A Devil in the Details: What are those strange genes in the Candida albicans genome?

BY RICHARD KESSIN, PH.D. ASSOCIATE DEAN FOR GRADUATE AFFAIRS
Dr. Aaron Mitchell, left, and his students, Clarissa Nobile and Vincent Bruno
SACCHAROMYCES CEREVISIAE, THAT WARHORSE OF BIOLOGY, is much beloved by bakers, brewers, and molecular geneticists. Candida albicans, which looks much like baker's yeast on a petri dish, is beloved by no one. A million Saccharomyces cells injected into a mouse cause no ill effect. A million Candida cells will kill it — and it takes far fewer to kill an immune-suppressed animal. Alas, we are not good at killing Candida — or any other pathogenic fungus.
C. albicans presents a lot of experimental difficulties for basic scientists. Baker's yeast is haploid and has a well defined sexual cycle to manipulate and combine mutations. C. albicans is diploid and if you want to make a mutant and ask whether the affected gene and the protein it codes for are essential for pathogenicity or would make a good drug target, you have to mutate two copies of the gene, which has been hard to do. And if you make these mutations, you still can't move them from one strain to another and make different combinations because the wretched Candida has no sexual cycle.
Because colonies of Candida and Saccharomyces look alike on petri dishes, biologists had been lulled into thinking that they share a great majority of genes. They looked to Saccharomyces, whose genome and genetics have both been worked out, for inspiration about Candida. But with the Candida genome newly sequenced and annotated — so that many of the genes are assigned a putative function — we are left with a large number that have no relatives (homologues) in Saccharomyces. How odd! But how to get at them and how to ask which are involved in infection and which can be attacked with new drugs?
Here the laboratory of Aaron Mitchell, Ph.D., professor of microbiology, has contributed mightily, first to the annotation of the 7,000 genes that make this organism its unpleasant self and, now, a method to mutate both copies of every gene in the genome and open the way to understanding more about the mechanisms of pathogenicity. Sometimes when the easy way fails, you have to back up, develop new methods, and try again.
Biofilms of Candida albicans are a major source of infection. In this pair of confocal micrographs, biofilms are compared from a wild-type strain (DAY185, right panel) and a biofilm-defective insertion mutant (GKO814, left panel). The images are depth views that show individual types of cell; a color scale indicates the distance of cells from the top of the sample. For the wild-type strain, long cylindrical cells (hyphae) are apparent  at 50-200 micrometers in depth (blue-yellow), and small round yeast cells lie at the base of the biofilm at 250 micrometers depth (red). In contrast, the mutant strain makes only a thin biofilm of total depth 20 micrometers and fails to produce long hyphae.
Vincent Bruno, a Ph.D. student in the Mitchell laboratory, building on earlier work by Brian Enloe and Aviva Diamond, has developed a clever genetic technique that allows researchers to insert random bits of DNA, each with a selectable sequence inserted into it. This DNA fragment enters the cell and inserts into a single gene according to the sequences at the ends. Cells in which this happens can be selected. But we have still only knocked out a single copy, or allele. We could have done this with existing methods. The beauty of the new technique is that occasionally a recombination event occurs that deletes the other gene in the diploid and because of the construction of the original insert, these rare double knockouts can be selected to grow on a petri dish. If a gene is essential for growth, double knockouts do not grow. If a gene is essential for pathogenicity, but not growth on simple media, strains carrying two defective versions will grow and can then be tested for infectivity. The technique can be used on a large scale, with lots of different genes disrupted. Now we can ask which genes, known or unknown, are required for this pathogen to do its work, whether by growing on a mucosal surface, or as nosocomial blood stream and deep tissue infections.
But what aspect of pathogenesis should one investigate? Anyone who has ever seen thrush, which is one infection caused by Candida albicans, realizes right away that these organisms do not grow in solitude, but rather form mats or biofilms. These also can be found on in-dwelling catheters. Biofilms are not just piles of cells, but rather differentiated structures that are produced in a temporally regulated way. During the process Candida extends hyphae (long filaments) and secretes an extracellular matrix of polysaccharide and protein. As in the case of bacterial biofilms, Candida structures keep out drugs and maintain the population of fungi.
Which of the 7,000 genes are dedicated to making biofilms and do any of them have an Achilles' heel? Using the new genetic techniques, Clarissa Nobile, Vincent Bruno, and postdoctoral fellow Mathias Richard have isolated mutants that are defective in the formation of biofilms. So far there are three mutants that block the beginning of biofilm formation and one that allows it to start but not to progress. The group calculates that something over 100 genes may be involved. The problem the lab now faces is to figure out what these genes do. One of the newly discovered genes is necessary for respiration, but the others are a mystery. Perhaps their exact functions can be identified and these will be sufficiently different for the processes of the host to allow novel drug development. We are, after all, starting with genes that are not present in humans or other organisms. We have not had a stellar history of success with controlling Candida or other fungi, especially in immune-compromised people, and the Mitchell laboratory is now returning to square one. Armed with the full genome sequence and their new genetic technology, they will not stay there long.
More information on Dr. Mitchell's work can be found at cumicro2.cpmc.columbia.edu/

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