Oxidative Stress Resistance Mechanisms in Filifactor Alocis

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A. PROJECT SUMMARY/ABSTRACT Recent oral microbiome studies have recognized a myriad of as-yet-culturable and fastidious organisms that have shown a strong correlation with periodontal disease severity. It is likely that the emerging new pathogens may play a more significant role in the disease compared to the traditional “red complex” bacteria Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. One such previously unrecognized organism, Filifactor alocis, is a Gram-positive, asaccharolytic, obligate anaerobic rod. Several recent studies have found this bacterium at significantly higher levels in adult or refractory periodontitis patients and have suggested that it could be included as a diagnostic indicator of periodontal disease. Currently, there is little or no information on survival mechanisms and virulence of F. alocis. Primarily, this is due to the unavailability of an efficient genetic system to allow genetic manipulations of the F. alocis genome. In response to environmental stress, our preliminary studies showed that F. alocis is relatively more resistant to H2O2-induced oxidative stress compared to P. gingivalis. Also, under H2O2-induced stress conditions, the survival of P. gingivalis is enhanced more than 4-fold in the presence of F. alocis. These observations suggest that F. alocis may have the ability to modify/reduce the oxidative stress environment and stabilize the microbial community of the periodontal pocket. In an RNA-seq analysis, the transcriptional profile of F. alocis showed that in coculture with P. gingivalis (compared to F. alocis monoculture) under H2O2-induced stress, the most highly upregulated genes in F. alocis encode for a putative manganese ABC transporter FA0894-FA0895-FA0896-FA0897. Manganese has been proposed to detoxify reactive oxygen species and protect bacteria from oxidative stress. It is our hypothesis that the F. alocis hypothetical ATP transporter, FA0894-FA0897, may play an important role in enhanced protection/survival of P. gingivalis against H2O2-induced stress. We wish to understand the modulation of F. alocis potential virulence factors in response to interaction with P. gingivalis and evaluate if they contribute to protection/persistence of P. gingivalis against the oxidative environment of the periodontal community. The proposed specific aims are: (1) to evaluate the specific role(s) of F. alocis putative ABC transporter FA0894-FA0897 in the protection/survival of P. gingivalis under H2O2-induced stress. (2) to develop an efficient genetic system for Filifactor that includes a markerless, in-frame deletion system and/or transposon mutagenesis system. Collectively, the data generated will facilitate a comprehensive assessment of the molecular mechanism(s) and overall interplay involving F. alocis and the ‘keystone’ pathogen P. gingivalis. It will also generate a polymicrobial model system that may facilitate the development of novel therapeutic interventions to aid in the control and prevention of periodontal disease. Furthermore, the new genetic manipulation system will help to uncover the relative significance of F. alocis in the etiology of periodontal disease.