JDRXXX10.1177/0022034520901738Journal of Dental ResearchE. gingivalis Causes Oral Inflammation and Tissue Destruction 901738research-article2020 Research Reports: Biological Journal of Dental Research 1 –7 © International & American Associations Entamoeba gingivalis Causes Oral for Dental Research 2020 Article reuse guidelines: Inflammation and Tissue Destruction sagepub.com/journals-permissions DOI:https://doi.org/10.1177/0022034520901738 10.1177/0022034520901738 journals.sagepub.com/home/jdr X. Bao1 , R. Wiehe1, H. Dommisch1 , and A.S. Schaefer1 Abstract A metagenomics analysis showed a strongly increased frequency of the protozoan Entamoeba gingivalis in inflamed periodontal pockets, where it contributed the second-most abundant rRNA after human rRNA. This observation and the close biological relationship to Entamoeba histolytica, which causes inflammation and tissue destruction in the colon of predisposed individuals, raised our concern about its putative role in the pathogenesis of periodontitis. Histochemical staining of gingival epithelium inflamed from generalized severe chronic periodontitis visualized the presence of E. gingivalis in conjunction with abundant neutrophils. We showed that on disruption of the epithelial barrier, E. gingivalis invaded gingival tissue, where it moved and fed on host cells. We validated the frequency of E. gingivalis in 158 patients with periodontitis and healthy controls by polymerase chain reaction and microscopy. In the cases, we detected the parasite in 77% of inflamed periodontal sites and 22% of healthy sites; 15% of healthy oral cavities were colonized by E. gingivalis. In primary gingival epithelial cells, we demonstrated by quantitative real-time polymerase chain reaction that infection with E. gingivalis but not with the oral bacterial pathogen Porphyromonas gingivalis strongly upregulated the inflammatory cytokine IL8 (1,900 fold, P = 2 × 10–4) and the epithelial barrier gene MUC21 (8-fold, P = 7 × 10–4). In gingival fibroblasts, we showed upregulation of the collagenase MMP13 (11-fold, P = 3 × 10–4). Direct contact of E. gingivalis to gingival epithelial cells inhibited cell proliferation. We indicated the strong virulence potential of E. gingivalis and showed that the mechanisms of tissue invasion and destruction are similar to the colonic protozoan parasite E. histolytica. In conjunction with abundant colonization of inflamed periodontal sites and the known resistance of Entamoeba species to neutrophils, antimicrobial peptides, and various antibiotics, our results raise the awareness of this protozoan as a potential and, to date, underrated microbial driver of destructive forms of periodontitis. Keywords: periodontal disease(s)/periodontitis, cytokines, MUC21, MMP13, mucosal immunity, host pathogen interactions Introduction amoeba into the lamina propria, but only 10% to 20% of infec- tions develop disease symptoms, with tremendous variation in Periodontitis is a very common complex inflammatory disease clinical outcome, such as colitis, diarrhea, vast intestinal tissue of the oral cavity with a frequency of 47% for adults >30 y of damage, and liver abscess (Walsh 1986; Haque et al. 2002; age in Western countries (Eke et al. 2012; Marcenes et al. Shirley et al. 2018). The wide variation in presentation of dis- 2013). Periodontitis is characterized by a disruption of the ease manifestations argues for additional susceptibility factors microbial homeostasis (Darveau 2010; Lamont et al. 2018), that determine parasite pathogenicity. The genetic constitution indicating misbalance of the complex network of interactions of the host is increasingly recognized as a contributor to patho- between the microbial community of the oral mucosa and host gen susceptibility and microbiome composition. E. histolytica epithelial and immune cells (Clemente et al. 2012). Likewise, virulence seems to both require and disrupt the microbiota a metagenomics analysis showed that the sites of oral inflam- during infection. Accordingly, characteristics of the host mation are characterized by reduced diversity in the taxonomic composition of active microbial communities (Deng et al. 2017). However, in contrast to this general reduction of taxo- 1Charité – Universitätsmedizin Berlin, corporate member of Freie nomical diversity, the prevalence of the protozoan Entamoeba Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute gingivalis was significantly increased in inflamed periodontal of Health, Institute for Dental and Craniofacial Sciences, Dept. of pockets, contributing the second-most abundant rRNA after Periodontology and Synoptic Dentistry, Berlin, Germany human rRNA. E. gingivalis is the only Entamoeba species that A supplemental appendix to this article is available online. is known to colonize the human oral cavity. Other human Entamoeba species colonize the intestinal lumen, including Corresponding Author: A.S. Schaefer, Parodontologie und Synoptische Zahnmedizin, Charité Entamoeba histolytica, which is considered a leading parasitic Centrum Zahn-, Mund- und Kieferheilkunde CC 3, Aßmannshauser cause of death worldwide and the causative agent of amebiasis. Straße 4-6, Berlin, 14197, Germany. This gastrointestinal disease is characterized by invasion of the Email: [email protected] 2 Journal of Dental Research 00(0) Table. Characteristics of the Study Samples. 2014). The invading amoeba also actively increases the pro- duction of host matrix metalloproteinases (MMPs) that break % (n) or Mean ± SD down the extracellular matrix (ECM; Thibeaux et al. 2014). Periodontitis Controls This results in tissue damage and translocation of intestinal Traits (n = 51) (n = 107) bacteria into the tissue, which may also promote dissemination Entamoeba gingivalis of bacteria to other organs with putative pathogenic conse- Inflamed periodontal pocket 77 (39) NA quences for systemic diseases. Notably, periodontitis is associ- Healthy site 22 (11) 15 (16) ated with other complex diseases, and long-lasting extensive Female sex 53 (27) 54 (58) oral inflammation may increase the risk for cardiovascular dis- Age, y 61 ± 15 42 ± 15 ease (Desvarieux et al. 2005), rheumatoid arthritis (Detert et al. Smokinga Never smoked 53 (27) — 2010), and oral cancer (Michaud et al. 2017). Nonsmoker ≤2 y 18 (9) — The high frequency of E. gingivalis at inflamed sites with the Nonsmoker >2 y 16 (8) — close biological relationship to E. histolytica raised our concern Current smoker 14 (7) — about its putative role in the pathogenesis of periodontitis. We Systemic diseasea hypothesized similar infection strategies as described for E. his- No self-reported systemic disease 88 (45) — tolytica. We also analyzed its virulence potential in contrast to the Diabetes 2 (1) — Cardiovascular disease 10 (5) — oral anaerobic bacterium Porphyromonas gingivalis, a successful CAL: affected sites, % colonizer of the oral epithelium that is strongly associated with 3 to 4 mm 36 ± 18 NA advanced periodontal lesions (Lamont et al. 2018). This study >5 mm 36 ± 24 NA was conducted in accordance with the principles of good clinical Pocket depth: affected sites, % practice and approved by the local Ethics Committee (EA4-207- 4 to 6 mm 26 ± 13 NA 17). Written informed consent was obtained from all subjects >7 mm 7 ± 13 NA according to the Declaration of Helsinki. Inflammation status BOP 96 (49) NA Sites with BOP, % 33 ± 28 NA Material and Methods BOP, bleeding on probing; CAL, clinical attachment loss; NA, not Description of the Study Population applicable. aControl data unknown. To determine the frequency of E. gingivalis in inflamed peri- odontal pockets and at noninflamed areas of the oral cavity, we microbiota shape the virulence potential of the parasite (Marie collected patients with clinically diagnosed periodontitis and and Petri 2014; Burgess and Petri 2016). The bacterial load of controls who self-reported to have no periodontal disease. subgingival biofilms from individuals with periodontitis accu- Cases (n = 51) were recruited at the Department of mulates with increasing clinical inflammation (Lamont et al. Periodontology, University Medicine Berlin, and diagnosed 2018). Therefore, inflammation appears to be an important according to the classification system released in 2018 (Caton ecologic change that can drive the outgrowth of periodontitis- et al. 2018). The controls (n = 107) consisted of Berlin associated microorganisms through tissue destruction and the University employees and students. All controls gave self- virulence potential of E. gingivalis. Similar to Entamoeba, the reports to be free of periodontal diseases, and none were seek- eukaryotic mucosal parasites Trichomonas vaginalis and tenax ing dental medical care. No bleeding on probing and no signs infect a large fraction of individuals without symptoms of reddened or swollen tissue determined absence of inflam- (Johnston and Mabey 2008; Benabdelkader et al. 2019) but are mation. A detailed description of the patient sample is pre- clearly associated with periodontal diseases, as recently sented in the Table. reviewed (Marty et al. 2017; Bonner et al. 2018). The pathogenesis of intestinal amebiasis is described as a E. gingivalis Collection, Culture, and Detection result of a sequel of specific steps (reviewed by Ghosh et al. 2019; Leon-Coria et al. 2020). The first barrier that E. histo- All procedures are described in detail in the Appendix. lytica must overcome to invade the colonic mucosa is the mucus layer, which covers the intestinal epithelium. Here,