Tracking down virulence in plague
PLAGUE 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 Europe's 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 DOE's National Nuclear Security Administration (NNSA).
At Livermore, the work on Y. pestis also receives support from Laboratory Directed
Research and Development.
Scientists at Livermore have developed DNA signatures for Y. pestis that can
be used to quickly detect and identify plague outbreaks. (See Uncovering
Bioterrorism,S&TR, May 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.
Livermore's DNA-based 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 Livermore's 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
Plague as prototype
The 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 host's defenses by injecting into host cells a series of virulence factors
that inhibit the response of the immune system.
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 of virulence.
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 are about
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 intestinal bug that is itself very much like Y. pseudotuberculosis. Y. enterocolitica is
currently being sequenced by the Sanger Center in Great Britain.
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, says Garcia.
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 disease's invasiveness.
Researchers have found that Salmonella has a similar plasmid, which one bacterium
probably obtained from the other.
The interesting thing is that if you insert the three pestis plasmids
into Y. pseudotuberculosis or Y. enterocolitica, you don't get pestis,
says Garcia. So something else is going on. Unfortunately, it's never
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 Yersinia's deadly kiss. Salmonella typhi, enteropathogenic
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.
Before Livermore's 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 team's
hypothesized pathway, from DNA to the host organism, is shown in the figure
in the next paragraph.
Expression and function
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 with virulence.
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 not determine.
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-Maloney's 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.
Differences are key
Before Garcia's 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
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 Kaposi's 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.
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 NNSA's 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.