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JDRXXX10.1177/0022034520901738Journal of Dental ResearchE. gingivalis Causes Oral Inflammation and Tissue Destruction

Research Reports: Biological

Journal of Dental Research
2020, Vol. 99(5) 561­–567
Entamoeba gingivalis Causes Oral © International & American Associations
for Dental Research 2020

Inflammation and Tissue Destruction Article reuse guidelines:

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DOI: 10.1177/0022034520901738
https://doi.org/10.1177/0022034520901738
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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
Periodontitis is a very common complex inflammatory disease
of the oral cavity with a frequency of 47% for adults >30 y of
age in Western countries (Eke et al. 2012; Marcenes et al.
2013). Periodontitis is characterized by a disruption of the
microbial homeostasis (Darveau 2010; Lamont et al. 2018),
indicating misbalance of the complex network of interactions
between the microbial community of the oral mucosa and host
epithelial and immune cells (Clemente et al. 2012). Likewise,
a metagenomics analysis showed that the sites of oral inflam-
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-
nomical diversity, the prevalence of the protozoan Entamoeba
gingivalis was significantly increased in inflamed periodontal
pockets, contributing the second-most abundant rRNA after
human rRNA. E. gingivalis is the only Entamoeba species that
is known to colonize the human oral cavity. Other human
Entamoeba species colonize the intestinal lumen, including
Entamoeba histolytica, which is considered a leading parasitic
cause of death worldwide and the causative agent of amebiasis.
This gastrointestinal disease is characterized by invasion of the
amoeba into the lamina propria, but only 10% to 20% of infec-
tions develop disease symptoms, with tremendous variation in
clinical outcome, such as colitis, diarrhea, vast intestinal tissue
damage, and liver abscess (Walsh 1986; Haque et al. 2002;
Shirley et al. 2018). The wide variation in presentation of dis-
ease manifestations argues for additional susceptibility factors
that determine parasite pathogenicity. The genetic constitution
of the host is increasingly recognized as a contributor to patho-
gen susceptibility and microbiome composition. E. histolytica
virulence seems to both require and disrupt the microbiota
during infection. Accordingly, characteristics of the host
1
Charité – Universitätsmedizin Berlin, corporate member of Freie
Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute
of Health, Institute for Dental and Craniofacial Sciences, Dept. of
Periodontology and Synoptic Dentistry, Berlin, Germany
A supplemental appendix to this article is available online.
Corresponding Author:
A.S. Schaefer, Parodontologie und Synoptische Zahnmedizin, Charité
Centrum Zahn-, Mund- und Kieferheilkunde CC 3, Aßmannshauser
Straße 4-6, Berlin, 14197, Germany.
Email: arne.schaefer@charite.de
562 Journal of Dental Research 99(5)

Table.  Characteristics of the Study Samples. 2014). The invading amoeba also actively increases the pro-
% (n) or Mean ± SD
duction of host matrix metalloproteinases (MMPs) that break
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
Smokinga  
ease (Desvarieux et al. 2005), rheumatoid arthritis (Detert et al.
  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.
a
Control 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, the In Vitro Infection of Gingival Cells
parasite modulates transcription of the colonic MUCIN2 gene with E. gingivalis and P. gingivalis
in host epithelial cells causing subsequent depletion and lead-
ing to breakdown of the mucus layer (Cornick et al. 2016). Preparation of amoebic cultures for infection is described in
This results in direct cell contact, upon which the amoeba detail in the Appendix. Cell cultures of primary gingival epi-
induces apoptosis of the epithelial cells, causing epithelial thelial cells (pGECs) and primary gingival fibroblasts (pGFBs)
damage (Cornick et al. 2017) and tissue invasion. The host from 2 independent donors were infected with amoeba, P. gin-
responds to amoebic infection by tremendous upregulation of givalis, and mock infection medium in triplicates for 2 h.
the inflammatory cytokine interleukin 8 (IL-8), which results E. gingivalis infection of healthy live gingiva is described in
in neutrophil infiltration (Yu and Chadee 1997; Mortimer et al. the Appendix.
E. gingivalis Causes Oral Inflammation and Tissue Destruction 563

Quantification of Gene Expression

and Cell Proliferation
The procedures for expression analysis
and cell proliferation are described in the
Appendix.

Tissue Staining
Gingival biopsies were fixed in 4% para-
formaldehyde for 48 h and embedded in
paraffin. Tissue sections (5 μm) were
stained with a periodic acid–Schiff (PAS)
staining kit (ab150680; Abcam) as previ-
ously described (Li et al. 2018).

Results
Frequency of E. gingivalis
Colonization of the Oral Cavity
E. gingivalis was detected in healthy oral
cavities in 15% of controls (n = 107; Table). Figure 1.  MUC1, MUC21, IL1β, and IL8 expression in primary gingival epithelial cells after
In patients (n = 51), we detected E. gingivalis Entamoeba gingivalis and Porphyromonas gingivalis infection for 2 h. For each gene, 2 biological
replicates with 3 technical replicates were conducted. P values were corrected for 18
in 77% of inflamed gingival pockets and independent tests (16 mucin and 2 interleukin genes). Genes with significant expression changes
22% of healthy sites. In the control group are shown. The smallest P value of an experiment is specified in the figure legend. (A) Of the
(n = 107), 15% of the healthy oral cavities tested mucin genes, MUC21 was 7.7-fold upregulated after E. gingivalis infection, P = 7 × 10–4
were colonized by E. gingivalis. (Pcorrected = 0.013). (B) P. gingivalis infection upregulated MUC1 expression >2.5-fold, P = 0.002
(Pcorrected = 0.043). This effect was not validated in the replication. (C) E. gingivalis infection
increased IL1β expression 9.5-fold, P = 6 × 10–5 (Pcorrected = 1 × 10–3). IL8 expression was
E. gingivalis Modulates increased 1,983.3-fold, P = 2 × 10–4 (Pcorrected = 3.6 × 10–3). (D) P. gingivalis infection increased
IL1β expression 1.9-fold, P = 2 × 10–4 (Pcorrected = 3.6 × 10–3). IL8 expression was increased 7-fold,
MUCIN21 Expression P = 0.002 (Pcorrected = 0.041). *P < 0.05, **P < 0.01, ***P < 0.001. Values are presented as means.
in Oral Epithelial Cells
Mucosal epithelial tissues form a physical and immunologic transcript levels was not significant after correction for multi-
barrier and, as such, have direct antimicrobial activity and the ple testing. The oral bacterium P. gingivalis did not induce
ability to opsonize microbes to aid clearance. Mucin glycopro- MUC21 expression in oral epithelial cells but showed upregu-
teins significantly contribute to these functions and are actively lation at a similar level as MUC1, as was observed after E.
targeted by mucosal pathogens such as E. histolytica (Linden gingivalis infection.
et al. 2008; Cornick et al. 2016). The oral mucosa and charac-
teristic of the mucus layer differ substantially from that in the
gut; however, numerous mucins are present in the oral cavity E. gingivalis Activates Interleukin 8 Expression
as secreted (e.g., MUC5B or MUC7) and as cell surface mucins
in Oral Epithelial and Fibroblast Cells
(e.g., MUC1; Corfield 2018). To test if E. gingivalis was capa-
ble of modulating mucin gene expression after infection of oral In colon epithelial cells, E. histolytica increases IL1β expres-
epithelial cells, we systematically screened the expression of sion moderately but upregulates IL8 >1,000-fold (Kim et al.
human mucin genes (MUC1, -2, -3A, -4, -5B, -5AC, -6, -7, -12, 1998). We tested, if E. gingivalis infection induced IL1β and
-13, -15, -16, -17, -19, -20, -21) in response to E. gingivalis IL8 expression in pGECs and pGFBs. IL8 was differentially
infection in pGECs and pGFBs. MUC1, -3A, -15, -4, -5B, expressed in pGECs and pGFBs, with a stronger expression in
-5AC, -6, -7, -13, -16, -19, -20, and -21 were expressed in pGECs (Cq = 26.2) as compared with pGFBs (Cq = 30.5;
pGECs at strong to moderate levels with quantification cycle Appendix Fig. 3). After coincubation of pGECs with E. gingi-
(Cq) values 25 < Cq < 35. MUC2, -12, and -17 were not valis, the expression of IL1ß was increased 9.5-fold (P = 6 ×
expressed (Cq > 35). After E. gingivalis infection for 2 h, 10–5; Pcorrected = 0.001), and IL8 was increased 1,983.3-fold
MUC21 was significantly upregulated (7.7-fold, P = 7 × 10–4; (P = 2 × 10–4; Pcorrected = 0.004; Fig. 1). After infection of
Pcorrected = 0.013; Fig. 1, Appendix Fig. 3). MUC1 showed pGECs with P. gingivalis, IL8 expression was 7.7-fold
nominal significant upregulation (P = 0.007) but the change of increased (P = 0.002; Pcorrected = 0.041).
564 Journal of Dental Research 99(5)

Pcorrected = 0.001) and 3.7-fold (P = 5 × 10–

5
; Pcorrected = 5 × 10–4), respectively (Fig. 2,
Appendix Fig. 4).

E­ . gingivalis Invades the

Inflamed and Wounded Oral
Mucosa
E. histolytica is commensal in 80% to 90%
of infected individuals, but in some cases,
it invades the colonic mucosa causing
amoebiasis. To see if E. gingivalis was
capable to invade the oral mucosa, we
microscopically analyzed the inflamed
gingiva of a patient with severe general-
ized chronic periodontitis (Fig. 3A, B).
After tissue staining, we observed amoeba
in various tissue layers that were sur-
rounded by abundant neutrophils. We vali-
dated the presence of E. gingivalis in
Figure 2.  MMP3, MMP13, IL1β, and IL8 expression in primary gingival fibroblasts after addition to microscopic visualization of
Entamoeba gingivalis and Porphyromonas gingivalis infection for 2 h. For each gene, 2 biological the tissue by polymerase chain reaction.
replicates with 3 technical replicates were conducted. P values were corrected for 10 Within the gingival epithelium of live,
independent tests (8 MMP and 2 interleukin genes). Genes with significant expression changes
are shown. The smallest P value of an experiment is specified in the figure legend. (A) Of the uninflamed ex vivo biopsies, microscope
tested MMP genes, MMP3 expression was 2-fold upregulated after E. gingivalis infection, P = analysis did not reveal E. gingivalis.
0.002 (Pcorrected = 0.018). MMP13 showed 11.2-fold upregulation after E. gingivalis infection, P = However, after wounding live, healthy gin-
3 × 10 (Pcorrected = 0.003). (B) P. gingivalis infection increased MMP3 expression 2.8-fold, P =
–4

1 × 10–4 (Pcorrected = 0.001). MMP13 expression was increased 3.7-fold, P = 5 × 10–5 (Pcorrected =
gival biopsies by slightly cutting the upper
5 × 10 ). (C) IL-1β expression in primary gingival fibroblasts was 11.9-fold upregulated after
–4 epithelial layer with a scalpel or punctur-
E. gingivalis infection, P = 1 × 10–4 (Pcorrected = 0.001), and IL-8 was 17.9-fold upregulated, P = 4 × ing it with sterile needles, we found the
10–5 (Pcorrected = 4 × 10–4). (D) After P. gingivalis infection, IL-1β was 15.0-fold upregulated, P = active and feeding stages of amoebic tro-
9 × 10–5 (Pcorrected = 9 × 10–4), and IL-8 showed 153.5-fold upregulation, P = 3 × 10–5 (Pcorrected =
3 × 10 ). Values are presented as mean ± SD.
–4 phozoites after 6 h of incubation.
Microscopy indicated moving of the
amoeba within gingival tissue, penetration
In pGFBs, IL1β expression in pGFBs was 11.9-fold upregu- into the cytoplasm of live host gingival epithelial cells (GECs),
lated after E. gingivalis infection (P = 1 × 10–4; Pcorrected = and ingestion of fragments from the nuclei of the host cells
0.001), and IL8 was 17.9-fold upregulated (P = 4 × 10–5; (Fig. 3C–G). In the unharmed gingiva, we did not observe
Pcorrected = 4 × 10–4). After P. gingivalis infection, IL1β was invaded amoeba after 6 h of incubation.
15.0-fold upregulated (P = 9 × 10–5; Pcorrected = 9 × 10–4), and
IL8 showed 153.5-fold upregulation (P = 3 × 10–5; Pcorrected = 3
× 10–4; Fig. 2, Appendix Fig. 4). E. gingivalis Inhibits Cell Proliferation
and Induces Cell Death of pGECs
E. gingivalis Modulates MMP13 Expression E. histolytica adherence to colonic epithelial cells induces
in Oral Fibroblast Cells apoptosis (Cornick et al. 2017). Likewise, P. gingivalis adher-
ence to GECs is associated with enhanced cell death through
To test if E. gingivalis was also capable to modulate MMP
apoptosis (Stathopoulou, Galicia, et al. 2009). We tested if
expression in gingival fibroblasts, the main cell type of the oral
E. gingivalis and P. gingivalis impaired growth of pGECs in
ECM, we screened the expression of MMP1, -2, -3, -7, -8, -9,
vitro. E. gingivalis and P. gingivalis both inhibited cell prolif-
-13, and -20 in pGFBs. MMP1, -2, and -3 showed strong
eration, whereas the mock-infected control cultures continued
expression (Cq < 25). MMP7, -8 and -13 were moderately
to grow until the end of the experiment after 72 h (Fig. 4).
expressed (25 < Cq < 35), and MMP9 and MMP20 were not
expressed (Cq > 35). After 2-h coincubation of pGFBs with
E. gingivalis, we observed 2.0-fold upregulation of MMP3 Discussion
(P = 1.8 × 10–3; Pcorrected = 0.018) and 11.2-fold upregulation of
MMP13 (P = 3 × 10–4; Pcorrected = 0.003). P. gingivalis infection Our study confirmed the reported high frequency of E. gingi-
upregulated MMP3 and MMP13 2.8-fold (P = 1 × 10–4; valis in the oral cavity. The clear abundance of E. gingivalis in
E. gingivalis Causes Oral Inflammation and Tissue Destruction 565

inflamed periodontal pockets suggests that

this ecologic niche is propitious for its sur-
vival and that the parasite might induce
changes that promote this environment.
Moreover, histochemical staining of
inflamed ex vivo biopsies from periodon-
titis visualized E. gingivalis within the
masticatory mucosa, where it was sur-
rounded by abundant neutrophils. It was
previously suggested that neutrophils are
likely unable to resolve E. gingivalis infec-
tion (Bonner et al. 2018), which will lead
to increase of neutrophil invasion, further
aggravating inflammation and tissue
destruction. Histochemical staining also
visualized moving and feeding amoeba
within various tissue layers of the mastica-
tory mucosa in an ex vivo model of amoe-
bic invasion. In this model, we showed
that the parasites were able to penetrate
the cytoplasm of live human GECs.
Internal materials moved through channels
between the host nucleus and the proto-
zoan, indicating feeding from host chro-
matin after contact between the amebic
pseudopod and host cells. The penetration
of E. gingivalis into various tissue layers
during 6 h of coincubation in our infection
model indicated efficient invasion mecha-
nisms. However, invaded amoeba were
observed only after the epithelium was
slightly punctured and not in intact tissue.
Similarly, the high frequency of asymp- Figure 3.  Entamoeba gingivalis invades, moves, and feeds within the oral mucosa. (A, B) Periodic
tomatic infection with E. histolytica indi- acid–Schiff staining reveals the presence of E. gingivalis in the inflamed gingiva of a patient with
cates that virulence to the human host severe generalized chronic periodontitis. (A) E. gingivalis colonized the inflamed oral mucosa
of a 36-year-old man with severe generalized periodontitis. (B) The amoeba (red cross) were
tissues is dispensable for Entamoeba sur- surrounded by abundant neutrophils, as distinguished by their segmented nuclei (orange asterisk;
vival, replication, and transmission. zoom of panel A). (C–G) Human live explants of uninflamed, manually injured oral mucosa
However, Entamoeba virulence probably showed moving and feeding amoeba in various tissue layers after 6 h incubation with E. gingivalis
depends on a complex interaction of para- (black arrows). The trophozoite is located below the squamous epithelium, left to the elongated
keratinized papillae (C). The amoeba penetrated the cytoplasm of a cell. Internal material
site, environmental factors, and host (possibly the nucleus) from the host cell elongated through a “channel” (red arrow) into the
immune response, where infections can be trophozoite. A hollow surrounds the amoeba, indicating that it modified cell morphology (D,
more severe in hosts with impaired zoom of panel C). The trophozoite is moving within the gingival epithelium; the moving direction
is indicated by the upward pseudopod (E). The pseudopod contacted a host cell, and its nucleus
immune function (as summarized by was half disintegrated and filled a food vacuole inside the amoeba (F). The amoeba contacted the
Marie and Petri 2014). nucleus of an endothelial cell, and streaks from another endothelial cell, possible chromatin, are
The first layer of defense that blocks observable in the hollow (G).
adherence of the amoeba to the epithelium
is the mucin barrier. Mucins are molecules
that bind to invading microorganisms, but they also act as a genome-wide expression screen that searched for differential
growth substrate and food source for commensal bacteria. transcription in the gingiva following surgical wounding iden-
Thus, some mucins are specifically upregulated by pathogens, tified MUC21 as 1 of 7 genes of the transcriptome that showed
and some are secreted constitutively. In the pathology of ≥30-fold upregulation after wounding (Wang and Tatakis
E. histolytica invasion, the first step of host defense involves 2017). The observation of strong MUC21 upregulation in
upregulation of MUC2 (Johansson et al. 2011). Our screen for response to disruption of the oral epithelium adds independent
differential expression of human mucins in response to E. gin- evidence to our finding that MUC21 plays an important role in
givalis infection in pGECs and pGFBs identified MUC21 to defense mechanisms of impaired oral barrier integrity. We note
have the strongest increase in expression. Noteworthy, a that the oral bacterium P. gingivalis did not induce MUC21
566 Journal of Dental Research 99(5)
Figure 4.  Entamoeba gingivalis and Porphyromonas gingivalis inhibit
proliferation of primary gingival epithelial cells (pGECs). Direct contact
of E. gingivalis and P. gingivalis to pGECs impaired cell proliferation as
compared with mock-infected cells. Values are presented as mean ± SD.
expression in GECs, indicating mucin upregulation to be
species-specific.
Entamoeba, bacteria, and their human host coevolved and
developed species and tissue-specific infection and defense
mechanisms. A known example is the modulation of cytokine
secretion of GECs by P. gingivalis. Generally, IL1β is expressed
early after bacterial challenge and then upregulates IL8, which
is the primary cytokine involved in the recruitment of neutro-
phils to the site of damage or infection (Eskan et al. 2008).
However, GECs challenged with live P. gingivalis mount a pri-
mary cytokine response of IL1β that is not followed by a sec-
ondary response of IL8 (Stathopoulou, Benakanakere, et al.
2009). In contrast, coculture of E. histolytica with HT-29 colon
epithelial cells for 3 h activated mRNA IL8 expression 1,000-
fold (Kim et al. 1998), and it was shown that E. histolytica com-
ponents directly induce IL8 gene expression in human colonic
epithelial cells (Yu and Chadee 1997). This is of interest in the
context of amoebiasis because IL8 stimulates the respiratory
burst, which allows the rapid release of reactive oxygen spe-
cies that are necessary to break down the ECM. In the current
study, we showed that coculture of E. gingivalis increased IL8
expression 1,900-fold, whereas coculture of P. gingivalis
increased IL8 expression 7-fold. An adaptive benefit of oppo-
site modulations of immune response by both pathogens seems
plausible. P. gingivalis is often part of the normal oral micro-
biota, and constitutive activation of IL8 in GECs would be dis-
advantageous for both bacteria and host. However, E. gingivalis
would profit from the effects of increased neutrophil invasion
and ECM breakdown. In contrast to the situation in epithelial
cells, we noticed that IL8 expression in GFBs was ~100-fold
upregulated by P. gingivalis infection. In the healthy situation,
P. gingivalis is not found in the connective tissue of the healthy
oral mucosa. Although speculative, the activated host response
in GFBs might be an advantageous immune response for the
human host to fend off tissue invasion. However, IL8 transcript
levels in GFBs were still significantly lower after P. gingivalis
infection as compared with E. gingivalis infection.
P. gingivalis adherence to GECs is associated with enhanced
cell death (Stathopoulou, Galicia, et al. 2009). Likewise,
E. histolytica adherence to colonic epithelial cells induced
apoptosis (Cornick et al. 2017). We showed that E. gingivalis
impaired cell proliferation and induced cell death of GECs to a
similar extent as P. gingivalis. Our data indicate that amoebic
infection impairs cell proliferation as severely as P. gingivalis.
We noticed that the mock-infected cells of our P. gingivalis
proliferation experiments grew slower as compared with the
mock-infected cells of the E. gingivalis experiments. We
explain this difference by the age of these cells. Whereas the
cells of the amoeba experiments were collected from patients
prior to the experiments, the cells used for bacterial stimulation
were stored 10 y at –80 °C. The long storage time might have
affected the proliferation rate.
Our screen for differential expression of human MMPs in
response to E. gingivalis infection identified MMP13 as the
most upregulated MMP, with >10-fold increase after 2 h of
stimulation. In contrast, P. gingivalis infection increased
MMP13 <4-fold. The strong activation of MMP13, which is
insignificantly expressed in healthy gingiva, indicates a role in
tissue invasion of E. gingivalis. It is of interest that the major
cytokine to induce MMP13 expression was IL1, which induced
a marked increase in MMP13 mRNA in mouse calvarial bone
cultures (Kusano et al. 1998). In primary human chondrocyte
cultures, MMP13 mRNA was also inducible by IL1 (Borden
et al. 1996). MMP13 plays a special role in wound healing, tis-
sue remodeling, cartilage degradation, bone mineralization,
and ossification (UniProtKB-P45452, MMP13_HUMAN, GO
biological process). Because of the unique role of MMP13 in
ossification and keratinocyte migration during wound healing,
a candidate gene study quantified MMP13 gene expression in
patients with untreated periodontitis and showed that MMP13
activity in gingival crevicular fluid was significantly increased
in inflamed periodontal sites (Hernandez et al. 2006).
In conclusion, the current study indicated strong virulence
potential of E. gingivalis. With the abundance at inflamed peri-
odontal sites and in the context of the known resistance of
Entamoeba species to neutrophils, antimicrobial peptides, and
antibiotics, this colonizer of the oral mucosa cannot be regarded
as an opportunistic microorganism. Instead, E. gingivalis
should be perceived as a potent microbial driver of destructive
forms of periodontitis that has remained, mostly (Bonner et al.
2014; Bonner et al. 2018), unappreciated to date. Practicing
dentists should make sure that inflamed periodontal pockets
and tissue are cleared of this protozoan.
Author Contributions
X. Bao, contributed to conception, design, data acquisition, and
analysis, drafted and critically revised the manuscript; R. Wiehe
and H. Dommisch, contributed to data acquisition, critically
revised the manuscript; A.S. Schaefer, contributed to conception,
design, data acquisition, and interpretation, drafted and critically
E. gingivalis Causes Oral Inflammation and Tissue Destruction 567
revised the manuscript. All authors gave final approval and agree
to be accountable for all aspects of the work.
Acknowledgments
This work was funded by the medical faculty of the Charité–
University Medicine Berlin and by the Chinese Scholarship
Council. We especially acknowledge the contribution of Katharina
Schildhauer with tissue acquisition and Marion von Zitzewitz for
her help in histochemistry. The authors declare no potential con-
flicts of interest with respect to the authorship and/or publication
of this article.
ORCID iDs
X. Bao https://orcid.org/0000-0003-0262-1719
H. Dommisch https://orcid.org/0000-0003-1043-2651
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