J R Soc Med 2002;95:77-80
doi:10.1258/jrsm.95.2.77
© 2002 Royal Society of Medicine
Oral bacterial disease and the science of cellular conversation
Brian Henderson BSc PhD
Cellular Microbiology Research Group, Eastman Dental Institute,
University College London, 256 Gray's Inn Road, London WC1X 8LD, UK
E-mail:
b.henderson{at}eastman.ucl.ac.uk
 |
INTRODUCTION
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An explosion of discovery between 1870 and the early 1900s identified
the
major bacterial pathogens of
Homo sapiens, and in the 1930s
Domagk's
work on sulpha compounds and Fleming's luck with
Penicillium notatum
initiated our love affair with antibiotics. The 1950s
brought apparent victory
in our war with bacteria, and so research
in microbiology switched to other
sources of human infection,
principally the virus; but the rapid escalation of
antibiotic
resistance in the past two decades has led to a renaissance
in
bacteriological research and the development of a new science,
cellular
microbiology
1,2,3,4—a
fusion of classic microbiology
and cell biology with major inputs from
molecular and structural
biology.
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CELLULAR MICROBIOLOGY: ENTERIC PATHOGENS LIGHT THE WAY
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Cellular microbiology has moved the focus from the study of
bacteria, or of
host cells, in isolation to the study of the
interactions between these cells
that cause bacterial diseases.
Many of our most successful pathogens target
the gut, and one
of the most interesting of these pathogens is
enteropathogenic
Escherichia coli or EPEC. This pathogen does what
all such organisms
must do to infect—it adheres to the walls of the
intestine.
The interaction involves one of the paradigms of modern cellular
microbiology—the
type III secretion system. In simple terms this is a
molecular
syringe with which the bacterium can pierce the host cell plasma
membrane
and inject proteins that alter the behaviour of the injected
cell. In
EPEC, as in the other bacteria possessing type III
secretion systems, the
genes encoding this syringe (injectisome)
are found on what is termed a
pathogenicity island. These islands
are large segments of the bacterial
chromosome which are associated
with virulence (strains lacking these islands
are generally
avirulent), and have a different nucleotide composition from
the
host bacterial chromosome. In EPEC the pathogenicity island
is called the
‘locus of enterocyte effacement’ and
consists of those genes
encoding the injectisome and those proteins
secreted by it. The basic trick of
EPEC is to manipulate the
actin cytoskeleton of the host enterocytes to induce
loss of
the microvilli and produce a raised area on the cell surface
(the
pedestal) to which the bacterium attaches, via a receptor.
The most remarkable
facet of the interaction of EPEC with enterocytes
is that the receptor to
which the bacteria bind—the translocated
intimin receptor—is not a
host protein but a bacterial
protein that has been injected into the host
cell
5.
Bacteria have evolved many strategies for interacting with key elements of
eukaryotic cell behaviour in order to infect their hosts or to live with them
in harmony as commensals. We are only beginning to understand the complexity
of the interactions which occur between bacterial pathogens and their infected
hosts and there is an enormous amount still to be learned. Much of our
ignorance concerns the interactions of oral bacteria with the many cells and
tissues that constitute the oral cavity.
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CELLULAR MICROBIOLOGY AND THE ORAL CAVITY
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As the accompanying
article
6 reveals,
the human oral cavity
is awash with bacteria, and the numerous habitats within
this
anatomical site contain many hundreds of bacterial species.
There are two
related questions about our interactions with
these oral bacteria. The first
concerns the enormous amount
of oral bacterial disease that
Homo
sapiens
endures
5. The whole
population
is subject to the ravages of caries, gingivitis and periodontitis.
Although
the gut contains probably as many bacterial species as does
the
mouth, we do not have to take daily prophylactic measures
to limit the
bacterial content of the gut. The very common oral
diseases are due to a small
number of Gram-positive or Gram-negative
bacteria. The related question
is—what prevents us responding
to all of our oral microflora? Our
understanding of the interaction
of oral bacteria with host cells and surfaces
is still in its
infancy, but the discipline of oral microbiology has begun to
show
how bacterial biofilm—a complex organization of bacteria
adherent
on a surface—contributes to the various oral
diseases.
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CELLULAR MICROBIOLOGY OF Actinobacillus
actinomycetemcomitans
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One example will be given of how cellular microbiology is being
used to
address the behaviour of oral pathogens.
Actinobacillus
actinomycetemcomitans is a Gramnegative bacterium which is either a
member of the
Haemophilus—Actinobacillus—Pasteurella
family or
closely related to
it
7. This family
contains major human and
animal pathogens such as
Haemophilus
influenzae, causing meningitis
in man, and
Pasteurella
multocida, causing a chronic inflammatory
condition of pigs in which the
bones of the snout are destroyed.
A. actinomycetemcomitans is
implicated as a causative organism
of periodontitis, particularly localized
juvenile
periodontitis
8.
This
organism is said to produce several unusual virulence
factors
9,10
and
to invade host
cells
10,11.
Virulence factors
Amongst the reported putative virulence factors of this bacterium are a 116
kDa leukotoxin which targets only human neutrophils and
monocytes12, a
secreted chaperonin 60 protein which stimulates bone
resorption13 by
acting as a growth factor for
osteoclasts14 and
promotes cytolysis of epithelial
cells15, and a
small peptide with unusual cytokine-network-inducing
properties16. The
organism possesses the three contiguous genes (cdtA, cdtB and
cdtC) which encode an activity known as cytolethal distending toxin
(CDT)17. This toxin
blocks cycling cells in the latter part of the cell cycle (termed
G2)—an action that seems to be due to the nuclease activity
exhibited by
CdtB18. Our own
work suggests that CdtC is required to allow CdtB to enter into cells
(Akifusa, Henderson, Stenbeck and Henderson, Unpublished). The role of CDT in
pathogenesis is unclear. One possibility is that it blocks lymphocyte
proliferation and is therefore
immunosuppressive19.
While these so-called virulence factors have effects on cells which could
give rise to tissue disease, their actual role in the diseases caused by
A. actinomycetemcomitans is uncertain. The clearest way of
determining this is to inactivate the gene of interest and assess whether the
bacterium has lost its pathogenicity. An organism related to A.
actinomycetemcomitans, Haemophilus ducreyi (which causes the sexually
transmitted disease, chancre) likewise produces a leukotoxin and CDT. The
assumption that both toxins contributed to its pathogenicity was shown to be
incorrect when inactivation of the genes did not block the
pathogenicity20,21.
A major goal of modern cellular microbiology is the identification of the
complete set of likely virulence genes. With bacteria that kill host cells
various mutagenesis techniques can be used to identify genes that contribute
to survival in the host and are likely to be involved in virulence. One way of
doing this is to introduce mobile genetic elements called transposons that, on
random insertion into the bacterial genome, inactivate individual genes. It is
only very recently that methods have become available to do this in A.
actinomycetemcomitans22
and this methodology has identified flp-1 as a pilin gene subfamily
required for the non-specific adherence of this
organism23. Many of
the virulence proteins produced by bacteria are secreted, and one global
method of identifying genes encoding secreted proteins is to make a gene
library in a vector which has a truncated version of the gene for alkaline
phosphatase. DNA fragments of the chromosome of the bacterium of interest
containing signal sequences required to export proteins to the bacterial
periplasm are fused to the alkaline phosphatase gene. The exported protein,
when it reaches the periplasm, can then be identified by assay for alkaline
phosphatase; and the responsible genes can be determined by screening of the
genome libraries for clones containing the AP gene. Two recent papers describe
identification, by this method, of several genes that could be important in
bacterial virulence. Another strategy for identifying secreted proteins is
termed proteomics. In this technique bacterial secreted proteins are separated
on two-dimensional gels on the basis of differences in their charge and mass.
Individual proteins can then be identified by direct sequencing or by in-gel
proteolysis and peptide mass fingerprinting by mass
spectrometry26.
Antibodies have been used for many years to identify gene products produced
by genomic DNA fragments in expression libraries. These antibodies can come
from the blood of patients with specific bacterial infections. A recent and
very exciting development of this technique, termed IVIAT (in-vivo
induced antibody technology), uses plate-grown bacteria to adsorb sera from
patients who are infected with the bacterium of interest. In theory, this
removes all antibodies directed to the components that bacteria produce when
they are not infecting their host, leaving behind those antibodies that
recognize only those gene products that bacteria generate when they are
producing disease in the host. This simple technique is now being applied to a
range of oral
bacteria27 and has
identified in A. actinomycetemcomitans a substantial number of genes
that may be responsible for virulence (Hillman JD, Personal
communication).
Cell invasion
We have long known that certain organisms, Mycobacterium
tuberculosis being a good example, are obligate intracellular bacteria.
One of the major discoveries of cellular microbiology has been that many
pathogenic bacteria can invade several sorts of host cell. Some strains of
A. actinomycetemcomitans can enter both vascular endothelial
cells28 and
epithelial cells29.
The receptor involved in invasion appears to be that which binds to the
pro-inflammatory lipid mediator platelet-activating
factor28. Most of
the bacteria that invade human cells utilize the actin cytoskeleton for
movement within and between
cells30. Seemingly,
A. actinomycetemcomitans in culture enters and moves through
epithelial cells by a novel mechanism involving
microtubules31.
Again, we do not know how important cell invasion is for the pathogenesis of
diseases caused by A. actinomycetemcomitans. However, it is clear
that bacteria within cells are no longer directly exposed to the immune system
or to some antibiotics, and that they gain distinct advantages from hiding
within cells.
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CONCLUSIONS
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To fully understand bacterial disease we must grapple with the
nature of
the two-way communication that exists between bacteria
and the cells of the
host. Such communication is, as far as
we can tell, normally beneficial for
both parties and this is
why, with perhaps 1000-3000 bacterial species
existing in and
on our bodies, only a few dozens cause disease. To understand
this
bacteria-host communication we need to study the interactions
in
situ. The most obvious arena for such study is the human
mouth. This
accessible organ, with its multiple habitats, rich
microflora and rapid
responsiveness to bacterial overgrowth,
should be the perfect test-bed for
identifying the rich complexity
of the conversations that must be occurring
between our own
bacteria and our tissues. The genetic and cellular techniques
needed
for this study exist, and the need is for clinicians who will
take up
the challenge. The importance of this challenge is highlighted
by the finding
that
A. actinomycetemcomitans and
Porphyromonas gingivalis
were present within buccal epithelial cells of 23
out of 24 individuals
examined
32. The
suggestion that periodontopathogens
are normally present inside oral
epithelial cells adds a new
dimension to the study of host—bacterial
interactions
in the mouth.
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REFERENCES
|
|---|
-
Cossart P, Boquet P, Normark S, Rappuoli R. Cellular microbiology
emerging. Science1996; 271:315
-16[Medline]
-
Henderson B, Wilson M, McNab R, Lax AJ. Cellular
Microbiology: Bacteria-Host Interactions in Health and Disease.
Chichester: Wiley, 1999
-
Cossart P, Boquet P, Normark S, Rappuoli R. Cellular
Microbiology. Washington, DC: ASM Press,2000
-
Wilson M, McNab R, Henderson B. Bacterial Disease
Mechanisms: An Introduction to Cellular Microbiology. Cambridge:
Cambridge University Press (in press)
-
Celli J, Deng W, Finlay BB. Enteropathogenic Escherichia
coli (EPEC) attachment to epithelial cells: exploiting the host cell
cytoskeleton from the outside. Cell Microbiol2000; 2:1
-9[CrossRef][Medline]
-
Wade W. Unculturable bacteria—the uncharacterized organisms
that cause oral infections. J R Soc Med2002; 95:83
-5
-
Olsen I, Shah, HN, Gharbia SE. Taxonomy and biochemical
characteristics of Actinobacillus actinomycetemcomitans and
Porphyromonas gingivalis. Periodontology 20001999; 20:14
-52[Medline]
-
Slots J, Ting M. Actinobacillus actinomycetemcomitans and
Porphyromonas gingivalis in human periodontal disease: occurrence and
treatment. Periodontology 20001999; 20:82
-121[Medline]
-
Wilson M, Henderson B. Virulence factors of Actinobacillus
actinomycetemcomitans relevant to the pathogenesis of inflammatory
periodontal diseases. FEMS Microb Rev1995; 17:365
-79[CrossRef][Medline]
-
Meyer DH, Fives-Taylor P. The role of Actinobacillus
actinomycetemcomitans in the pathogenesis of periodontal diseases.
Trends Microbiol1997; 5:224
-8[CrossRef][Medline]
-
Fives-Taylor PM, Meyer DH, Mintz JP, Brissette C. Virulence factors
of Actinobacillus actinomycetemcomitans. Periodontology
2000 1999;20:136
-67[Medline]
-
Korostoff J, Fei Wang J, Kieba I, Miller M, Shenker BJ, Lalley ET.
Actinobacillus actinomycetemcomitans leukotoxin induces apoptosis in
HL60 cells. Infect Immun1998; 66:4474
-83[Abstract/Free Full Text]
-
Kirby AC, Meghji S, Nair SP, et al. The potent bone
resorbing mediator of Actinobacillus actinomycetemcomitans is
homologous to the molecular chaperone, groEL. J Clin
Invest 1995;96:1185
-94
-
Reddi K, Meghji S, Nair SP, et al. The Escherichia
coli chaperonin 60 (groEL) is a potent stimulator of osteoclast
formation. J Bone Miner Res1998; 13:1260
-6[CrossRef][Medline]
-
Gouhlen F, Hafezi A, Uitto V-J, et al. Subcellular
localisation and cytotoxic activity of the GroEL-like protein isolated from
Actinobacillus actinomycetemcomitans. Infect Immun1998; 66:5307
-13[Abstract/Free Full Text]
-
Reddi K, Nair SP, White PA, et al. Surface-associated
material from the bacterium Actinobacillus actinomycetemcomitans
contains a peptide which, in contrast to lipopolysaccharide, directly
stimulates fibroblast interleukin-6 gene transcription. Eur J
Biochem 1996;236:871
-6[Medline]
-
Pickett CL, Whitehouse CA. The cytolethal distending toxin family.
Trends Microbiol1999; 7:292
-7[CrossRef][Medline]
-
Lara-Tejero M, Galan JE. A bacterial toxin that controls cell cycle
progression as a deoxyribonuclease protein. Science2000; 290:354
-7[Abstract/Free Full Text]
-
Shenker BJ, Hoffmaster RH, McKay TL, Demuth DR. Expression of the
cytolethal distending toxin (Cdt) operon in Actinobacillus
actinomycetemcomitans: evidence that the CdtB protein is responsible for
G2 arrest of the cell cycle in human T cells. J
Immunol 2000;165:2612
-18[Abstract/Free Full Text]
-
Stevens MK, Latiner JL, Lumbley SR, et al.
Characterisation of a Haemophilus ducreyi mutant deficient in
expression of cytolethal distending toxin. Infect
Immun 1999;67:3900
-8[Abstract/Free Full Text]
-
Young RS, Fortney KR, Gelfanova V, et al. Expression of
cytolethal distending toxin and hemolysin is not required for pustule
formation by Haemophilus ducreyi in human volunteers.
Infect Immun2001; 69:1938
-42[Abstract/Free Full Text]
-
Thomson VJ, Bhattarcharjee MK, Fine DH, Derbyshire KM, Figurski DH.
Direct selection of IS903 transposon insertions by use of a broad-host range
vector: isolation of catalase-deficient mutants of Actinobacillus
actinomycetemcomitans. J Bacteriol1999; 181:7298
-307[Abstract/Free Full Text]
-
Kachlany SC, Planet PJ, DeSalle R, Fine DH, Figurski DH, Kaplan JB.
flp-1, the first representative of a new pilin gene subfamily, is
required for non-specific adherence of Actinobacillus
actinomycetemcomitans. Mol Microbiol2001; 40:542
-54[CrossRef][Medline]
-
Mintz KP, Fives-Taylor PM. Identification of genes encoding
exported proteins of Actinobacillus actinomycetemcomitans. Infect
Immun 1999;67:6217
-20[Abstract/Free Full Text]
-
Ward JM, Fletcher J, Nair SP, et al. Identification, using
alkaline phosphatase fusions, of the exported proteins of the oral
opportunistic pathogen, Actinobacillus actinomycetemcomitans.Infect Immun2001; 69:2748
-52[Abstract/Free Full Text]
-
Fletcher JM, Nair SP, Ward JM, Henderson B, Wilson M. Analysis of
the effect of changing environmental conditions on expression patterns of
exported surface-associated proteins of the oral pathogen Actinobacillus
actinomycetemcomitans. Microb Pathog2001; 30:359
-68[CrossRef][Medline]
-
Handfield M, Brady LJ, Progulske-Fox A, Hillman JD. IVIAT: a novel
method to identify microbial genes expressed specifically during human
infections. Trends Microbiol2000; 8:336
-9[CrossRef][Medline]
-
Schenkein H, Barbour SE, Berry CR, Kripps B, Tew G. Invasion of
human vascular endothelial cells by Actinobacillus
actinomycetemcomitans via the receptor for platelet activating factor.
Infect Immun2001; 68:5416
-19[Abstract/Free Full Text]
-
Meyer D, Lippmann JE, Fives-Taylor PM. Invasion of epithelial cells
by Actinobacillus actinomycetemcomitans: a dynamic multistep process.
Infect Immun1996; 64:2988
-97[Abstract]
-
Oelschlaeger TA, Hacker J. Bacterial Invasion into
Eukaryotic Cells. New York: Kluwer Academic/Plenum,2000
-
Meyer DH, Rose JE, Lippmann JE, Fives-Taylor PM. Microtubules are
associated with intracellular movement and spread of the periodontopathogen
Actinobacillus actinomycetemcomitans. Infect Immun1999; 67:6518
-25[Abstract/Free Full Text]
-
Rudney JD, Chen R, Sedgewick GJ. Intracellular Actinobacillus
actinomycetemcomitans and Porphyromonas gingivalis in buccal
epithelial cells collected from human subjects. Infect
Immun 2001;69:2700
-7[Abstract/Free Full Text]
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INTRODUCTION
CELLULAR MICROBIOLOGY: ENTERIC...
