is potentially a deadly agent of bioterrorism. Unlike anthrax, which
has been so much in the news lately, plague is highly infectious
and can be readily passed from one person to another. The bite of
a plague-infected flea or the inhalation of just a few cells of
plague bacterium can kill. Like smallpox, plague can spread and
kill large numbers of people very quickly. Fortunately, it can usually
be treated with antibiotics.
History tells us how devastating
a plague epidemic can be. In what is known as the Justinian epidemic,
from 540 to
590 AD, plague spread from Lower Egypt to Alexandria to Palestine
and on to the Middle East and Asia. At its peak,
10,000 deaths occurred every day in Byzantium. Eight hundred years
later, in 1347, plague came to Italy from Asia or Africa, probably
by ship. By 1351, fully one-third of Europes population had
died from bubonic plague.
This European epidemic is
known as the Black Death or the Great Pestilence. In 1894, when
Andre Yersin identified the tiny bacterium that causes plague, he
named it pestis after the Great Pestilence. He tried to name the
genus Pasteurella after his mentor, Louis Pasteur. But Yersinia,
after its discoverer, is the name that stuck.
Today, Yersinia pestis
is one of several infectious diseases and agents of bioterrorism
that researchers across the Department of Energy complex are studying
as part of the Chemical and Biological National Security Program.
This program comes under the purview of DOEs National Nuclear
Security Administration (NNSA). At Livermore, the work on Y.
pestis also receives support from Laboratory Directed Research
Scientists at Livermore have
developed DNA signatures for Y. pestis that can be used to
quickly detect and identify plague outbreaks. (See Uncovering
2000.) Signatures for nine strains of the disease have been
submitted to the Centers for Disease Control and Prevention in Atlanta,
Georgia, where they are undergoing a rigorous validation process.
detection method proved its mettle in northern Arizona last June
when it was used to identify a plague outbreak in prairie dogs in
just four hours. Standard detection processes, which require growing
the suspected bacteria in a laboratory, take 36 to 48 hours.
For a plague detector to
be truly effective, it must do more than simply indicate the presence
of a specific organism known to cause plague, says Pat Fitch, who
leads Livermores Chemical and Biological National Security
Program. The detector also must be able to identify the specific
traits found in atypical plague-causing organisms. Scientists know
of several hundred strains (or isolates) of Y. pestis, and
they do not all behave in precisely the same way. A few strains
are believed to have been genetically modified or engineered to
be more deadly. There have also been two clinical cases of naturally
occurring antibiotic-resistant plague. Knowing the precise identity
of a strain of plagueor of any infectious disease, for that
mattercould help physicians treat a patient properly.
Plague research at Livermore
currently is focusing on what makes Y. pestis so virulent
and able to overcome the defenses of a host organism. Fitch is leading
the Pathogen Pathway Project, using plague as a prototype for the
functional genomics of a larger set of pathogenic agents that could
be used in biological terrorism. (See the box below for more information
on functional genomics.)
Besides building better detectors,
work on the Y. pestis genome will also lead to a better understanding
of pathogenicity and better vaccines and treatments for the disease.
The ultimate goal, says Fitch, is to produce a computer model
that simulates the workings of a cell so that we can better manage
exposure to pathogens.
Sequencing to Functional Genomics
DNA decoding, known as sequencing, is the process that
determines the precise order
of the four nucleic acid basesadenine (A), thymine
(T), guanine (G), and cytosine (C)
that comprise the DNA of all living things. In
the case of Yersinia pestis, its DNA sequence comprises
4.66 million bases. The Sanger Institute in Great Britain
recently published the complete and annotated sequence
of the Y. pestis genome. For three years before
that, a preliminary version of the sequence had been available
for use by researchers throughout the world.
As the DNA genome,
or parts list, for an organism becomes availablewhether
it be for plague, mice, or humansresearchers begin
to examine the accumulated sequence data very closely.
They are trying to identify what makes this particular
genome work the way it doesits wiring diagram, so
to speak. They search for specific genes. They also study
how DNA works with proteins and the environment to create
complex, dynamic living systems. Proteins are large molecules
composed of amino acids that perform most life functions
and make up the majority of cellular structures.
as this field of research is known, encompasses many topics.
Some researchers examine when, where, and under what conditions
genes are expressed that is, turned on. Others study
the expression and function of the proteins encoded by
certain genes. Still others use x-ray crystallography
and other methods to generate three-dimensional structures
of proteins, which offer clues to their
function. Researchers may inactivate or knock out genes
and study the results to learn what specific genes do.
Other researchers compare the DNA sequence of several
organisms in an effort to identify unique genes and
interpret their function. Research is under way in several
of these areas as Livermore examines what makes Y.
pestis so virulent.
highly contagious Y. pestis is an excellent model for studying
the interactions of a pathogen and its host. In the case of Y.
pestis, the host may be a flea, a rodent, or a human. Fleas
carry plague bacteria and help transmit the disease. Once an infected
flea bites a rodent or human, the bacteria begin to multiply in
the new host, and their virulence shifts into high gear. Y. pestis
circumvents the hosts defenses by injecting into host cells
a series of virulence factors that inhibit the response of the immune
Earlier research has shown
that when Y. pestis is grown at the body temperature of a
flea (26°C), its cells divide, but it does not express (turn
on) many of the genes that make it virulent in rodents and humans.
When the temperature increases to 37°C (human or rodent body
temperature), the bacterium begins to produce the proteins essential
to its virulence. This virulence mechanism can be induced in the
laboratory, making plague relatively easy to study.
Examination of the Y.
pestis genome before and after virulence has been induced shows
what genes have been turned on. But that information is not enough
to show precisely which genes are responsible for various aspects
For comparative purposes,
a Livermore team led by microbiologist Emilio Garcia collaborated
with the Institut Pasteur in France to sequence Y. pseudotuberculosis,
the parent organism of Y. pestis. Although their DNA sequences
95 percent identical, Y. pestis and Y. pseudotuberculosis
behave differently. Y. pseudotuberculosis lodges in the intestine
and causes flulike intestinal distress. Y. pestis is also
closely related to the mild-mannered Y. enterocolitica, an
that is itself very much like Y. pseudotuberculosis. Y.
enterocolitica is currently being sequenced by the Sanger Center
in Great Britain.
Yersinia pestis, which causes plague, is a pathogen likely
to be used by terrorists. (b) Its DNA forms loops, unlike human
DNA, which forms strands. Scientists studying it screen between
two to five million of its nucleic acid bases to find unique
regions (circled). Using polymerase chain reaction technology,
the unique regions can be amplified thousands of times and processed
to identify and characterize Y. pestis.
Bacteria evolve very
efficiently and make use of about 80 percent of their DNA,
says Fitch. By comparison, humans use only about 30 percent of their
DNA. Aiding speedy evolution are the many insertion sequences in
a bacterial genome. Insertion sequences are bits of DNA that allow
large regions of DNA to replicate themselves and move around the
genome, relocating themselves somewhere else. When an insertion
sequence lands within a gene, it deactivates that gene. These transfers
can also occur across species, and it is not difficult for a bacterium
to grab DNA from another bacterium.
Y. pestis evolved
from Y. pseudotuberculosis within the past 15,000 years,
a rapid evolution even for bacteria. Something happened then
to cause Y. pestis to learn how to live in a flea,
In addition to their normal
chromosomal DNA, bacteria may have smaller circles of DNA known
as plasmids. Plasmids replicate separately from chromosomal DNA
and often house genes that encode enzymes critical to the host cell
or organism. For example, when a bacterium has become resistant
to antibiotic drugs, it is usually because the bacterium has acquired
a new plasmid.
One Y. pestis plasmid
encodes at least two genes that allow Y. pestis to survive
in fleas. Another plasmid is home to the gene that activates the
diseases invasiveness. Researchers have found that Salmonella
has a similar plasmid, which one bacterium probably obtained from
The interesting thing
is that if you insert the three pestis plasmids into Y.
pseudotuberculosis or Y. enterocolitica, you dont
get pestis, says Garcia. So something else is
going on. Unfortunately, its never simple.
Once its virulence genes
have been turned on, plague infects its host using what is known
as Type III secretion, an injection mechanism more colorfully called
Yersinias deadly kiss. Salmonella typhi,
Escherichia coli, Chlamydia psittaci, various species
of Bordetella, and other pathogenic bacteria appear to share
this syringelike injection mechanism. This common trait may indicate
another area of transferred genomic material.
research on plague started, many of the genes critical for virulence
had been identified but were poorly understood. The same was true
for the underlying mechanisms of virulence. There was also little
understanding of the gene and protein interactions that take place
between the pathogenic bacteria and its host.
The Pathogen Pathway Project
is using functional genomics tools to identify genes important to
virulence and understand the pathways of virulence. The teams
hypothesized pathway, from DNA to the host organism, is shown in
the figure in the next paragraph.
Type III secretion, a syringelike injection mechanism more colorfully
called Yersinias deadly kiss, which is how
plague infects a host once its virulence genes have been turned
An early task for Livermore
bioscientists and computations experts was to develop a relational
database of the DNA sequence of Y. pestis. In collaboration
with the DOE Genome Consortium at Oak Ridge National Laboratory,
these data were used to computationally predict where the 4,500
genes in Y. pestis are located and which genes might be associated
Next, Livermore bioscientist
Vladimir Motin and colleagues designed chemical reagents for extracting
over 300 genes from Y. pestis DNA, including all known virulence-associated
genes on the plasmids. In an initial test, they extracted 85 genes
associated with virulence and spotted them on a glass microscope
slide alongside 11 control spots, making up a 96-spot microarray.
A microarray permits scientists
to study the response of thousands of genes or other pieces of DNA
quickly and efficiently in a process known as transcript profiling.
In the process, each gene receives some kind of stimulus, causing
it to turn on and produce messenger RNA (mRNA). In the case of plague,
the stimuli are changes in temperature and calcium concentration.
The production of mRNA leads, in turn, to the synthesis of unique
proteins. The level of mRNA can be measured for each individual
gene. The more active or expressed genes there are, the more mRNA
will be present.
For the 96-spot microarray,
the team developed a protocol to study the response of Y. pestis
genes under conditions that mimic the infection process: at both
flea and human/rodent body temperatures, 26°C and 37°C,
and at calcium levels that correspond to those of blood (higher
level) and organs (lower level), the latter location being where
more virulence genes are expressed.
More recently, they developed
a microarray for all 4,500 Y. pestis genes. All of the genes
are being mapped at six time intervals as temperature rises and
calcium concentration drops. The team is thus beginning to establish
a timeline for how and when genes change and are expressed while
the plague bacterium is infecting a human host. Some genes are expressed
early, while others are late-onset genes. A detailed picture of
how the bacterium behaves during the infection process will provide
useful information for the development of diagnostic techniques
and treatment methods.
Garcia and other researchers
also completed a detailed analysis of three Y. pestis plasmids,
which allowed them to confirm the location of several known virulence
genes and to uncover four novel ones believed to contribute to virulence.
Computerized comparisons with other genomic databases indicated
the presence of a large number of virulence-related genes that are
similar in both closely related bacteria such as Y. pseudotuberculosis
and distantly related bacteria such as E. coli. The team
also found numerous gene coding regions whose function they could
Using a proteomic approach
of protein separation techniques and mass spectrometry (MS), Livermore
researchers led by Sandra McCutchen-Maloney are analyzing complex
mixtures of proteins isolated from Y. pestis. By comparing
samples grown at the two physiological conditions mimicking the
flea and the human (at 26°C and 37°C, respectively) and
at low calcium concentration to induce virulence, the team is detecting
differential protein expression to identify candidate proteins important
for Y. pestis pathogenicity. Comparisons are also being made
between human cells that have and have not been exposed to Y.
pestis in order to understand the host immune response. Because
it is the proteins that are actually responsible for virulence effects,
the group is also working to correlate their proteomic data with
genomic data obtained from microarray experiments. To learn more
about the individual proteins responsible for virulence, the team
is using various biochemical assays to test functional models of
the candidate virulence factors. In addition, McCutchen-Maloneys
group is looking at hostpathogen interactions by using surface-enhanced
laser desorption ionization (SELDI) MS to study various proteinDNA
and proteinprotein interactions within Y. pestis and
between Y. pestis and the human host. For example, regulatory
proteins that bind to genes and control differential expression
are under investigation, as are the specific proteinprotein
interactions of suspected virulence factors. These molecular interactions
are key to the genetic feedback that occurs as a pathogen infects
its host, as shown in the figure below.
schematic diagram of information that is hypothesized to describe
the pathways of virulence in a pathogen. The regulatory (feedback)
loop is often nonlinear, and there can be multiple feedback
paths with complex interactions.
Before Garcias team
completed its comparative sequencing of Y. pseudotuberculosis,
microbiologists Gary Andersen, Lyndsay Radnedge, and others examined
the differences between Y. pseudotuberculosis and Y. pestis
using a different technique. This process, developed in Russia,
is known as suppression subtractive hybridization (SSH). SSH identifies
regions of DNA that are present in one species but absent in another.
SSH has the advantage of
requiring only small amounts of genomic DNA. It can be used with
any genome, even one that has not yet been characterized. It is
especially useful for identifying the large genomic differences
typically found between bacterial genomes. For example, SSH identified
the genetic material that causes Kaposis sarcoma, a skin lesion
associated with HIV and AIDS. At Livermore, SSH has been useful
for finding differences among anthrax strains and other potential
agents of bioterrorism.
Comparison of Y. pestis
and Y. pseudotuberculosis revealed seven DNA regions in Y.
pestis that do not occur in Y. pseudotuberculosis. Four
of them occur very closely to one another on the Y. pestis
genome. It is fair to assume that pestis acquired this
region during its evolution from Y. pseudotuberculosis,
To learn more about the
function of genes in these areas, Garcia and others are beginning
knock-out studies. They will inactivate, or knock out,
one gene at a time and test the resulting bacterium on an animal
to see how the host and its genes respond. This is slow, laborious
work, but it will help to determine what the function of each Y.
pestis gene is, if any, and what gene or genes in the host are
expressed as a response. This detailed examination of pathogenhost
interaction for plague will be the first of its kind.
96-spot microarray for transcript profiling of 85 genes of Yersinia
pestis; 11 of the spots are controls. Genes tagged with fluorescent
dyes change color in response to various stimuli.
Research to date on plague
lays the groundwork for additional work planned at Livermore in
the areas of microbiology, proteomics (the global study of proteins),
bioinformatics (the integration and analysis of biological data),
and biological modeling for the NNSAs Chemical and Biological
National Security Program. Some of the research will elaborate on
plague, some will examine a broader spectrum of human pathogens,
and some will further the development and use of biodetectors, mass
spectrometry, and other technologies.
In the U.S. today, plague
pops out of the rodent population and into the human populace occasionally
in the desert Southwest. It is a larger problem in a few other countries.
But the real fear is that plague could be used as an agent of mass
destruction. At least in industrialized countries, it is unlikely
that plague would cause the huge number of deaths that occurred
during earlier epidemics. Better sanitation, a more educated populace,
and a far superior medical system would likely prevent that. But
the world needs to be prepared.
Key Words: bioterrorism
agents, Chemical and Biological National Security Program, plague,
surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS),
suppression subtractive hybridization (SSH), virulence, Yersinia
pestis, Yersinia pseudotuberculosis.
information, contact Pat Fitch (925) 422-3276 (firstname.lastname@example.org).