Microbial Pathogenesis 144 (2020) 104193

Contents lists available at ScienceDirect

Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Oral spirochetes: Pathogenic mechanisms in periodontal disease T

a b c d
Leila Yousefi , Hamed Ebrahimzadeh Leylabadlo , Tala Pourlak , Hosein Eslami ,
Sepehr Taghizadehb, Khudaverdi Ganbarove, Mehdi Yousefif, Asghar Tanomandg,
Bahman Yousefif, Hossein Samadi Kafilh,∗
a
Students' Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
b
Tuberculosis and Lung Diseases Research Center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
c
Connective Tissues Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
d
Dental and Periodontal Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
e
Department of Microbiology, Baku State University, Baku, Azerbaijan
f
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
g
Department of Microbiology, Maragheh University of Medical Sciences, Maragheh, Iran
h
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

ARTICLE INFO ABSTRACT

Keywords: Periodontitis is an infectious inflammatory disease resulting from infection of biofilm forming bacteria. Several
Oral spirochetes bacterial factors regulate inflammatory response and cause to tissue damage and loss of connection between
Periodontitis gingival and tooth. Since bacterial virulence factors and also host immune responses have role, understanding of
Treponema denticola periodontal disease is complex, in overall we can say that in this disease epithelium is deleted by bacteria. Oral
Oral infection
spirochetes are related to periodontitis, among them, Treponema denticola, have been associated with periodontal
diseases such as early-onset periodontitis, necrotizing ulcerative gingivitis, and acute pericoronitis. This review
will analyse mechanisms of pathogenesis of spirochetes in periodontitis. Microorganisms cause destruction of
gingival tissue by two mechanisms. In one, damage results from the direct action of bacterial enzymes and
cytotoxic products of bacterial metabolism. In the other, only bacterial components have role, and tissue de-
struction is the inevitable side effect of a subverted and exaggerated host inflammatory response to plaque
antigens.

1. Introduction sub-acute and chronic [4,5]. Periodontitis is an infectious inflammatory

Spirochetes are bacteria that have importance in medical because of
causing disease, but they have not been understood well [1]. Some of
them cause disease like Lyme disease (Borrelia burgdorferi), Leptos-
pirosis (Leptospira interrogans), Syphilis (Treponema Pallidum) in humans
[2]. Various spirochetal morphotypes can be observed in periodontal
pockets, but many of these morphotypes are as yet uncultivable. Three
organisms always present in periodontal pockets in chronic form of
periodontitis, namely Treponema denticola (T. denticola), Porphyromonas
gingivalis and Tannerella forsythiaand [3]. One of the most studied oral
spirochetes, T. denticola, possesses the features needed for adherence,
invasion, and damage of the periodontal tissues. Spirochetes are pre-
valent in another oral infections like pericoronitis in its degree of acute,
disease resulting from infection of biofilm forming bacteria, the site of
infection is gingivae and underlying connective tissue and bone around
the tooth [6]. In this disease, inflammatory response, that has caused
plaque around the teeth, leads to destruction of collagen attachment
between tooth and bone and in these conditions bacteria have im-
portant role [6]. Chronic forms of disease has prevalence in both de-
veloped and developing countries, between 10% and 15% of the world
population are infected [7,8]. On the other hand, periodontitis influ-
ences on risk of systemic diseases such as diabetes, artherovascular,
rheumatoid arthritis and some kinds of cancers [3,9,10]. So, this review
will analyse mechanisms of pathogenesis of oral spirochetes in peri-
odontitis, therefore we can produce significant treatment for these
diseases. Also, it is tried to design some figures for better

Corresponding author. Drug Applied Research Center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
∗

E-mail addresses: yousefileila94@gmail.com (L. Yousefi), hamedebr7@gmail.com (H.E. Leylabadlo), t.pourlak@gmail.com (T. Pourlak),
eslamihosein56@yahoo.com (H. Eslami), sepehrtaghizadeh10@gmail.com (S. Taghizadeh), khuda1949@mail.ru (K. Ganbarov),
yousefime@tbzmed.ac.ir (M. Yousefi), tanomanda@yahoo.com (A. Tanomand), yousefib@tbzmed.ac.ir (B. Yousefi), Kafilhs@tbzmed.ac.ir,
kafilhs@tbzmed.ac.ir (H.S. Kafil).

https://doi.org/10.1016/j.micpath.2020.104193
Received 21 February 2020; Accepted 7 April 2020
Available online 15 April 2020
0882-4010/ © 2020 Elsevier Ltd. All rights reserved.
L. Yousefi, et al. Microbial Pathogenesis 144 (2020) 104193

Table 1 this disease is result of deleting epithelium due to bacteria presentation

Used methods for detecting some of motility genes.
Motility genes Method
flgE, tdpA, prtB PFGE, DNA hybridization
flgE Electronmicroscopy
flgE Southern blot hybridization was
conducted with the ECL system
flgB, flgC, fliE, FliF, fliG, RT-PCR
fliH, FliI, fliJ
dmcA inverse PCR Western blot,
fliG RT-PCR
FlaB1, FlaB2, FlaB3 nLC-MS/MS analysis
understanding. (see Tables 1 and 2, Fig. 1)
2. Oral spirochetes
Presumably rotation of the flagella have important role in its
Treponems that live in oral cavity are in forms of small, medium and
large. Four species of oral spirochetes has been identified: T. denticola,
T. socranskii, T. vincentii, and T. pectinovorum [11]. In other studies have
been reported another four species in subgingival, T.maltophilum,
T.medium, T.amylovorum and T.lecithinolyticum [12]. Oral spirochetes
are related to periodontitis [13], but T. denticola, particularly is asso-
ciated with prevalence and severity of periodontitis [14]. T.maltophilum
has been found in the root canals it is tiny and has motility due to
possessing two periplasmic flagella. This organism has been mostly
isolated from individuals with severe periodontitis [15,16]. Also pre-
sence of this bacterium has been reported in endodontic infections. For
the first time, presence of T. lecithinolyticum, T. amylovorum and T.
medium has been reported in primary infections of root canal and pre-
sence of T. maltophilum and T. lecithinolyticum have been reported from
subgingival plaque and periodontal diseases [17].
2.1. Mechanism of infection
When number of bacteria increase in gingival crevice, create con-
ditions that are appropriate for growth of Gram-negative anaerobic
organisms, and presumably help initiating of periodontal disease. Many
of microflora are involved in periodontal disease and proteolytic Gram-
negative anaerobes compose most of them [18]. Several bacterial fac-
tors regulate inflammatory response and cause to tissue damage and
loss of connection between gingival and tooth [19]. Since bacterial
virulence factors and also host immune responses have role, under-
standing of periodontal disease is complex, in overall we can say that
[16,20]. Oral spirochetes, especially T. denticola have been associated
References with severe periodontal disease including acute necrotizing ulcerative
gingivitis and early onset periodontitis [21]. Developments in detection
[101] techniques by nucleic acid have helped researchers to investigate the
[102]
complex microflora of disease. By using hybridization technique was
[102]
identified that three organisms, Porphyromonas gingivali, Bacteroides
[103] forsythus, and T. denticola, are most related to periodontal disease [22].
Amplified 16SrRNA of gingival plaque from a patient with severe per-
[104] iodontitis showed that Oral Spirochetal population is complex [23], in a
[105]
research twenty-three species were obtained and belonged to eight
[1]
groups. From these groups only T.denticola and T. vincentii were de-
tected. Since T. denticola simply is isolated from clinical samples, most
of biochemical, clinical and molecular researches have concentrated on
this species. A study has focused on components of outer membrane
that has role in adherence, cytotoxic and proteolyticactivity [24]. At
first the gingival crevicular fluid (GCF) in subgingival crevice makes
alkaline or neutral environment because of nitrogenous compounds like
peptides and amino acids and this condition favours growth of asac-
charolytic and proteolytic and anaerobic organisms [25]. These kind of
bacteria degrade nitrogenous compounds by their proteases for serving
as substrates in metabolism, in result, these enzymes cause im-
munoreactions and inflammation [25]. Microorganisms cause destruc-
tion of gingival tissue by two mechanisms. In one, damage results from
the direct action of bacterial enzymes and cytotoxic products of bac-
terial metabolism [26]. In the other, only bacterial components in-
directly cause, and tissue destruction is the inevitable side effect of a
subverted and exaggerated host inflammatory response to plaque an-
tigens. Both innate and adaptive immune system are involved in re-
sponse to the invasion [27]. Molecular analysis of oral spirochetes
virulence has identified virulence factors: motility, chemotaxis, ad-
herence, cytotoxicity, iron acquisition, chymotrypsin-like protease ac-
tivity, peptidase enzymes, hemolytic activity, phospholipase C, toxic
metabolites, antibiotic resistance and plasmids. Some of genes and
proteins influence in these factors [28,29]. T. denticola has a lot of
virulence characteristics, as follows: adhesins, pore forming toxins,
proteases, proteins for invading to immune (resistance to defensins),
inhibition, major outer sheath protein (Msp), for activation of immune
(Msp, peptidoglycan, lipoprotein) and transporting metal (haemin
binding protein, lactoferrin binding protein) [3]. Many Gram-negative
organisms also, secrete virulence factors by outer membrane vesicles
(OMVs). For the first time has been reported that these OMVs weaken
epithelial cell functions [30]. OMVs by their small size go into epithelial
layers and might be destructive to lower tissue. These also hold true for
T. denticola OMVs and furthermore migrate through endothelial layers,

Table 2
Summary of virulence factors of T. denticola.
Virulence factors Activity References

Motility and Chemotaxis dmcA gene encodes homologue methyl-accepting chemotaxis proteins [106,107]
T. denticola preplasmic flagella in addition to motility has influence on helical morphology of bacteria
Adhesins A53 kDa weight protein Binding to fibronectin [5]
OppA A 72 KDa protein that binds to fibronectin [5,108]
Flagellin Binding to fibronectin [5]
Lectin-like adhesion Has affinity to mannose and galactose on fibroblast surface [5]
Factor present in host serum Binding the bacterium to fibroblasts [5]
Msp Mediates the adhesion to extracellular matrix (ECM) components [56]
Transporting Metal Haemin binding protein Iron acqusition [85,109]
Lactoferrin binding protein Iron acqusition [85,109]
Hemolysin Lyses erythrocytes for causing heme compounds [85]
Enzyme Dentilisin Degrades fibronectin, laminin and type IV collagen and serum proteins including fibrinogen, transferrin, [32]
IgA, IgG and α 1-antitrypsin
PLC Hydrolyzing membrane phospholipids [110,111]
Serine protease Has chymotrypsin-like activity [24]
Phospholipase C Hydrolyzing phosphatidylcholine [110]
Trypsin-like Hydrolysis of benzoyl-DL- arginine-2-naphthylamide (BANA) [101]
Chymotrypsinlike Degrades immunoglobulin A, immunoglobulin G, α1-antitrypsin, BSA, transferrin, gelatin and fibrinogen [79]

2
L. Yousefi, et al.
showing that OMVs presumably penetrate blood vessels [31]. This
might be in relation with the recent subject that periodontitis may in-
fluences on systemic diseases [32,33]. We summarize some related
mechanism action and metabolites used by spirochetes that lead to
elucidation of spirochete virulence properties including motility, che-
motaxis, adherence to host cells, colonization, Lipopolysaccharides,
toxins, enzymes and transporting metals. The mechanism action of as-
sociated with periodontitis as shown in Figure- 1.
2.1.1. Motility and chemotaxis
Motility and chemotaxis is a kind of virulence factor and their role
in spirochetes is important. When T. denticola is present in a tissue,
intracellular contact destroys [34]. Oral spirochetes by mechanisms like
other spirochetes invade to tissue by immigrating between intracellular
[35]. Motility systems of oral spirochetes is suited for high viscosity
environments, and motility of T. denticola is depended on viscosity
[36,37]. There are some genes that are involved in motility and che-
motaxis of T. denticola, for example, the fliG, fliF, fliH, flgB, flgC and fliE
genes although these genes present also in other bacteria [38,39], also,
dmcA gene encodes homologue methyl-accepting chemotaxis proteins
and is located in upstream of prtB gene which encodes chymotrypsin
like activity in T. denticola [40,41]. T. denticola preplasmic flagella in
addition to motility has influence on helical morphology of bacteria
[42]. Periodontitis can be predicted by the flagella [3]. Table- 1 shows
used methods for detecting some of the motility genes and some of
these genes that are involved in the periodontitis.
2.1.2. Adherence to host cells
It has been established that adherence of bacteria to host cells is
initial step in development of infection. In most of bacteria this is
3
Microbial Pathogenesis 144 (2020) 104193
Fig. 1. Schematic view of mechanisms ac-
tion of oral spirochetes in periodontitis.
Adherence of bacteria to host cells is initial
step in development of infection. T. denti-
cola, particularly is associated with pre-
valence and severity of periodontitis, binds
to epithelial cells, fibroblasts and ery-
throcytes in different sites. OppA and MSP
serve as an adhesion in it's surface. MSP
causes secretion of proteinase from PMLs
and regulates pro-inflammatory cytokines
in various cells and in the outer membrane
combines with CTLP. Porphyromonas gingi-
valis and Fusobacterium spp aggregate with
T. denticola in order to facilitate coloniza-
tion. PMNs, by releasing free radicals kill
ingested microorganisms. In PMNs oxygen
consumption increases with happening re-
spiratory burst. Nearly 90% of that oxygen
use for producing superoxide free radicals
(O2ˉ) that is origin of H202. Phagocytes de-
stroy bacteria by H202 and its metabolites.
TNF-α and IL-1 in site of infection increase
and have immunomodulatory impacts on
PMNs, so they could prevent from chemo-
taxis of PMNs and lower FMLP receptors. In
Periodontitis lesions plasma cells are pre-
dominant among leukocytes. MSP: major
surface protein, CTLP: chymotrypsin-like
protein, PMNs: polymorphonuclear leuko-
cytes, H202: hydrogen peroxide, TNF-α:
tumor necrosis factor alpha, IL-1: inter-
leukin 1.
specific and requires receptors on the surface of the host cells [43].
Other non-specific factors like charge and hydrophobic attractions have
also important role in adherence of bacteria to host cells. Binding of
bacteria to extracellular matrix molecules, like collagen, fibronectin,
and laminin, also are of importance to infective process [44]. Some
bacteria use specialized structures like fimbriae for binding to host
surface or other bacteria, but spirochetes attachment has not been un-
derstood [45]. Although, attachment of T. denticola to specific host cells
is important for causing disease, but we have little information about its
mechanism [46]. Studies have shown that attachment to cultured cells
is a characteristic that is seen just in pathogenic organisms, since ad-
herence of the Reiter treponeme and T. denticola to fibroblast cell lines
and testicular cells was not seen [47]. T. denticola attach to fibroblasts
and epithelial cells, and mucopolysaccharides such as hyaluronidase is
receptor on epithelial cells. T. denticola and T. vincentii have enzymatic
activity that is essential in invading of the organism to gingival epi-
thelium, also Treponemes could attach to matrix proteins like fi-
bronectin in pellicle [48]. T. denticola binds to hydroxyapatite beads
coated with lysosome or fluids. T. denticola also binds to laminin. A
four-amino-acids site including arg-gly-asp-ser on fibronectin is binding
region of spirochetes. Binding site for laminin on T.denticola is the same
for fibronectin and this leads to steric hindrance [49].
2.1.3. Adhesin proteins
In addition to enzymatic activities, toxic effects of peptidoglycan are
important in binding of the organism to eukaryotic cells [50]. T. den-
ticola has a 56–58 kDa weight protein on its surface and at least four
various proteins have been involved as adhesins of T. denticola [39].
Three proteins in T. denticola (53 and 72 kDa and flagellin) have been
identified for binding to fibronectin [51]. T. denticola binds to
L. Yousefi, et al.
fibroblasts by two ways: lectin-like adhesion on its surface that has
affinity to mannose and galactose on fibroblast surface and a factor
present in host serum binding the bacterium to fibroblasts [52].
2.1.3.1. OppA protein. OppA protein is a 70 or 72 KDa protein that is
located in T.denticola surface and serves as an adhesion. This protein
has a role in interaction between the organism and host tissue in
periodontitis [53]. An investigation has found that it is involved in
binding the organism to soluble proteins not to receptors on host cells
[54]. OppA was present in representative strains of T. denticola and in
T.vincentii but was not detected in T. pectinovorum or T.socranskii [54].
Binding of soluble host proteins by OppA may be important both for
spirochete-host interactions in the subgingival environment and for
uptake of peptide nutrients.
2.1.3.2. Major surface protein. Major surface protein (MSP) is involved
in interaction between the organism the host cells, and so play a role in
periodontal disease [55]. MSP control the attachment and also
cytopathic effects of the bacterium on host cells and is like porins
that has pore-forming activity [56]. In cell membrane of the bacterium,
this protein has oligomeric form and is homologous to Tpr proteins in T.
pallidum subsp. Pallidum, which reacts with antibody during syphilis.
Has been reported that MSP can mediate the adhesion to extracellular
matrix (ECM) components that causes secretion of proteinase from
polymorphonuclear leukocytes (PMLs) and regulates pro-inflammatory
cytokines in various cells. In the outer membrane of the bacterium, MSP
combines with chymotrypsin-like protein (CTLP) and this complex has
molecular mass of about 150 kDa [56]. Sequencing of Msp of T.
maltophilum and T. lecithinolyticum has determined that probably it has
role in adherence as well as Msp in T. denticola [57]. Msp and CTLP are
important in nutrient acquisition in these bacteria. Also, Msp prevent
the cell from degradation by presenting CTLP on its surface [58].
2.1.4. Colonization
Adherence plays an important role in cytopathology of oral spir-
ochetes. T. denticola binds to hydroxyapatite covered with saliva, serum
or crevicular fluid. This shows that Treponema can colonizes dental
hard surfaces like gingival tissue [59]. T. denticola binds to epithelial
cells, fibroblasts and erythrocytes in different sites [60]. Surface and
secreted proteins of T. denticola are necessary in colonization and cy-
totoxicity and host immunological responses in periodontal diseases
[61]. These bacteria have interaction with other bacteria especially
Fusobacterium spp. Porphyromonas gingivalis and Fusobacterium spp ag-
gregate with T. denticola in order to facilitate colonization [62,63].
Coaggregating of P. gingivalis and T. denticola shows that they have
symbiotic life. When they are cultured with each other, P. gingivalis
presumably produces glycine and thiamin for T. denticola, this has been
understood from expression of their genes in culturing them alone [58].
2.1.5. Lipopolysaccharides
Lipopolysaccharides (LPS), also known as lipoglycans and en-
dotoxins, are large molecules consisting of a lipid and a polysaccharide
composed of O-antigen, outer core and inner core joined by a covalent
bond; they are found in the outer membrane of Gram-negative bacteria
[64]. Lipid A is a portion of LPS in Gram-negative organisms that in-
duces inflammatory response. LPS activates innate immune system by
inducing TLR-4, a surface protein for identifying bacterial products
[65]. As LPS comes into blood circulation, it causes several biological
reactions such as fever, shock, intravascular coagulation. Concentration
of LPS in serum is increased in periodontal diseases and causes systemic
complications [66]. LPS has a notable effect on most of cells in peri-
odontal tissue compromising macrophages, lymphocytes, fibroblasts
and osteoclasts. Most of the pathogenesis of periodontal diseases is
associated to biological activity of LPS [67]. LPS binds to its receptor,
cluster of differentiation-14 (CD14) in membrane of monocytes/mac-
rophages, via LPS binding protein (LBP) [68].
4
Microbial Pathogenesis 144 (2020) 104193
By activation CD14 through TLR4-dependent pathway, monocytes
and endothelial cells are activated and release pro-inflammatory mo-
lecules including Interleukin 1 (IL-1), tumor necrosis factor (TNF),
Prostaglandin E2 (PGE2) and IL-6. These molecules produce in-
flammatory mediators such as histamine and prostaglandins [69].
Studies have shown that in periodontal diseases level of CD14 is in-
creased due to presence of LPS in blood circulation [70].
2.1.6. Toxins and enzymes
Spirochetes contain enzymes or substances that directly damage to
tissue. Oral spirochetes in periodontal diseases release toxic substances
and enzymes that are important in development of periodontitis. These
products damage to tissue by activating immune responses [71]. This
organisms have different proteolytic enzymes like collagenolytic en-
zyme. T. denticola, T. vincentii, and the unspeciated strain, contain
collagenolytic enzymes. T. denticola contains a fibrinolytic enzyme and
peptidases for hydrolyzing synthetic trypsin substrates [72]. Surface
and secreted enzymes of T. denticola are as virulence factors. One of
these is serine protease that has chymotrypsin-like activity [73].
Synthesis and secretion of phosphatidylcholine-hydrolyzing phos-
pholipase C (PLC) by T. denticola, T. vincentii, and T. phagedenis has been
investigated. The assay for PLC activity is based on the hydrolysis of p-
nitrophenylphosphorylcholine (NPPC) with producing the chromogen,
p-nitrophenol (NP) [74]. T. denticola contains a trypsin-like enzyme that
can be identified by the hydrolysis of benzoyl-DL-arginine-2-naphthy-
lamide (BANA). This enzyme could also be identified in subgingival
plaque and was associated to the plaque levels and proportions of
spirochetes [75]. A, BANA-positive plaque shows about 30% spir-
ochetes in subgingival plaque. Ability of subgingival plaque for hy-
drolyzing BANA is a reliable sign for presence of high proportions of
spirochetes and could be used clinically to identify those sites and in-
dividuals who require treatment for reduction of spirochetal over-
growth [75]. T. denticola contains enzymes that can degrade peptides.
Two of these enzymes catalyze the hydrolysis of substrates that have
arginine [76]. Two other peptidases in T. denticola have tendency for
catalyzing proline. This bacterium also is active against phenylalanine
containing substrates and these enzymes aren't very active against na-
tive proteins [77]. The organism releases real protease that degrades
elastin, fibrin, keratin and collagen in basement membrane. Their re-
sults were that T. vincentii has low activity for enzymes but T. denticola
has strong activity for N-a-benzoyl-L-arginine-p-nitroanilide (BAPNA)
and succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide
(SAAPNA) [78]. They degraded N-acetyl-tyrosine-p-nitroanilide very
little and did not degrade other synthetic substrates. The enzyme de-
graded azocasein and azocoll but not rhemazo brilliant blue elastin
[79]. The chymotrypsinlike enzyme is a real protease because it de-
grades different proteins like immunoglobulin A, immunoglobulin G,
α1-antitrypsin, BSA, transferrin, gelatin and fibrinogen, and it cleavages
polypeptides in one or several sites. T. denticola has also been reported
to has enzymes for degrading chondroitin sulfate and hyaluronic acid
that are kinds of glycosaminoglycans [79]. Dentilisin, another enzyme,
has a role in degradation of miscellaneous proteins like fibronectin,
laminin and type IV collagen and also serum proteins including fi-
brinogen, transferrin, Immunoglobulin A (IgA), IgG and α 1-antitrypsin.
Also, this proteinase contributes to adhesion of organism to epithelial
cells. Dentilisin is the most important factor in penetrating of T. denti-
cola to tissue. It has been shown that dentilisin activates host pro-
collagennases and so, host enzymes presumably cooperate with T.
denticola in tissue degradation [80].
2.1.7. Transporting metals
Most bacteria for acquiring iron release siderophores, that get iron
from host proteins and after interaction with receptors on bacterial
membranes transfer iron [81]. Also, some organisms have outer mem-
brane proteins that are induced during low iron, bind to transferrin and
lactoferrin for getting iron. At last, some gram negative bacteria provide
L. Yousefi, et al.
their required iron from compounds containing heme [82]. T.denticola
does not secret siderophores, it can attach lactoferrin and haemin and
also lyses erythrocytes by hemolysin for causing heme compounds [83].
T.denticola has two haemin binding proteins, a 47-kDa from strain
ATCC 35405 [84] and a 44-kDa from strains GM-1, MS-25, ATCC
33520, and ATCC 33404, but their role in iron acquisition have not
been confirmed and their mechanisms have not been determined [85].
Table- 2 shows summary of virulence factors of T. denticola.
2.2. Host tissue responses and immunological interactions
Toxic substances and enzymes released from oral spirochetes da-
mage to tissue by activating immune responses [86]. Spirochetes by
increasing their number cause inflammation. They have endotoxin and
this endotoxin is present in subgingival plaque. Their small size and
motility are factors of invading to periodontal tissues and at this site
release their endotoxin near to fibroblasts and epithelial cells [87]. The
most important host defense cells are polymorphonuclear leukocytes
(PMNs), so their functions like chemotaxis, enzyme secretion and
phagocytosis have been investigated. Obviously, phagocytes after in-
teracting with bacteria by releasing free radicals kill ingested micro-
organisms [88]. In PMNs oxygen consumption increases with hap-
pening respiratory burst. Nearly 90% of that oxygen use for producing
superoxide free radicals (O2ˉ). O2 alone could not be a bactericidal
agent, O2ˉ is origin of hydrogen peroxide (H202). Phagocytes destroy
bacteria by H202 and its metabolites [89]. T. denticola and T. vincentii
prevent from polymorphonuclear Leukocyte (PMNL) function invitro
and thus could not be phagocyted [90]. Simultaneously, lysosomal
granules aren't degranulated and suggest that fusion of lysosomes with
phagosomes might be limited. Degranulation decreases by zymosan
when PMNs accost with spirochetes [91].
In various forms of periodontitis the chemotactic characteristic of
crevicular fluid polymorphonuclear neutrophils (CF-PMN) has been
investigated. Most of researches have showed that neutrophils reduce
their phagocytotic and chemotactic behaviour in early onset period-
ontitis [92]. It is not fully obvious whether chemotactic disorders
happen in early or late stages of infection. Cytokines function has been
surveyed in periodontitis patients. Specially, TNF-a and IL-1 in site of
infection increase. These cytokines have immunomodulatory impacts
on immune cells and also nonimmune cells consist of PMNs, so they
could prevent from chemotaxis of PMNs and lower N-formyl-methionyl
peptides (fMLP) receptors [92]. Immunoregulatory mechanisms
monitor Leukocytes and their products, and severity of the periodontitis
depends on inflammatory response and biofilm. In Periodontitis lesions
there are a lot of leukocytes and among them plasma cells are pre-
dominant cells- accompanied by lymphocytes form almost 75–80% of
inflammatory cells [93].
B cells are a part of adaptive immune system. It's mechanism include
converting into plasma cells and generating antibodies. B cells have
immuno-regulatory importance consist of direct and indirect influence
on other cells by antigen presentation and releasing cytokines [94].
Plasma cells generate cytokines like TNF-α, IL-6, IL-10 and TGF-β. TNF-
α through stimulating matrix metalloproteinases (MMPs) increases
extracellular matrix, but TGF-β reduce production of these MMPs [95].
Plasma cells located near blood vessels induce vascular endothelial
growth factor, that activate MMPs. So, plasma cells are a lot in the
inflammation site and MMP-mediated tissue degradation is obvious.
MMP-mediated tissue destruction have been studied in oral diseases.
So, MMP is active in gingivitis and is related to periodontitis [96].
MMPs are released by neutrophils and fibroblasts and support de-
gradation of collagen. MMP-8 and MMP-13 (collagenases 2 and 3) were
also produced by macrophage-like cells and plasma cells [96]. PLC
secreted by T. denticola, T. vincentii, and T. phagedenis has ability of
hydrolyzing membrane phospholipids and this results in destruction of
5
Microbial Pathogenesis 144 (2020) 104193
crevicular epithelial cells or producing of arachidonic acid that is used
in the production of prostaglandins and leukotrienes. The eicosanoids
are agent of inflammation, destroy of tooth attachment and bone lose
[97].
T. denticola can cancel oxidative burst of neutrophils in vitro. Some
strains of T. denticola prevented lymphocyte that was acting against
phytohemagglutinin, phorbol myristate acetate, concanavalin A and
antigen SKSD [98]. The inhibition was dependent on the presence of
monocytes and it is possible to reverse by using catalase and in-
domethacin, showing that hydrogen peroxide and prostaglandins had
role as mediators. T. denticola prevent from proliferation of fibroblast by
a cell-bound factor with about 50000 molecular weight [99]. Some
strains of T. denticola suppress growing of fibroblast without affecting
on cell viability. If these bacteria do in vivo like in vitro, they could
change fibroblast proliferation and involve in normal tissue home-
ostasis. These findings show that endotoxin do not interfere in inhibi-
tion [7]. T. denticola shows keratinolytic activity in tissue culture by
binding to epithelial cells. If any of these enzymes and factors release in
subgingival environment, can have contact with host tissue and cells
and this contact is increased by penetrating the organisms into epi-
thelial spaces and inside connective tissue and this ability, especially for
T. denticola, causes to prevent PML function. Invading to host causes
immune system to cross with spirochetal antigens [100]. Has been re-
ported that in individual with moderate periodontitis there is high titers
of antibodies against spirochetes but about individuals with severe
disease have not been reported. In a study have been found that both
healthy and periodontitis patients have high titers of antibody against
T. denticola, T. vincentii, and T. phagedenis [99]. In another survey, same
people with the most severe periodontitis showed a lower antibody ti-
ters to T. denticola and T. socranskii compared with people with less
periodontal morbidity and healthy controls. These findings show that
spirochetes are immunogenicity, but did not use from antigens that
identify diagnostic antibody response. Alternately, in presence of high
antigens, these organisms cause immune suppression and thereby can
escape from host immune system [99].
3. Conclusion
Spirochetes specially T. denticola play notable role in periodontal
diseases and also are prevalent in other oral infections. Periodontitis
influences on risk of systemic diseases such as artherovascular, rheu-
matoid arthritis and some kinds of cancers. So this review has analysed
mechanisms of pathogenesis of oral spirochetes in periodontitis and
oral infections, therefore it will help us to significantly treat these
diseases. The review of the included articles has showed that oral
spirochetes established infections and periodontitis by its virulence
factors. There is a need for more studies investigating the known me-
chanisms of pathogenesis in oral infections and periodontitis associated
with oral spirochetes. In addition, this study has not shown all of genes
encoding virulence factors of related bacteria. Future reviews might
provide further insight into the understanding of the disease, as well as
new possible mechanisms to eliminate oral spirochetes in primary in-
fections.
Declaration of competing interest
None to declare.
Acknowledgment
This study was supported by Faculty of Medicine, Tabriz University
of Medical Sciences with reference number 63656 and ethic committee
approved with registration number IR.TBZMED.REC.1398.1003.
L. Yousefi, et al.
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