Genetically Engineered Bacteria to Fight Tooth Decay
Science Research by: Ruba Mulki
A
Florida researcher is hoping to soon begin clinical trials for his bacterial
rinse that's designed to stave off tooth decay for a person's lifetime. So
far, the rinse has worked in rats and early prototypes have been tested in
three people.
"You would just need to squirt onto tooth
surfaces once," said Jeffrey Hillman a professor of oral biology at the
University of Florida in Gainesville.
Bacteria, he explains, take care of the rest.
The rinse could be a wonderfully simple approach
to dental care, although some worry that introducing a modified microbe
directly into the body could lead to trouble.
Most tooth decay is
caused by a particular strain of bacteria called Streptococcus mutans
(S. mutans). While 500-600 different kinds of bacteria thrive on
mucus and food remnants in the mouth, S. mutans is particularly
damaging because it consumes sugar (mostly refined sugars) on the surface of
teeth and converts it to lactic acid.
The
lactic acid is what eats away at a tooth's enamel
In the early 1980s, Hillman set out to find a
bacterium that might destroy the decay-causing strain. After taking hundreds
of sample swabs from patients' mouths, he found a bacterium that secretes a
toxin that kills S. mutans.
Hillman and his colleagues then altered a gene in
the bacterium so it would not secrete lactic acid of its own. Recently they
tweaked the bacterium again so it would only survive if fed a particular
nutritional supplement. That ensures the bacterium won't spread from one
person to another while kissing or sharing utensils.
"Subjects will have to chew gum or use mouthwash
to provide the bacteria with its nutritional supplement," said Hillman.
When Hillman squirted the strain on rats, the
substance appeared to prevent tooth decay in the animals for the entire
six-month period of the tests. He has also squirted a version of the
bacterium on three human volunteers.
The strain these people harbor in their mouths
kills off the S. mutans bacterium, but does not prevent decay since
it also produces lactic acid. Tests show the strain has successfully warded
off all S. mutans bacteria since the early 1980s. And none of the
three subjects have passed on their unique mouth bacteria to their spouses
or children.
Some might worry that releasing a genetically
altered creature inside the human body could lead to trouble. But Hillman
claims he's just speeding up evolution. The decay-causing bacterium was
probably innocuous until people began eating large quantities of refined
sugar. Another 1,000 years and people might have shed the S. mutans
bacterium anyway, he says.
Other dental experts warn it can be dangerous
tinkering with the body's complex balance of bacteria.
"There are many
varieties of bacteria in the mouth and they live in a kind of ecosystem
there," said Kenneth Burrell, senior director of the American Dental
Association's Council on Scientific Affairs. "There's a balance there if you
upset it, you can throw off the bacterial population. And some bacteria may
be necessary to maintain a healthy mouth."
Burrell adds, however, if tests show the rinse
does not upset this balance it could be a boon to dental hygiene. The rinse
would only need to be applied once — preferably when a person is very young
— and then the bacterium would settle into the patient's mouth for life.
Adult patients could also use the rinse to prevent any further dental decay
they may have already experienced.
Bacteria is often thought of as a target when it
comes to cleaning, but recently, researchers have found strains that work
well as cleaning tools. Companies like BioOne and Eco-Save provide cleaners
that employ bacteria to eat through plumbing and bathroom scum. And
environmentalists have found certain bacteria are effective in cleaning up
toxic waste.
Now Hillman's bacterial strain, known as BCS3-L1,
could take up a similar role in the mouth. Hillman presented his findings at
this year's meeting of the American Association for the Advancement of
Science in Boston.
Just because a bacterium may be fighting tooth
decay in your mouth doesn't mean you'll be free of the task of tooth
brushing and flossing, Hillman says. Those daily practices are still needed
for preventing gum disease and bad breath.
And while the thought of a decay-ending agent
may cause unease among some dentists who make a living on the problem,
Burrell points out the rinse could actually end up improving business.
"If this rinse really works, it could mean the
average person will have their teeth for a longer time," he said. "Then they
might have various gum infections that they wouldn't have experienced if
they lost their teeth to tooth decay, and they'll need dentists for that."
The oral cavity is
home to many different species of streptococci and it is not surprising,
considering they share the same habitat, that they have many features in
common. This can pose problems in identification and in sorting out the
relationship between the various species. The application of a wide range of
biochemical tests (particularly for sugar fermentation and glycosidase
enzymes) and, more recently, the analysis of ribosomal RNA sequences has led
to general agreement about the species boundaries and 19 distinct species
are recognised. Nevertheless, much remains to be resolved as new discoveries
are made about the exchange of genetic material between bacteria, which
leads to mosaic chromosomes.
One group of oral
streptococci is closely related to S. mutans and is referred to as
the 'mutans group' or the 'mutans streptococci'. Note that the species name
is written in italics while the group name is not.
S. mutans is
carried by virtually everyone and the only other species common in man is
S. sobrinus, carried by between 8 and 35% of people in different
countries. Although S. mutans and S. sobrinus can be
distinguished by appropriate laboratory tests, these are expensive and
time-consuming so it is not always practicable to identify down to the
species level in large-scale epidemiological studies.
Nor has anyone
managed to invent a selective medium that would allow us to look for the
presence of a single species, in saliva samples for example. As a
consequence, most work on the relationship of bacteria to caries has lumped
the two species together as the mutans streptococci (MS).
Because of its
greater prevalence, most of the isolates will in fact be S. mutans
and some authors erroneously use the single name S. mutans even
though they could not tell if is S. sobrinus was also present. Of
course, all older papers published before the mid-80s refer to S. mutans
because S. sobrinus was not officially recognised then.
The two selective
media that are widely used for isolating caries-related streptococci are
based on Mitis-Salivarius agar and TYC agar to which the antibiotic
Bacitracin is added (TYCSB). This suppresses the growth of most species but
allows S. mutans and S. sobrinus to grow.
The inclusion of
sucrose leads to the formation of glucans and distinctive colony appearance
that aids identification. Diagnostic kits designed for use in the dental
clinic are also based on similar selective media so note that they are
measuring total MS, not just S. mutans.
Streptococcus mutans
is a causative agent of dental caries. It's ability to inflict damage is
strongly linked to the production of long chain glucose polymers (glucans)
which allow the bacteria to successfully colonize the smooth surface of
teeth.
To synthesize these
glucans the bacteria possesses a family of genes that express enzymes called
glucosyltransferases. Expression of the glucosyltransferase genes, gtfB,
gtfC and gtfD are strongly regulated. The long term goal of this project is
to identify elements that regulate their expression and then to subsequently
characterize them. The first modulator identified was the global prokaryotic
regulator, Integration Host Factor (IHF).
IHF's net effect is
to repress cloned S. mutans gtf genes as much as 6 fold when expressed in E.
coil. IHF is intimately involved in virtually all forms of nucleoid
manipulation i.e. replication, recombination and gene expression. In E. coil
and other gram negative bacteria, IHF can affect gene expression through
either transcriptional regulation through its ability to bind DNA at its
cognate binding site.
Before a formal
analysis of IHF function of gtf expression can be undertaken it will be
necessary to demonstrate that the phenotypes observed in E. coli are similar
in S. mutans. Thus the first step is to establish the genetics of IHF
homologs in S. mutans.
This will include
the cloning and sequencing of the IHF genes in addition to constructing IHF
mutans in S. mutans. No IHF from any gram positive genera has yet been
characterized.
Once the genes that
code for IHF have been cloned, a preliminary set of complementation
experiments will be performed to compare E. coli and S. mutans IHF directly.
Finally the S. mutans strain constructed to be deficient in IHF can be
assessed to determine their ability to express their gtf genes relative to
wild type S. mutans.
Armed with toothbrushes, toothpastes, and
floss, people wage a daily war against cavity-causing bacteria. Now,
researchers in England have found another way to defeat those microscopic
foes. Teeth treated with a new synthetic molecule remain free of the feared
bacteria for up to 4 months, they report.
Most cavities are caused by the bacterium
Streptococcus mutans, which binds to receptor proteins on the surface of
teeth and collects into the film of plaque that dentists warn their patients
about. Unlike other bacteria in the mouth, S. mutans produces lactic
acid, which erodes tooth enamel.
"If you can prevent infection with
Streptococcus mutans, you will actually prevent tooth decay," says
Charles G. Kelly of the Guy's, King's, and St. Thomas' Hospitals Medical and
Dental School in London.
Kelly and his colleagues pursued this goal by
creating a peptide, or short sequence of amino acids, that blocks the
receptors and thus prevents S. mutans from sticking to teeth.
Earlier work had shown that S. mutans
possesses a large protein, called adhesin, that binds to receptors. Kelly's
team identified and synthesized a critical 20amino acid portion of adhesin
that, in the test tube, successfully binds to the receptors.
The researchers then tested how well the
synthetic peptide prevents S. mutans from colonizing human teeth.
The researchers first treated three groups of
four volunteers with an antiseptic mouthwash for 9 days to remove all
microbes from their mouths. Over the following 3 weeks, a solution
containing the peptide was dripped twice a week onto the teeth of one group,
which also used a daily mouthwash with the peptide.
The other two groups received similar
treatments with a different peptide or no peptide at all. The researchers
then monitored growth of S. mutans on the volunteers' teeth.
Those who received the binding peptide remained
free of S. mutans for at least 3 months. The bacteria appeared on the
teeth of the others within 3 weeks, however. Kelly and his colleagues report
their findings in the January Nature Biotechnology.
After treatment, the peptide remains in the
mouth for only about 6 hours, Kelly says, but it appears to exert long-term
antimicrobial effects. "If you can hinder [S. mutans] colonization
initially, other bacteria occupy the niche," Kelly says.
Plaque formed by harmless bacteria acts as a
protective film, crowding out the acid-producing S. mutans.
The results of the study are "quite striking,"
says Randall T. Irvin of the University of Alberta in Edmonton. "If this is
indicative of what will happen in a larger group, it's encouraging." He
expects that bacteria subjected to this treatment would evolve resistance
less readily than when attacked with antibiotics.
This approach could be applied to other
microbial targets, Irvin says, "but it will take a lot of work." Receptor
binding often triggers normal cell processes, so the peptides would have to
be designed to deflect bacteria without interfering with those effects.
