Cell Scenario: A New Look at Microarrays The field generally known as cell-based analysis started
in the 1970s with two-dimensional gel electrophoresis,
which tracked levels of proteins in cells. Next came DNA
microarrays that measured thousands of genes simultaneously.
Today researchers are exploring the potential of a new
tool, the Phenotype MicroArray™ (PM), which offers
a panoramic view of cellular events. With the recent introduction
of a mammalian version of the PM, the tool is poised to
provide even more insight into how cells behave when affected
by environmental agents.
Just like a battery of tests on a person’s blood
can scan the health of vital organs, the PM can scan the
physiology of cells, yielding data on hundreds of traits
at once. Typical cell-based assays measure only one trait
at a time (for example, cell death or DNA synthesis), but
the PM can measure up to 2,000 traits--or phenotypes--under
hundreds of growth conditions. The PM can be used to fingerprint
cell lines used in research or biomanufacturing to ensure
stability, or monitor the effects of drugs or toxicants
on cells. Researchers can also compare normal and diseased
cells to see how cell functions are altered.
Building on Success
PM developer Barry Bochner, a bioengineer, patented a
simple dye method to measure cellular respiration while
a graduate student in the 1970s. Ten years later, he started
operations at Biolog Incorporated in Hayward, California,
to commercialize the technology as a tool to identify more
than 1,900 species of bacteria and fungi based on patterns
of carbon metabolism. These first products, a series of
five kits that each can identify about 300-600 species,
have been a chief tool of microbiologists since 1988.
Microbes grow on many carbon sources, including glucose
and other sugars, amino acids, and carboxylic acids. The
original microbial identification kits were based on redox
reactions that produce a color change in microwells; that
is, when cells utilize carbon for energy, they turn a colorless
dye purple. Each well of the microbial identification kits
contains a different assay--a different carbon fuel
and a tetrazolium dye--dried on the bottom. When a
microorganism metabolizes a carbon source, an irreversible
chemical reaction occurs, and the intensity of the purple
color formed in each well over time is analyzed and compared
to a database for identification.
Tetrazolium dye has long been used by toxicologists to
measure cell viability. Bochner’s team improved the
older dye chemistry by making it water-soluble and less
toxic to cells. In addition, they eliminated high background
color that results from serum in culture media reacting
with older tetrazolium dyes. With the improved dye, researchers
can measure as few as 100 or up to 20,000 cells in one
well.
Bochner’s next invention came during the genomics
era, when DNA microarrays allowed scientists to measure
the expression of thousands of genes simultaneously. “I
had this idea that we could go beyond carbon metabolism,” says
Bochner, now chairman and vice president of research and
development at Biolog. So he created the PM.
The PM uses the same technology as the microbial identification
kits, except that the PM measures nearly 2,000 chemical
reactions related to carbon, nitrogen, phosphorus, and
sulphur metabolism, as well as pH, growth range, and sensitivity
to antibiotics and stress factors. The reactions reflect
key cell pathways, including cell surface binding, biosynthesis
of molecules, stress and repair processes, and the metabolism
of carbon and nitrogen. “With two thousand phenotypes
we can detect most of the important changes in cellular
physiology,” says Bochner.
Few technical skills are required to run any of the Biolog
kits. A researcher simply adds a cell suspension to the
wells to start the reactions. The data generated are captured
and interpreted by Biolog’s OmniLog® system,
a combination incubator and scanner that monitors, analyzes,
records, and graphs changes in each well with proprietary
bioinformatic software. Data are collected in 15-minute
intervals for up to 48 hours.
PM Applications
Data from the PM and DNA microarrays complement each
other and bridge the gap between molecular changes and
biological outcomes. “Just because a gene is turned
on or off doesn’t mean that a biological pathway
gets turned on,” says Bochner. The PM gives a global
view of cellular processes by detecting how gene changes
alter one or many biological properties of cells.
The PM can therefore help researchers assess the effects
of environmental toxicants on cells. Toxicants work by
interfering with cellular respiration, damaging the pathways
that cells need to live and grow, so the formation of the
purple dye color is either reduced or totally prevented.
Similarly, pharmaceutical companies can use the PM to monitor
toxicity of new drugs. “This is a general method
for studying the effects of any chemical on cell pathways,” says
Bochner.
The function of unknown genes can be identified with
knockout experiments to see what cell phenotypes appear,
disappear, or get stronger or weaker. In knockout mutants
of Pseudomonas aeruginosa, which infects the lungs
of patients with cystic fibrosis, investigator Ian Paulsen
of The Institute for Genomic Research uncovered unusual
transport functions for genes. “Biolog lets us pin
functions to novel genes,” says Paulsen, who is mapping
the physiology of this pathogen. “There is no comparable
product on the market,” he adds, “that lets
you do high-throughput physiological screening. ”
The PM can also highlight pathways linked to a pathogen’s
virulence. At Lawrence Livermore National Laboratory, Sandra
McCutchen-Maloney studies Yersinia pestis, the cause
of bubonic plague. Y. pestis, one of the most virulent
bacteria known, is feared as a possible bioterrorism threat.
The microbe infects rodents in North America, and fleas
can transmit Y. pestis to humans. When tested with
the PM under biologically relevant conditions that occur
in fleas and humans, Y. pestis proved tougher
than expected and less vulnerable to antibiotics. The data “uncovered
new pathways involved in virulence that could be targets
for future therapeutics,” says McCutchen-Maloney,
who presented these findings at the 44th annual meeting
of the American Society for Cell Biology in December 2004.
 |
| |
image: Biolog, Matt Ray/EHP |
In another application, veterinarian Jean Guard-Bouldin
of the USDA worked to solve the mystery of how eggs with
uncracked shells become contaminated with Salmonella
enteriditis. The PM was used to “identify a number
of physiological capabilities in Salmonella that
we would not have otherwise predicted,” says Guard-Bouldin.
It turned out that the egg-contaminating strains had evolved
metabolic capabilities that adapted the pathogen to grow
in the reproductive tracts of hens that otherwise appeared
healthy. “Defining the characteristics of egg-contaminating
strains by a PM approach also accelerated our ability to
locate the genes that were undergoing rapid evolution,” she
says.
Like DNA microarrays, the PM generates massive amounts
of data. “The challenge is to find ways to make sense
of large data sets,” says Paulsen. It is up to bioinformatics
experts to develop statistical methods to analyze data
in a way that makes sense for their application.
With the new mammalian PM, introduced in September 2005
at the Society for Biomolecular Screening conference held
in Geneva, Switzerland, researchers now have the ability
to work with a variety of human cell lines, ranging from
blood to liver cells, as well as primary rat hepatocytes,
which toxicologists prefer. The first mammalian PM contains
384 assays for energy-producing pathways shared by a range
of cell types.
“It was a big leap to mammalian cells,” says
Bochner, who spent three years adapting the microbial methods
to work in more complex mammalian cells. The goal is to
expand the mammalian PM to 2,000 phenotypes as the methods
are perfected.
Carol Potera
| New Applications
for the Tried-and-True |
Environmental scientists are finding
new applications for Biolog’s tried-and-true microbial
identification kits. Ken Cullings, an evolutionary ecologist
at NASA’s Ames Research Center, used one kit to evaluate
the effects of defoliation on soil fungal diversity. In a
mixed lodgepole pine and Engelmann spruce forest in Yellowstone
National Park, Cullings’s team removed half the needles
on naturally reseeded pine seedlings. A year later, soil samples
showed a significant increase in the physiological diversity
of soil fungi. “Fungi specific to pines jumped ship
to the nondefoliated spruces when the pine was defoliated,” says
Cullings.
The results, reported in the April 2005 issue of Applied
and Environmental Microbiology, translate directly
to forestry practices: after harvesting old-growth forests,
replant with mixed tree species to improve future productivity.
The same advice holds for replanting after forest fire
devastation. “We showed in a pine-spruce ecosystem
that mixed species moderate detrimental effects,” says
Cullings.
Another NASA scientist, microbial ecologist Jay Garland
of the Kennedy Space Center, is seeking beneficial microbes
to prevent Salmonella contamination of alfalfa sprouts.
NASA hopes to someday send alfalfa sprouts on space missions
to produce oxygen and water and also serve as a food source
for astronauts. But sprouts are vulnerable to Salmonella contamination.
Since 1995, at least 21 food poisoning outbreaks due to
eating contaminated sprouts have been reported in the United
States, according to Garland.
To prevent infection, seeds are soaked in bleach, but
the practice is neither completely effective nor palatable
to health-conscious consumers. “An alternative is
to manipulate the numbers of good microbes that live naturally
on sprouts to stop Salmonella from growing,” says
Garland. He uses one of the microbial identification kits
to screen bacteria growing naturally on sprouts to identify
mixtures that rapidly block the growth of harmful Salmonella and
other pathogens, as described in the January 2005 issue
of Journal of Food Protection. This biocontrol strategy
could protect both Earth-bound consumers and astronauts.
James Thomas, a molecular biologist at Canada’s
University of Lethbridge, performs source tracking of fecal
indicator bacteria in southern Alberta, where an intense
livestock industry generates enormous amounts of waste.
During the summer, smaller communities often must boil water
due to high levels of fecal bacteria. In collaboration with
Health Canada, Thomas screens environmental water samples
from the city of Lethbridge and surrounding rural communities
with Biolog kits to find out how the contamination is happening,
and why it especially occurs in the hot weather.
Thomas and his colleagues used a microbial identification
kit to monitor 37 sites within the Oldman River Basin, where
a network of irrigation canals and three reservoirs provide
water to the agricultural region. The results showed that
the lowest levels of the enteric bacteria Escherichia
coli and Enterococcus faecalis are detected at
the outflow of reservoirs. “Reservoirs remove microbial
pollutants in agricultural wastewaters,” says Thomas.
The construction of more in-stream reservoirs or wetlands
could significantly improve the water quality of rural watersheds,
they concluded in the 15 September 2005 issue of Science
of the Total Environment. -Carol Potera
|
|
