Periodontology 2000, Vol. 0, 2017, 1–12 © 2017 John Wiley & Sons A/S.
ley & Sons A/S. Published by John Wiley & Sons Ltd
Printed in Singapore. All rights reserved
Bacterial modulators of bone
remodeling in the periodontal
pocket
BRIAN HENDERSON & FRANK KAISER
Bone is one of the most fascinating of natural
composite materials, consisting of a largely collage-
nous organic matrix in which hydroxyapatite crystals
are embedded, giving bone its rigidity and strength,
allowing it to be sculpted into its complex shapes and
enabling it to be superbly sensitive to stress (63). The
mechanical properties of bone are, in turn, controlled
by its major cellular elements. The major cell popula-
tion in bone is one that is only now coming to the
attention of the biomedical community. The osteo-
cyte comprises some 95% of the cells of bone and is,
in appearance, a dendritic-like cell whose processes
fill the canaliculi of bone. Osteocytes are responsible
for bone being able to recognize, and respond to,
mechanical stress and strain (73). It is also being rec-
ognized that osteocytes are key bone-regulatory cells
as a result of: (i) their interactions with other bone
cells, principally osteoclasts; and (ii) their synthesis
and secretion of key modulatory proteins (cytokine/
endocrine-like molecules) that influence bone
remodeling and also phosphate kinetics in the
circulation (68).
The osteocyte originates from the osteoblast lin-
eages of bone, with the osteoblast being the synthetic
cell of bone responsible for generating the bone
matrix (65). The osteoblast is a mesenchymal cell, as
must be the osteocyte. The third major bone-modu-
lating cell population – the osteoclast – arises from
the myeloid cell lineage and is one of only two types
of multinucleate cell in mammals. The major function
of the osteoclast is to remove bone matrix and its
functions intermesh with those of the osteoblast and
the osteocyte (51).
Bone remodeling is the normal result of the inter-
meshing of these three cell populations with a range
of cell-surface and secreted mediators (plus
PERIODONTOLOGY 2000
mechanical stress signals) to ensure that the constant
fatigue fracturing of bone, which occurs as a result of
the stresses and strains of living on a 1G planet (or
through other mechanical forces, such as mastica-
tion), is constrained and that bone maintains its opti-
mal mechanical strength (37). Unfortunately, this
natural process often goes wrong and creates local
and systemic pathology in the form of diseases, such
as osteoporosis, rheumatoid arthritis and, of course,
periodontitis. A paradigm shift in thinking about the
skeleton, and the cellular basis of its turnover in
health and disease, has given rise to a new discipline
over the past decade. This is the discipline of
osteoimmunology, which is based on growing evi-
dence for a role of both innate and adaptive immune
mechanisms in controlling normal and pathological
skeletal turnover and the cells responsible for bone
remodeling. Osteoimmunological mechanisms have
now been suggested to underpin the bone pathology
in osteoporosis (17), rheumatoid arthritis (70) and
periodontitis (29). This new paradigm, in the context
of periodontitis, also has to encompass the role of
bacteria in the bone loss of this disease. This is the
point where there starts to be a ‘blurring’ of the
pathogenic mechanisms in skeletal diseases, with the
recent finding that the major periodontopathogenic
bacterium, Porphyromonas gingivalis, may play a
major role in the pathogenesis of rheumatoid arthritis
(14). This novel finding will be returned to at a later
stage in this review.
Bacteria and periodontitis
With our new understanding of how enormously col-
onized are the epithelial surfaces of the skin and the
1
Henderson & Kaiser
mucosal membranes of the oral cavity, gut and
urogenital tract, comes a newly developing paradigm
of the role played by the bacterial microbiota in both
health and disease. This developing paradigm has lar-
gely focused on the gut, with the evidence suggesting
that the colonic microbiota plays a major role in con-
trolling the energetics of the mammal (79) and from
this context the colonic microbiota is considered a
bonus for mammals. Indeed, in spite of the huge
numbers of diverse bacteria in the colon, the amount
of tissue pathology thought to be caused by these
bacteria is actually rather small. This contrasts with
the much larger association between tissue pathology
and bacteria in bacterial vaginosis (54) and in peri-
odontitis. Indeed, a recent National Health and Nutri-
tion Examination Study (NHANES) estimated that
47% of the population of the USA suffers from peri-
odontitis (12). This is a truly staggering amount of
pathology, particularly as periodontitis is a chronic
disease state.
This raises the obvious question – why do such
large proportions of the world’s population respond
to their oral bacteria and develop a chronic inflamma-
tory disease of the gums that also causes destruction
of the periodontal ligament and alveolar bone? The
obvious answer is that the periodontium is the major
weak spot in the protective mucosa of the body, as a
result of the interruption of this mucosal layer by the
teeth. In other tissues, the argument would continue,
the surface mucosa/epithelium, on which the micro-
biota is being supported, prevents the ingress of bac-
teria into tissues and the consequent induction of
inflammation. A counter to this argument is the
recent report that in normal human skin, members of
the skin microbiota can breach the basement mem-
brane of the skin and are found within the dermis
and even within the adipose tissues of the subdermal
layer (52). If bacteria can enter the submucosal space
in all mucosal sites, and interact with the submucosal
cells of the host, it counters the specific argument for
the ‘weakness’ of the gingival mucosal barrier (caused
by protrusion of the teeth) and also changes the pos-
sible hypotheses that can be forwarded to explain
how bone loss occurs in periodontitis.
Mechanisms which explain
bacterially-induced bone
breakdown in periodontitis
It is surprising that, even at the time of writing, it is
still not completely clear what mechanism, or mecha-
nisms, drive the bone loss in periodontitis. As this
2
disease is, it is believed, caused by a response to
members of the oral microbiota, it might be expected
that bacteria would have a key role to play in driving
bone destruction. However, it is not known if these
bacteria cause bone breakdown because they directly
or indirectly induce inflammation, or because they
release factors that can directly interact with bone or,
indeed, if they actually are able to interact with bone
cells. The idea that bacteria can induce bone destruc-
tion because they are able to induce gingival inflam-
mation has to be looked at in more detail. Clearly,
gingivitis does not induce alveolar bone resorption, so
we may need to look more deeply at the relationship
between local inflammation and bone loss. How
much more deeply can be seen from experimental
arthritis studies in which it is well known that it is pos-
sible to inhibit inflammation of the synovial lining of
the joint (a tissue that can be considered equivalent to
the gingiva) without inhibiting bone resorption and,
more recently, it has been shown that it is possible to
inhibit bone resorption in the joint without inhibiting
synovitis. This has been shown with osteoprotegerin,
the natural inhibitor of the cytokine, RANKL, which is
the major inducer of osteoclast formation (35), and by
use of the Mycobacterium tuberculosis cell stress pro-
tein, chaperonin 60.1 (86). So, the precise relationship
between local inflammation and bone destruction is
still not mapped. The next three sections of this
review cover possible mechanisms linking periodontal
bacteria with alveolar bone loss.
Indirect actions of bacteria inducing
bone destruction
What possible mechanisms can link oral bacteria with
alveolar bone resorption? The role of immune cells in
controlling bone remodeling has already been
alluded to, and it is now clear that most cellular com-
ponents of the innate and, particularly, the adaptive,
immune response (e.g. T-lymphocytes) (55), have
marked influence on bone cells and can produce
inappropriate bone remodeling. As such, the genera-
tion of chronic inflammation in the gingiva, in
response to members of the subgingival microbiota,
may attract and activate appropriate T-cell popula-
tions (particularly T-helper 1 and T-helper 17 cells)
and other leukocytes that are able to drive local alveo-
lar bone destruction. The role of the T-helper 17 lym-
phocyte (36) in periodontitis is probably to link up
bacterial signaling with bone resorption, and it is
interesting that this lymphocyte subpopulation is also
implicated in the destruction of bone observed in
rheumatoid arthritis (40). It would be assumed that

Fig. 1. Cytokines activating osteoclastogenesis in rheuma-
toid arthritis. Tumor necrosis factor, interleukin-1, inter-
leukin-6 and interleukin-17 up-regulate expression of
RANKL in osteoblasts and synovial fibroblasts. RANKL
mediates differentiation, survival and activation of osteo-
clasts. Tumor necrosis factor, produced by fibroblasts and
macrophages, promotes differentiation and survival of
osteoclasts. Interleukin-1 supports differentiation, survival
and activation of osteoclasts. Interleukin-6 and interleukin-
17 promote osteoclastogenesis indirectly. Interleukin-6 is
largely produced by fibroblasts and macrophages; it
enhances the expression of RANKL and contributes to the
inflammation and associated tissue destruction are
caused by the release, by the subgingival bacteria
within the periodontal pocket, of one or more of the
pathogen-associated molecular pattern molecules
that are the evolutionarily stable components of bac-
teria which are recognized by the growing number of
host pattern-recognition receptors found on immune
cells (34) and on the epithelial cells supporting the
microbiotas (57). Binding of pathogen-associated
molecular patterns to their pattern-recognition
receptors, such as the toll-like receptors, results in the
production of a range of proinflammatory cytokines,
such as the early response cytokines interleukin-1
beta and tumor necrosis factor alpha, which are
potent osteolytic agents and could drive bone resorp-
tion. Indeed, it was Dewhirst & Stashenko who
revealed the osteolytic activity of interleukin-1 (11).
Since this time, a growing number of nonspecific and
specific cytokines have been found which can stimu-
late the formation/activation of osteoclasts and pro-
mote bone resorption (Fig. 1). It should be noted that
most of our information on the cytokine biology of
Bacterial modulators of bone remodeling in periodontal pocket
induction of T-helper 17 cells. T-helper 17 cells secrete
interleukin-17, but a main source of synovial interleukin-17
is probably mast cells. Interleukin-17 induces the expres-
sion of RANKL in osteoblasts and fibroblasts and enhances
secretion of proinflammatory cytokines by macrophages.
Macrophage colony-stimulating factor and interleukin-34
promote differentiation and activation of osteoclasts;
interleukin-33 supports osteoclast differentiation. IL,
interleukin; M-CSF, macrophage colony-stimulating factor;
TGF-b, transforming growth factor-beta; Th, T-helper; TNF,
tumor necrosis factor. (From: Braun & Zwerina (5). With
permission).
bone resorption comes from the study of rheumatoid
arthritis (5) (Fig. 1). As the signals coming from the
bacteria in the periodontal pocket, and causing alveo-
lar bone resorption, will be different from the induc-
ing signals in the autoimmune disease, rheumatoid
arthritis, it is to be expected that these two diseases
would exhibit different mechanisms of bone break-
down. However, as stated above, the T-helper 17
lymphocyte subpopulation is implicated in both
diseases (9), suggesting some commonality of bone
pathogenesis.
Bacterially induced local autoimmunity
and bone destruction
The contention above would be different if there
was evidence that periodontitis is an autoimmune
disease. Currently, there is only minimal evidence
for aggressive periodontitis showing some autoim-
mune signature (21). However, before discussing
bacterial modulators of bone cells that could be
directly responsible for aberrant bone remodeling
3
Henderson & Kaiser

in periodontitis, it is important to mention briefly Direct actions of periodontopathic

the emerging evidence for a role for P. gingivalis in bacteria on alveolar bone
autoimmunity and the possibility of its role in peri-
In this section, the role of molecules secreted by bac-
odontal autoimmunity. The amino acid arginine
teria, which can modulate bone-cell function and
can be converted within proteins to citrulline by
which may contribute to pathological bone remodel-
the enzymes known as peptidylarginine deiminases
(80). This protein decoration, known as protein ing in periodontitis, will be discussed. Until recently,
the general finding was that pathogen-associated
citrullination, changes the physicochemical proper-
molecular patterns and other molecules emanating
ties of proteins. Protein citrullination was first
from bacteria promoted bone breakdown generally
shown to enhance immune responsiveness to mye-
by a mechanism that induced the formation of osteo-
lin basic protein, suggesting a role for this protein
decoration in autoimmune encephalomyelitis (94). clasts. Less is known about bacterial molecules that
can inhibit osteoblast bone synthesis. However, in
However, it was quickly established that citrulli-
the past decade, a slow trickle of papers has appeared
nated residues in proteins are essential parts of the
in which a variety of bacterial molecules, including
antigenic determinants recognized by autoantibod-
homologues of well-known osteolytic bacterial patho-
ies present in the blood of patients with rheuma-
gen-associated molecular patterns, have been
toid arthritis (78). It was Travis & Potempa (45),
described to inhibit bone breakdown and osteoclast
who performed pioneering work on the virulence
formation. Thus, it would appear that bacteria can
factors of P. gingivalis known as gingipains, who
promote or inhibit bone resorption depending on the
discovered that this bacterium also produced a
molecules they express; however, it is not known if
peptidylarginine deiminase and suggested that it
this is part of an evolved control system employed by
was probably a virulence factor. However, it was
the rheumatologist, Gerald Weissmann who put bacteria, or whether it has any role in the induction
or pathogenesis of periodontitis.
both strands of this story together to suggest that it
The rest of this section will describe the bone-mod-
was the humoral immune response to the products
ulating properties of the best-known bacterial ‘oste-
of the peptidylarginine deiminase of P. gingivalis
olytic’ components, with an emphasis on those
that could have initiated rheumatoid arthritis (66).
emanating from periodontopathic bacteria.
Biochemical studies have shown that, among the
oral bacteria tested, only P. gingivalis can citrulli-
nate proteins/peptides, and that a combination of Lipopolysaccharides
arginine gingipains and peptidylarginine deiminase
As suggested above, gram-positive and gram-negative
results in the formation of citrullinated peptides
bacteria have a wide range of pathogen-associated
from human fibrinogen and a-enolase. These pep-
molecular patterns and other molecules that can
tides are then proposed to drive the autoimmune
influence bone remodeling. Unfortunately, there is
pathology of rheumatoid arthritis (81). Potentially
increasing evidence for the same apparent class of
the strongest evidence for this hypothesis comes
pathogen-associated molecular pattern having oppos-
from an experimental study showing that wild-type
ing actions on bone. This makes for significant confu-
P. gingivalis exacerbates the pathology of collagen-
sion in the literature and could either be caused by
induced arthritis in mice, while an isogenic mutant
experimental error or because the workers publishing
lacking the gene encoding the P. gingivalis peptidy-
the opposing data are actually not using the same
larginine deiminase did not induce any
molecules. This problem has been best explored using
exacerbation of this experimental autoimmune
the gram-negative outer amphiphilic molecule,
condition (43).
lipopolysaccharide. This forms the outer layer of the
These findings clearly raise the possibility that
outer membrane of gram-negative bacteria and the
autoimmunity to locally induced citrullinated pro-
molecule is a complex consisting of an inner and
teins could be a driving factor in periodontitis and
outer core region and an O-specific side chain. The
therefore the mechanisms of bone destruction in
biologically active component of the lipopolysaccha-
rheumatoid arthritis and periodontitis are likely to be
ride polymer is a small phosphoglycolipid, called lipid
similar. Two recent clinical studies have failed to sup-
A, which has a unique and highly polymorphic struc-
port this contention directly (38, 59) but further stud-
ture and it is in this ability of bacteria to generate vari-
ies are needed in case the autoantigenic proteins
ations on the theme of lipid A that leads to different
driving periodontitis differ from those known to
biological actions of this key pathogen-associated
induce rheumatoid arthritis.

4

molecular pattern (20). Briefly, lipid A is a disaccha-
ride, which can vary in the composition of its saccha-
ride components, and these sugars are bound to four
(R)-3-hydroxyl fatty acids at positions 2,3 and 20 ,30 with
phosphate groups at positions 1 and 40 . Esterification
of these fatty acids can occur at the 20 and 30 positions,
and in this way the number of fatty acyl chains can
increase to six (and sometimes even to seven). All
these modifications of lipid A alter its biological activ-
ity and its effects on bone cells. A rough rule of thumb
is that the more acyl chains a lipopolysaccharide
molecule has, the more biologically active it is. Thus,
seven- and six-chain lipid A molecules are extremely
potent molecules; potency decreases with five-chain
molecules; and with four acyl chains the lipopolysac-
charides can be almost inactive or even act as
lipopolysaccharide antagonists (20).
The first evidence for lipopolysaccharide being able
to promote bone resorption appeared in 1977, when
it was shown that lipopolysaccharide induced the
formation of multinucleate osteoclasts, suggesting
its bone-resorbing mechanism was different to
osteoclastic resorption induced by parathyroid hor-
mone (67). This stimulated research into the oste-
olytic effects of lipopolysaccharide molecules from
periodontopathogens (Table 1). It was established, in
the 1990s, that lipopolysaccharide signals to cells
mainly through binding to toll-like receptor 4 and
such knowledge was aided by the fact that
Table 1. Lipopolysaccharides from periodontopathogens and their effect on bone and osteoclast formation/activation
Organism Bone resorption stimulated in
culture
Aggregatibacter Yes
actinomycetemcomitans
Aggregatibacter (Not detected)
actinomycetemcomitans
Aggregatibacter Stimulates bone loss in vivo
actinomycetemcomitans
Aggregatibacter Lipopolysaccharide-induced bone (Not detected)
actinomycetemcomitans resorption is Myd88-dependent
Capnocytophaga ochracea Yes
Eikenalla corrodens Yes
Fusobacterium nucleatum Yes
Porphyromonas gingivalis (Not detected)
Treponema denticola (Not detected)
Veillonella spp. Yes
Bacterial modulators of bone remodeling in periodontal pocket
lipopolysaccharide-unresponsive strains of mice (e.g.
C3H/HeJ) exist (60). Lipopolysaccharide-induced
bone resorption has been shown to be induced both
in in vitro and in vivo systems. In in vivo experiments,
in which lipopolysaccharide is injected into the gin-
giva, it has been shown that toll-like receptor 4-defi-
cient mice exhibit much less bone resorption than do
lipopolysaccharide-responsive mice, and this is asso-
ciated with lower expression of the key osteoclast
inducer/activator, RANKL (50).
Curiously, in spite of the wealth of data showing
that lipopolysaccharide promotes bone resorption by
inducing/activating osteoclasts, the story is actually
more complex, with a growing number of reports
that, in given circumstances, lipopolysaccharide can
actually inhibit osteoclast formation and can have
bifunctional properties. This was first reported by
Zou & Bar-Shavit (95), who used bone marrow-
derived osteoclast precursors purified of other cell
types (e.g. osteoblasts and stromal cells). Lipopolysac-
charide not only failed to promote osteoclast differen-
tiation, but it also blocked the osteoclastogenesis
promoted by RANKL. However, with RANKL pre-
treated cells, lipopolysaccharide did indeed promote
osteoclast formation. The induction of osteoclasts
appeared not to be caused by RANKL, but by the
activity of tumor necrosis factor alpha (75). A key
transcription factor in osteoclast differentiation is
nuclear factor of activated T-cells 1 (92). This is now
Influence on osteoclasts References
(Not detected) Kiley & Holt (30)
Stimulates osteoclast Ueda et al. (77)
formation
(Not detected) Rogers et al. (64)
Madeira et al. (42)
(Not detected) Iino & Hopps (25)
(Not detected) Progulske et al. (58)
(Not detected) Sveen & Skaug (74)
Stimulates osteoclastic pit Sismey-Durrrant & Hopps (72)
formation in dentine slices
Stimulates osteoclast Choi et al. (7)
formation
(Not detected) Sveen & Skaug (74)
5
Henderson & Kaiser
seen to be a pivotal factor in this bifunctional
response of osteoclasts to lipopolysaccharide (41). In
osteoclast precursors that have not been exposed to
RANKL, lipopolysaccharide inhibits nuclear factor of
activated T-cells 1. In contrast, if the same cells have
been stimulated by RANKL, then lipopolysaccharide
promotes the expression of nuclear factor of activated
T-cells 1. This reveals the complexity of the cell net-
works involved in osteoclast differentiation.
Not much is known about the periodontopathic
lipopolysaccharides that promote bone resorption
through toll-like receptor 4 and osteoclastogenesis.
However, there is growing knowledge about the
unusual properties of the lipopolysaccharide of the
major periodontopathogen, P. gingivalis, whose
lipopolysaccharide interacts with toll-like receptor 2
(2) as well as toll-like receptor 4. This bacterium
can make penta-acylated lipopolysaccharide which,
surprisingly, is 10- to 100-times less active than an
almost identical penta-acylated lipopolysaccharide
from Bacteroides spp. (4). There is also evidence
that the penta- and tetra-acylated lipopolysaccha-
rides made by P. gingivalis can differentially target
toll-like receptor 4 and downstream signaling path-
ways, suggesting that if this bacterium can modu-
late its outer lipopolysaccharide, it could markedly
alter its virulence potential, including its effects on
bone (22). This may explain, in part, the differential
effects of P. gingivalis lipopolysaccharide on osteo-
clast formation (69).
Thus far, the discussion has been based on the abil-
ity of lipopolysaccharide to induce osteoclast forma-
tion and activate mature osteoclasts. Does
lipopolysaccharide also inhibit the bone-forming
actions of osteoblasts or the bone-controlling activity
of the major osteocyte population? It was reported
that lipopolysaccharide from P. gingivalis was cap-
able of inhibiting the differentiation of osteoblasts in
culture, as assessed by measuring markers of cellular
differentiation, such as alkaline phosphatase, osteo-
calcin and osteopontin (26). The mechanism of this
effect is activation of Notch signaling (89). In in vivo
studies, the inhibition of osteogenesis by lipopolysac-
charide has been shown to be linked to induction of
tumor necrosis factor alpha synthesis (76). Finally,
there is evidence that lipopolysaccharide is able to
inhibit the differentiation of osteoblasts into osteo-
cytes (3). It will be interesting to find out more details
of the role of bacterial lipopolysaccharide on ‘osteo-
cytogenesis’.
This review of the literature reveals that the small
number of lipopolysaccharide species examined is
capable of providing different cellular signaling
6
mechanisms for this extremely heterogeneous
amphiphilic molecule to promote bone remodeling.
Given that the periodontal pocket may contain
lipopolysaccharide species from several hundred
bacteria means that it is difficult to predict what the
overall influence of the lipopolysaccharide molecules
in their entirety will be. For all we know, there may
be synergy or antagonism between all of these differ-
ent lipopolysaccharides, which could lead to very dif-
ferent biological outcomes for the patients involved.
Clearly, we need to know much more about the
lipopolysaccharide molecule as it relates to bone
remodeling.
Wall teichoic acids and lipoteichoic acids
Unlike gram-negative bacteria, gram-positive organ-
isms lack an outer membrane, with its key
lipopolysaccharide component, and a periplasm. In
contrast, the layers of peptidoglycan enveloping the
cytoplasm are very thick and contain anionic gly-
copolymers that can exit onto the cell surface. One of
the best studied of these are the teichoic acids – phos-
phate-rich polymers that come in two varieties: the
lipoteichoic acids, which anchor to the plasma mem-
brane and extend into the peptidoglycan layers; and
the wall teichoic acids, which are bound to the pepti-
doglycan and extend onto the bacterial surface (6).
These molecules are relatively well-studied pathogen-
associated molecular patterns with a perceived role in
host immune and inflammatory responses to gram-
positive bacteria (82). While being important mole-
cules and, like lipopolysaccharide, with significant
structural diversity, the wall teichoic acids and lipote-
ichoic acids have received relatively scant attention,
compared with lipopolysaccharide, in terms of their
influence on bone remodeling.
Given the similarities in the general response of
cells to lipopolysaccharide and teichoic acids, the
expectation is that the wall teichoic acids and lipotei-
choic acids would exert osteolytic activity, presum-
ably by promoting osteoclastogenesis. There are only
two reports that teichoic acids could promote bone
breakdown, the first dating back to the mid-1970s
(19). The teichoic acids of the gram-positive organism
Streptococcus mutans (1) have been reported to pro-
mote bone breakdown in vivo. The most important
gram-positive bacterium, in relation to bone pathol-
ogy, is clearly Staphylococcus aureus (88). It would be
assumed that the teichoic acids of this organism had
osteolytic activity. However, the lipoteichoic acid
from S. aureus turned out to be inhibitory to osteo-
clast formation, working via a toll-like receptor 2-

sensitive mechanism (90). This may be specific for
this form of lipoteichoic acid, and clearly a more
detailed analysis of wall teichoic acids and lipotei-
choic acids from gram-positive periodontopathogens
is required to give a clearer understanding of the role
of this gram-positive component in periodontal bone
destruction.
Lipoproteins and lipopeptides
Bacterial lipoproteins and lipopeptides are another
class of pathogen-associated molecular pattern gen-
erated by all forms of bacteria (61) and for which
some evidence exists for a role in bone breakdown
(48). The Omp19 lipoprotein of Brucella abortus was
shown to induce osteoclast formation (10). The direct
role of lipoprotein in S. aureus-induced bone loss
in vitro and in vivo has recently been shown using a
lipoprotein-deficient mutant, which failed, unlike the
wild-type organism, to stimulate bone breakdown
(32). An interesting bacterial lipopeptide with a grow-
ing list of biological actions is mycoplasmal macro-
phage activating lipopeptide-2, which interacts with
toll-like receptor 2/6 (71, 84). When present at pico-
molar concentrations, this 2-kDa peptide demon-
strated the ability to induce murine calvarial bone
resorption, being one of the most active bone-resorb-
ing agonists recorded (56).
Miscellaneous bacterial osteolytic
molecules
In addition to the classes of osteolytic molecules
described above, there is a diverse literature on the
ability of other bacterial molecules to either stimu-
late bone resorption or inhibit osteoblast-induced
bone synthesis. For example, the capsular polysac-
charide of Aggregatibacter actinomycetemcomitans
promotes osteoclast formation via a mechanism
involving interleukin-1 production (53). The fimbriae
of P. gingivalis have been reported to stimulate bone
resorption in vitro (28) and have been shown to
stimulate osteoclastic bone resorption in a toll-like
receptor 2-dependent manner (24). Bacterial porins
have also been shown to promote calvarial bone
resorption in vitro (46). The Treponema denticola
cell-surface protein, Td92, induces osteoclast forma-
tion and inhibits osteoprotegerin synthesis (31).
Another class of protein reported to be a potent
stimulator of bone resorption are the molecular
chaperones chaperonin 60 (33, 62) and chaperonin
10 (47). That chaperonin 60, also known as heat
shock protein 60, could stimulate bone resorption
Bacterial modulators of bone remodeling in periodontal pocket
was an unexpected finding of a potent osteolytic
protein released by the major periodontopathogen,
A. actinomycetemcomitans (33). Of interest, this pro-
tein stimulated bone resorption in the C3H/HeJ
mouse strain, revealing that the mode of action of
this chaperonin 60 protein was not through activat-
ing toll-like receptor 4 (33). These chaperonins are
examples of moonlighting proteins, as might be a
number of the bacterial osteolytic proteins. Of inter-
est, and as will be discussed in the next section,
some chaperonin 60 proteins can inhibit bone
breakdown. Possibly the most potent osteolytic bac-
terial component is the eponymous toxin from Pas-
teurella multocida (39). This toxin is responsible for
atrophic rhinitis in pigs, a disease in which the turbi-
nate bones are destroyed (15). The P. multocida
toxin was shown to be a potent stimulator of calvar-
ial bone resorption in vitro (13) and to promote
osteoclast formation when administered to rats (44),
suggesting a direct influence on osteoclastogenesis.
However, it turns out that this toxin, potent as it is,
has an indirect effect on osteoclast differentiation,
which has been claimed to depend on the action of
osteoblasts (49) and, surprisingly, B-lymphocytes
(23).
One of the intriguing facets of bone-cell biology is
the sensitivity of this complex, interrelated cellular
system to molecules that would not be expected to
have any influence on bone remodeling. One such
example is protein A from S. aureus. For most biolo-
gists, all they would know about this molecule is that
it is used for purifying IgA. However, in 2007, staphy-
lococcal protein A has been shown to have a wide
range of additional functions, including acting as
a B-cell superantigen able to subvert immune
responses (96) and as a von Willebrand factor-binding
protein (18). Curiously, this protein has recently been
suggested to be critical in mediating the bone loss
that occurs in S. aureus-induced osteomyelitis by
inhibiting osteoblasts and stimulating osteoclastoge-
nesis (8, 83). This is an unexpected effect, and may be
the sign of things to come as we explore, in more
detail, the bacterial mediators of bone remodeling.
Bacterial molecules that inhibit
bone remodeling and breakdown
Thus far, the discussion has been within the para-
digm that bacteria and their skeleton-modulating
molecules act to stimulate bone destruction. How-
ever, evidence is emerging for bacterial factors that
can actually inhibit the cellular processes of bone
7
Henderson & Kaiser
Fig. 2. Bacteria–host and bacteria–bacteria signaling at
mucosal surfaces. Complex reciprocal (and higher level)
dynamic signaling must occur on mucosal surfaces
between bacteria and associated mucosal epithelial cells
remodeling. This has already been touched upon
with lipopolysaccharide and lipoteichoic acid, and
these molecules will not be dealt with again.
There are also a number of bacterial proteins
emerging that inhibit the stimulation of bone remo-
deling.
A number of low-molecular-mass compounds
emanating from bacteria, such as reveromycin A,
destruxins, mevastatin, FK506, cyclosporin A, prodi-
giosins, concanamycins and symbioimine, some of
which are well-known immunosuppressants, have
been shown to inhibit osteoclast differentiation by
acting at different stages in the process (87). This
same research group has also reported that a macro-
lide compound, biselyngbyaside, from a marine
cyanobacterium, inhibits osteoclastogenesis and can
induce mature osteoclasts to apoptose (91). Anti-
coumarins from Streptomyces spp. can also inhibit
osteoclast differentiation by inhibiting the synthesis
of nuclear factor of activated T-cells 1 (93).
8
and between these epithelial cells and the microbiota. In
addition, the stability of the composition of the microbiota
must be controlled by complex dynamic signaling between
these different bacteria.
Likewise, it has been shown that several bacterial
proteins, from bacteria implicated in bone-remodel-
ing pathology, can inhibit osteoclastogenesis. The
first example of this was the hemoglobin receptor
protein of P. gingivalis, which is a porphyrin-binding
protein found on the cell surface and a major pro-
tein in the supernatant of this organism (27). This
protein was found to inhibit RANKL-induced osteo-
clastogenesis when added within 24 h of stimulation
with RANKL, but not at later time points, suggesting
that it interfered with the early events in osteoclast
formation. This effect was associated with inhibition
of the production of nuclear factor of activated T-
cells 1 (NFATc1) (16). The M. tuberculosis chaper-
onin 60.1 protein also inhibited osteoclastogenesis
and this was associated with longer-term inhibition
of nuclear factor of activated T-cells 1. Indeed, this
protein could be added up to 4 days after the addi-
tion of RANKL and still be able to inhibit osteoclast
formation, suggesting a more comprehensive mode

of action than that of the hemoglobin receptor pro-
tein (86). Again, like the hemoglobin receptor pro-
tein, inhibition of osteoclast formation, induced by
M. tuberculosis chaperonin 60.1, was associated with
inhibition of the transcription of nuclear factor of
activated T-cells 1 (86). It is interesting that the M.
tuberculosis chaperonin 60.1 paralogue, chaperonin
60.2, is unable to inhibit or stimulate bone resorp-
tion, despite the fact that both proteins share 63%
sequence identity (86).
Summary
Homo sapiens share a dynamic existence with 1014
bacteria, with these prokaryotes existing on the
exterior epithelial and mucosal surfaces of the body.
Not only are the exterior surfaces of our bodies
awash with bacteria, they are awash with an amaz-
ing diversity of bacteria, with each individual proba-
bly having a different set of stably colonizing
prokaryotes as their life companions (85). These
bacteria are not simply inert organisms disassoci-
ated from their host, but must be part of a rich sig-
naling nexus in which reciprocal signaling between
bacteria and associated epithelial cells occurs and
exists within a further network of reciprocal signal-
ing between members of the microbiota. It can be
argued that these mucosal surfaces represent the
most complex signaling systems outside the nervous
system (Fig. 2). With, say, each individual having
200 bacterial species on the gingival mucosa, this
equates to almost 1 million bacterial proteins
(200 9 3000–6000 genes per bacterium) that are
potentially able to act as signals between bacteria
and between bacteria and the host epithelium. This
does not include the multitude of low-molecular-
mass signals generated by bacteria. In turn, the sig-
nals received by the mucosal epithelium can be
directed to local immune cells to control local
immune responsiveness and to maintain normal
alveolar bone status. We know almost nothing
about this immensely complex signaling system,
other than it goes awry in a large proportion of
individuals, resulting in bone pathology. Such
pathology could be driven either by colonization
with organisms which significantly influence bone
remodeling or by some form of complex genetic
susceptibility to the normal gingival microbiota, or
by a combination of both factors. It is likely that an
understanding of the mechanism of bone destruc-
tion in periodontitis can only be achieved by having
full knowledge of the identity of the bacterial
Bacterial modulators of bone remodeling in periodontal pocket
populations in the periodontal pockets in patients
with various forms of periodontitis, linked with a
complete genetic profile of the patients with peri-
odontitis (plus their clinical status). It is only with
the foundation of these two interrelated complex
data sets for a sufficient population of patients that
it will be possible to tease out the general relation-
ships between colonization with periodontopathic
bacteria, ‘genetic susceptibilities in periodontitis’
and the generation of bone-destructive behavior.
One of the most likely scenarios for explaining the
bacteria/host/bone breakdown equation that is
periodontitis, is that patients with periodontitis have
some complex multigenetic aberration in their abil-
ity to perceive and respond to bacteria. This could
either be a deficit or an enhanced ability to recog-
nize/respond to bacteria. In either case, the out-
come is that local immune responses to oral
bacteria are greater than normal and eventually lead
to aberrant signaling that causes pathological bone
remodeling. It is indeed fortunate that the technol-
ogy, largely in the form of high-throughput nucleic
acid sequencing (next-generation sequencing), now
exists to probe human and bacterial genomes, or
even metagenomes, to identify mutations in the
human genome and relate these to the composition
of the microbiota. This can be aided by quantitative
transcriptomic analysis of the inflamed gingival and
oral microbiota using RNA sequencing. The entirety
of the information derived from such use of next-
generation sequencing, if it can be appropriately
analyzed, is likely to provide a clear indication of
the probable signaling pathways that result in the
aberrant bone breakdown that is periodontitis. All
this requires is the will, and the funding organiza-
tions capable of taking up this challenge.
Acknowledgements
B.H. and F.K. acknowledge the support of the Medical
Research Council on their ongoing research.
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