk of severe form(s) of periodontitis. A descriptive cross-sectional study of 156 consecutive patients with periodontitis was conducted. Subgingival plaque samples were collected from the participants. The identification of A. actinomycetemcomitans was performed using quantitative polymerase chain reaction. A descriptive analysis, a chi-square test, and a binary logistic regression statistical evaluation were performed. The prevalence of A. actinomycetemcomitans in this population of 156 participants was 17.30% (27 patients). The prevalence of stage-III periodontitis was 75.6% and greater in older men, while the prevalence of stage-IV periodontitis was 22.4% and greater in younger women. We observed a significant relation between the risk of severe periodontitis (stage-IV) and poor oral hygiene (p = 0.006), attendance at dental appointments (p ≤ 0.001), and familial history of periodontitis (p = 0.032). In conclusion, twenty-seven individuals were positive for A. actinomycetemcomitans. Poor oral hygiene, family history of periodontitis, and irregular attendance at dental appointments were identified as potential risk factors for severe periodontitis in this cohort.

Here, we present a protocol for evaluating type VI secretion system (T6SS)dependent fitness of the oral symbiont A. aphrophilus using biofilm competition assays and metaproteomics. We describe steps for designing T6SS-specific mutants. We then detail procedures for using them in competition assays with the pathobiont A.actinomycetemcomitans and in biofilm models, analyzing metaproteomes to assess the impact of the T6SS on multiple pathobionts. The biofilm modelis designed to mimic the oral plaque ecosystem and includes seven species. For complete details on the use and execution of this protocol, please refer to Oscarsson et al.

The objective was to evaluate using unstimulated saliva in detecting Aggregatibacter actinomycetemcomitans and to compare the saliva and subgingival and mucosa membrane occurrence of this periodontal pathogen in patients diagnosed with advanced periodontitis. Patients with advanced forms of periodontitis (n = 220; mean age: 54.03 ± 03 years) at stage III/IV were sampled. Unstimulated saliva, buccal cheek mucosa, and pooled subgingival plaque samples were collected. The identification of A. actinomycetemcomitans was performed using qPCR. A descriptive analysis and Wilcoxon test and analysis of variance were performed. A. actinomycetemcomitans was isolated from 28.18% of the subjects. A total of 660 samples were obtained, 220 from unstimulated saliva, 220 from buccal cheek mucosa surfaces, and 220 from pooled subgingival plaque samples. A. actinomycetemcomitans was isolated from 21.80% of unstimulated saliva, 19.50% of buccal cheek swabs, and17.70% of subgingival samples. There was a statistically significant difference between the presenceof A. actinomycetemcomitans in the unstimulated saliva samples and in the buccal cheek mucosa swab samples and pooled subgingival plaque samples (p < 0.001). These results suggest that in advanced periodontitis, unstimulated saliva is representative of pooled subgingival plaque/buccal cheek mucosa samples and its use is adequate in the oral detection of A. actinomycetemcomitans in a cohort of patients with stage III and IV periodontitis.