Phenotype MicroArrays for Microbial Cells

Pharmaceutical companies and biotech companies spend much time, effort, and money screening and assessing drug candidates. This process could be made much more efficient and less costly with tools that can quickly and accurately assess issues such as (1) Sites and modes of action of drugs, (2) Drugs with high specificity versus drugs with side effects, and (3) Beneficial as well as detrimental interactions with other drugs. PM technology provides an ideal tool and can be used in all these ways to evaluate potential new drugs.

The experimental approach is very analogous to testing cells with genetic changes. In the case of genetic changes, a gene can be "knocked out" which usually means that a protein is not made (i.e., also "knocked out") and consequently some cellular function is blocked. Most drugs are targeted to "knock out" the function of a specific protein, so adding a drug to a cell should result in phenotypic effects that are very similar to "knocking" out the gene.

Drugs can be added to cells prior to inoculation into PMs. By looking at the phenotypes altered by the drug one can determine the physiological functions in the cell that are affected. This information will indicate: (1) the site and/or mode of action of a drug, (2) whether the drug is specifically hitting one target or whether it is interfering also with other cellular processes and therefore likely to cause side-effects, (3) potentially beneficial as well as detrimental interactions with other drugs (many of the phenotypes in the PMs test for increased sensitivity or resistance to existing drugs).

Toxicological testing based on the use of cell lines is gradually replacing animal testing as a more cost-effective and humane approach. Using PMs, testing would be performed essentially as described in the preceding paragraph for drug testing. One simply adds a chemical to the cells and inoculates into PMs. Interference with an aspect of cell physiology will be manifested as an altered response in the PMs. The information from PMs will indicate toxicity levels as well as mechanisms of toxicity. Some chemical agents may be toxic only under specific growth conditions or only in combination with other toxic chemicals. Since PMs contain thousands of different testing conditions, they provide toxicological information that is much more thorough and comprehensive.

Cells have on the order of 2,000 to 40,000 genes. Even in the simplest and most studied microbial cells, only about half of the genes have a known function. Through a variety of genetic and biochemical techniques, scientists are identifying many genes as being "especially interesting". For example, from studies of hereditary human genetics, these genes may be implicated in an important disease or syndrome. Alternatively, they may be involved in cancer or microbially-induced or chronic diseases and thereby be potential targets for new drugs. Genes of great importance (both biologically and commercially) are also being identified in other animals, plants, and microorganisms. However, with current technology it is very difficult, expensive, and not-at-all straightforward to determine the function of these important genes.

The method most commonly proposed for determining the function of unknown genes relies on the use of nucleic acid- based microarrays. DNA microarrays are used to measure mRNA levels under several growth conditions, and then the data are analyzed to see if the mRNA levels of the unknown gene go up and down in correlation with the mRNA levels of a known gene. The hope is that by grouping genes that are regulated in the same manner, biologists will be able to discern genes that are members of the same functional pathways. This approach is rather lengthy, expensive, and complex and it relies on making numerous assumptions that may be incorrect.

PMs make it possible to go directly from a gene of interest to a cellular function. The experimental approach is to simply "knock out" the gene of interest in a cell line to create an isogenic pair of strains. The biologist simply inoculates the isogenic cell lines into the PMs and looks for one or more phenotypic differences. Alternatively one can do a "knock in" genetic construction in which a gene of interest is added to a cell line. Here again, the isogenic cell lines are assayed in PMs and one looks for discernable phenotypic differences. By analyzing isogenic strains and using PMs in large scale, high-throughput studies we can start from a genomic map and generate a virtual phenotypic map. PM technology is fast, inexpensive, and simple, and it does not make assumptions about gene transcription, translation, or post-translational modifications.

Finding Genes that Code for New Drug Targets

PM technology can be used in comparisons of cell lines to find new drug targets. For example, in antimicrobial R&D, pairs of pathogenic and non-pathogenic microbial strains can be compared. Pathogenic strains are known to contain additional genes such as drug resistance genes and pathogenicity islands. These extra genes code for proteins that convey additional phenotypes to the microbe and may be useful as drug targets.

Another example is the search for new anti-cancer agents. Cancerous cells can be compared to non-cancerous cells in PMs to look for phenotypic sites of potential vulnerability.

A major objective of many animal and plant genetic projects is the improvement of targeted cell lines or seed lines. After genetic manipulation, cell lines must be evaluated to see if they picked up the desired phenotypic traits and also to see if they picked up any undesired secondary phenotypic traits. PM technology will clearly be a very useful tool in these types of developments.

Cell lines can and do change when they are subcultured. This is due, at least in part, to unstable genetic constructions and to selective pressures that biologists unknowingly apply to cultured cells. Cell line stability is an important issue in basic research (e.g. cancer research) and in important medical applications such as vaccine and recombinant protein production. It is essential to know when and in what ways cell lines are changing

There is a great deal of interest in basic research and applied development in optimizing conditions for growing cells, especially animal and plant cells. In basic research it is important to understand the growth requirements of cells so that they can be handled properly and cultured rapidly. In commercial ventures, cells are cultured in vitro for many applications including cell transplantation and gene therapy, tissue replacement, vaccine production, and recombinant protein production. Many of the PMs contain biochemicals that may act as nutrients for certain cell types. A stimulatory effect on the cells will be detected as an increase in respiration and therefore an increase in color from the redox dye. This will point the scientist toward nutrients that improve the growth and health of the cells.

Many plants and microorganisms can go through stages in their life cycle where they form seeds or spores and later germinate. The conditions that trigger these changes are often very difficult to discern, requiring a very precise combination of culture conditions. Knowing optimal conditions for sporulation and germination can have important economic benefits. Sporulation is important in basic research and genetic and physiological studies, for example with actinomycetes, yeasts, and filamentous fungi. Germination is important in agriculture for cultivation of mushrooms and plants. PM's will offer biologists a very simple and straightforward means to streamline this testing.

Secondary metabolites such as antibiotics and pigments typically are produced under very specific growth conditions, often involving some special limitation of growth. PMs can provide thousands of growth conditions and allow a very rapid and easy way to look for optimal production conditions.

Many of the phenotypes that are measured with PMs indicate the presence of enzymatic activities that may have potential commercial use. Examples are enzymes involved in catabolism of carbon, nitrogen, phosphorus, and sulfur, and enzymes that protect cells against toxic chemicals. Therefore PMs can be useful for screening microorganism collections to look for these activities.

Other Cell-Based Assays

PMs provide an easy and highly efficient means for testing cells under thousands of diverse growth conditions. It provides a flexible and versatile format for many other types of cell-based experiments and assays. The limits are defined by the creativity and imagination of the scientist using them.