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year are exceptionally low; in comparison, temperate grasslands and prairies have production rates of around 1 kilogram of carbon per squared meter per year. However, the hypolithic production is an important food source for grazing nematodes and protozoans, and therefore the basis for the survival of a whole ecosystem in an extreme environment [17]. Presumably the polygonal patterning of primary production will result in a corresponding distribution of grazing organisms. The Polar deserts are extreme cold habitats, but the hypoliths in hot deserts undergo a much greater temperature variation, with annual temperatures ranging between 58C and 658C, and diurnal variations can also be similarly severe [18]. The comparison of the physiological capabilities of hypolithic species from both desert extremes is a compelling one for future studies. Multidisciplinary approach One of the most striking aspects of this study of hypolith productivity in polar deserts was the physical determination of the productivity of the polygon stone fields [1]. Without the periglacial activity, there would be very reduced, or no primary production under the stones. This is a superb example of why an understanding of how the environment is structured is so fundamental to our understanding of microbial ecology. Increasingly, extremophile investigators need to form consortia with researchers from wide-ranging disciplines to fully appreciate how and why life persists at extremes. The hypolith researcher needs to work with the geologist and meteorologist, just like a biologist studying psychrophiles cannot proceed very far without coordinating research goals with the glaciologist and physicist. We now know that hypoliths can grow under stones in deserts, and by definition these are habitats of low irradiance and limited water supply. Rates of primary production and growth rates can never be high under such environmental conditions. The exciting next phase will be to elucidate how this diverse group of cyanobacteria and algae can live in these conditions and what biochemical and physiological adaptations they have. Then we will understand more about their evolution, and potentially about what life on an earlier earth was like, and who

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knows, they might be a good proxy for life in extraterrestrial systems. References 1 Cockell, C.S. and Stokes, M.D. (2004) Widespread colonization by polar hypoliths. Nature 431, 414 2 Cannone, N. et al. (2004) Relationships between vegetation patterns and periglacial landforms in northwestern Svalbard. Polar Biol. 27, 562–571 3 Russell, N.J. (1997) Psychrophilic bacteria - Molecular adaptations of membrane lipids. Comp. Biochem. Physiol. A Physiol. 118, 489–493 4 Cavicchioli, R. et al. (2002) Low-temperature extremophiles and their applications. Curr. Opin. Biotechnol. 13, 253–261 5 Horneck, G. et al. (2001) Protection of bacterial spores in space, a contribution to the discussion on panspermia. Orig. Life Evol. Biosph. 31, 527–547 6 Schulze-Makuch, D. et al. (2004) Life in the Universe - Expectations and Constraints, Springer-Verlag Berlin Heidelberg 7 Cavicchioli, R. (2002) Extremophiles and the search for extraterrestrial life. Astrobiology 2, 281–292 8 Chyba, F.F. and Phillips, C.B. (2001) Possible ecosystems and the search for life on Europa. Proc. Natl. Acad. Sci. U. S. A. 98, 801–804 9 Chyba, F.F. and Phillips, C.B. (2002) Europa as an abode of life. Orig. Life Evol. Biosph. 32, 47–68 10 Marion, G.M. et al. (2003) The search for life on Europa: Limiting environmental factors, potential habitats, and earth analogues. Astrobiology 3, 785–811 11 Littler, M.M. et al. (1985) Deepest known plant life discovered on an uncharted seamount. Science 227, 57–59 12 Nansen, F. (1897) Farthest North: Being a Record of a Voyage of Exploration of the Ship ‘Fram’ 1893-96 and of a Fifteen Months’s Sleigh Journey, Archibald Constable and Co., Westminster, London 13 Committee on Frontiers in Polar Biology Polar Research Board (2003) Frontiers in Polar Biology in the Genomic Era, The National Academies Press, Washington, DC, USA 14 Clark, M.S. et al. (2004) Antarctic genomics. Comp. Funct. Genomics 5, 230–238 15 Goodchild, A. et al. (2004) A proteomic determination of cold adaptation in the Antarctic archaeon. Methanococcoides burtonii. Mol. Microbiol. 53, 309–321 16 Lohan, D. and Johnston, S. (2003) The International Regime for Bioprospecting. Existing Policies and Emerging Issues for Antarctica, The United Nations University Institute of Advanced Studies, Tokyo 17 Freckman, D.W. and Virginia, R.A. (1997) Low-diversity Antarctic soil nematode communities: distribution and response to disturbance. Ecology 78, 363–369 18 Schlesinger, W.H. et al. (2003) Community composition and photosynthesis by photoautotrophs under quartz pebbles, southern Mojave Desert. Ecology 84, 3222–3231 0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.11.002

Histoplasma hairpins herald hopefulness Jon P. Woods Department of Medical Microbiology & Immunology, University of Wisconsin Medical School, 1300 University Avenue, 420 SMI, Madison, Wisconsin 53706-1532, WI, USA

Histoplasma capsulatum is a significant respiratory and systemic fungal pathogen. Although many molecular tools have been developed, the fulfillment of Koch’s Corresponding author: Woods, J.P. ([email protected]). Available online 25 January 2005 www.sciencedirect.com

postulates to determine gene function has been hampered by obstacles to homologous gene targeting. Because H. capsulatum displays a considerable array of virulence mechanisms and has a 40-Mb genome that is currently being sequenced, the capability to perform high-throughput molecular manipulations would clearly

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be beneficial. Recent demonstration and application of experimental RNA interference (RNAi) technology promises a major contribution to advances in this area.

Introduction Fulfilling the molecular version of Koch’s postulates by gene inactivation and resupply has been perhaps the most important approach for determining gene function and phenotypic causation in pathogenesis and, more broadly, biology. No eukaryote has been subjected to gene disruption studies as comprehensively as the model yeast Saccharomyces cerevisiae. This can be explained by two contributing factors: (i) the practical efficiency of experimental allelic replacement, which requires only a few homologous nucleotides in this organism, and (ii) the large number of investigators in the field. These two aspects are probably linked. During the past 20 years, increasing attention has been devoted to pathogenic fungi that cause more and more disease associated with the burgeoning immunocompromise and host susceptibility of the human population, and for which molecular manipulation approaches have been progressively developed. The effort to develop molecular approaches is not trivial or easy. Across a broad phylogenetic spectrum, including fungi, protozoa, plants and mammals, the difficulty in constructing null mutants is probably related not so much to the inability of an organism to practice homologous recombination but to the allowed occurrence of alternate fates for introduced DNA, such as illegitimate recombination or episome formation, or other organism-specific technical challenges [1–8]. A paradigm for pathogenic fungi (and everything else) is that experimental obstacles are identified and crucial reagents and techniques are developed that address them, leading to markedly increased studies and findings. Pathogenic fungal challenges and advances Candida albicans is relatively closely related to S. cerevisiae and undertakes homologous gene targeting relatively efficiently, but the general diploidy and frequent aneuploidy of this organism presents the requirement for two or more disruptions to achieve a null phenotype. The development of the ‘ura-blasting’ approach and an appropriate transformation recipient strain [3] led to an explosion of gene disruptions and molecular studies with this fungus. The recent demonstration of RNA interference [RNAi; posttranscriptional suppression of gene expression via transcript degradation, mediated by short double-stranded RNA (dsRNA) molecules] in H. capsulatum has the potential for being such a seminal technical advance [9]. Cumulatively, there have been perhaps hundreds of gene disruptions in the fungal pathogens Candida albicans, Cryptococcus neoformans, and most recently Aspergillus fumigatus, which are all important pathogens in specific patient populations or host microenvironmental settings [10]. However, only a handful of genes have been disrupted in systemic dimorphic fungal pathogens, including Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, www.sciencedirect.com

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Penicillium marneffei and Sporothrix schenckii. Of these organisms, H. capsulatum is the best-studied and presents a model of intracellular fungal pathogenesis, having several biological, pathogenic and clinical features that make it the fungal homolog of the bacterial pathogen Mycobacterium tuberculosis. Many pathogenic mechanisms and virulence-associated traits have been described, including dimorphism, intracellular survival, microenvironmental pH manipulation, a morphotype-specific calcium-binding protein, and iron acquisition [11,12]. The H. capsulatum genome sequence has been largely determined and is now being assembled and annotated (http:// genome.wustl.edu/projects/hcapsulatum/) [13]. However, disruption and subsequent extensive phenotypic analysis has been accomplished for only three genes in this organism: URA5 [1,14], CBP1 [15] and MS8 [16]. At this rate, comprehensive analysis of the estimated 6000–12 000 genes in the w40-Mb genome would take some time. H. capsulatum clearly undergoes homologous recombination: it is alive, it mates, and allelic replacements have been performed. However, it also allows illegitimate recombination of transforming DNA – there is always more non-homologous DNA than homologous genomic DNA – and displays spontaneous generation of linear episomes with the addition of terminal telomeric sequences [2], both of which are obstacles for homologous targeting. Histoplasma capsulatum RNAi Telomeric plasmids – potentially a nuisance because they interfere with allelic replacement – were put to good use as an alternative approach to gene inactivation by the ‘knockdown’ of expression using episomal RNAi constructs. Rappleye et al. [9] focused initially on green fluorescent protein (GFP) expressed from a genomically integrated gene. Using an optimized construct, approximately a sixfold reduction in the level of fluorescence was achieved, corresponding to a sixfold reduction in transcript level. Important technical features that were necessary to enable the process to work most effectively included: (i) directed expression of a hairpin loop dsRNA molecule rather than using convergent promoters to express separate sense and antisense strands; (ii) including a small spacer loop (87 n) between the sense and antisense regions; and (iii) expressing a target dsRNA molecule of at least 500 bp. The approach was then applied to two native nutritional genes – ADE2 and URA5 – and revealed suppression of expression sufficient to achieve auxotrophic phenotypes. Finally, RNAi was used to knock down expression of the a-1,3-glucan synthase gene AGS1, followed by phenotypic testing, in comparison to a traditional ags1 null mutant constructed via allelic replacement. Both RNAi and gene-disruption mutants showed identical and important phenotypes: loss of detectable cellwall a-1,3-glucan and diminished virulence in both macrophage and mouse infection models. A very attractive feature of supplying the RNAi construct episomally is easy reversibility. Any genetic manipulation, be it traditional gene disruption or RNAi, could result in unintended genetic changes that artifactually influence the observed phenotypes. To attribute genotype-phenotype causality definitively, reversal of the process is necessary to fulfill

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molecular Koch’s postulates. With traditional gene disruption, this feat is accomplished by resupply of the functional gene and detection of reversal of phenotypic changes. With episomal RNAi, loss of the plasmid carrying the inactivating construct functionally achieves the same result (Figure 1). There are some caveats and questions still to be addressed, for the technique in general and for the application of this technique to any particular gene (Box 1). For instance, RNAi is a gene expression knockdown approach rather than a gene deletion approach [17]. As noted by Rappleye et al. [9] and recognized for RNAi in general, efficacy for any particular gene and any phenotypic effect of that gene will depend on the individual situation. Gene function can require either a lot of expression or only a little, and RNAi might suppress gene expression a lot or a little. Of course, deletion is impossible for essential genes; therefore, an approach, such as RNAi, which leaves some residual gene expression, might be the best or even the only technique for analyzing the function of these genes. The development of a regulatable expression system for H. capsulatum would be very useful for such an approach and remains to be done. Another caveat from this study is the variability in knock-down efficacy in different transformants carrying the RNAi episomes; some showed suppression adequate to observe phenotypic effects, whereas some showed less or no detectable phenotypic change. An important point is that Rappleye et al. [9] did find RNAi transformants with significant phenotypic effects for all four genes examined, and they showed stability of these phenotypes over prolonged laboratory passage of individual transformants. They hypothesized that some epigenetic phenomenon causing variable RNAi construct expression in different transformants might be responsible. This explanation is reasonable, as is variation in plasmid copy number, which might be functionally the same explanation. Both of these hypotheses are testable, although measuring RNAi construct transcript level could be challenging because the point of RNAi is transcript degradation; a regulatable expression system would also provide a useful alternative here. Determining the source of the variable

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efficacy and/or being able to control it would be useful for increasing the efficient application of RNAi and to avoid loss of RNAi suppression occurring unexpectedly or worse, undetectably, in a transformant under particular conditions or randomly. Regardless, the utility of the approach is clear, having been demonstrated for four genes and potentially useful for other genes in H. capsulatum. The application of this approach to other eukaryotic pathogens, for which gene disruption is not trivial, might be feasible, although the twist of episomal supply of the RNAi construct might not always be possible. However, integrative transformation with an RNAi construct might suffice, although the advantage of ready reversibility through the loss of an episome would be lost. AGS1 as a virulence determinant Aside from the technical accomplishment, this study also added AGS1 to the short list of demonstrated H. capsulatum virulence determinants and shed light on a long-recognized virulence-associated feature in several pathogenic fungi. Cell wall a-1,3-glucan has been associated with virulence in H. capsulatum [18], P. brasiliensis [19], and B. dermatitidis [20], based largely on the occurrence of spontaneous or induced – but never molecularly targeted – variants that differ in its expression and concomitantly in virulence. Rappleye et al. [9] demonstrated that deletion or RNAi suppression of AGS1 expression resulted in loss of this cell-wall polysaccharide and reduction in virulence in macrophage and mouse infection models. One relative caveat in assessing this notable achievement is that apparently the alterations were not reversed to fulfill molecular Koch’s postulates for the virulence phenotype, either by complementation of the null disruptant or by loss of the RNAi plasmid. Still, support for a role of this gene in pathogenesis has been bolstered and important new questions raised (Box 1). Concluding remarks RNAi provides a promising alternative to allelic replacement for manipulating gene expression levels in H. capsulatum, with potential for high-throughput Episomal RNAi suppression

Traditional gene disruption Wild-type genomic locus: phenotype +

Allelic replacement with selectable marker: phenotype −

Wild-type genomic locus: phenotype +

Episomal RNAi transformant: phenotype − transcription Sense fragment

Antisense fragment

Episomal (or integrative) complementation: phenotype + Episome lost: phenotype +

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Figure 1. Molecular Koch’s postulates: traditional gene disruption versus episomal RNAi suppression. www.sciencedirect.com

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Box 1. Questions for future research Experimental RNAi for H. capsulatum † Why does variability in the effectiveness of RNAi suppression occur in different transformants? † Can a regulatable expression system be developed for controlled expression of RNAi constructs? † Are the parameters established for effective RNAi by Rappleye et al. [9] (hairpin construct, spacer length between sense and antisense fragments, and the length of dsRNA expressed) best for suppression of any target gene? † Will RNAi suppression be effective for other genes in relevant environments, for example, during host infection? † Can RNAi be used as a high-throughput screening approach to identify genes influencing virulence or other phenotypes?

a-1,3-glucan in H. capsulatum and other dimorphic systemic fungal pathogens † Is a defect in AGS1 expression or Ags1p function responsible for the lack of cell wall a-1,3-glucan expression in previously observed spontaneous or induced variants of H. capsulatum, P. brasiliensis and B. dermatitidis? † Are there other molecular mechanisms for this phenotypic variation not involving AGS1? † How is AGS1 expression regulated? † How does AGS1 and/or a-1,3-glucan expression affect pathogenesis and host interactions? † Are AGS1 and/or a-1,3-glucan ever expressed in H. capsulatum strains that possess a gene homolog but have not been observed to make a-1,3-glucan, but are fully virulent? † Is a-1,3-glucan synthesis a good antifungal drug target, for H. capsulatum strains that display it, for all H. capsulatum strains, for all dimorphic systemic fungi, or for all fungal pathogens?

application. A gene influencing expression of a fungal cellwall polysaccharide long associated with virulence has been identified and manipulated to yield results supporting its role in pathogenesis. Both the technological achievement and the biological findings are harbingers for future advances. Acknowledgements Work in our laboratory is supported by NIH R01s HL55949 and AI52303, and NIH R37 AI42747 (to George Deepe).

References 1 Woods, J.P. et al. (1998) Rare homologous gene targeting in Histoplasma capsulatum: disruption of the URA5Hc gene by allelic replacement. J. Bacteriol. 180, 5135–5143

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2 Woods, J.P. and Goldman, W.E. (1992) In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum. Mol. Microbiol. 6, 3603–3610 3 Fonzi, W.A. and Irwin, M.Y. (1993) Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728 4 Cormack, B.P. and Falkow, S. (1999) Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata. Genetics 151, 979–987 5 Mullins, E.D. and Kang, S. (2001) Transformation: a tool for studying fungal pathogens of plants. Cell. Mol. Life Sci. 58, 2043–2052 6 Gumpel, N.J. et al. (1994) Studies on homologous recombination in the green alga Chlamydomonas reinhardtii. Curr. Genet. 26, 438–442 7 Hohe, A. and Reski, R. (2003) A tool for understanding homologous recombination in plants. Plant Cell Rep. 21, 1135–1142 8 Wurtele, H. et al. (2003) Illegitimate DNA integration in mammalian cells. Gene Ther. 10, 1791–1799 9 Rappleye, C.A. et al. (2004) RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence. Mol. Microbiol. 53, 153–165 10 Magee, P.T. et al. (2003) Molecular genetic and genomic approaches to the study of medically important fungi. Infect. Immun. 71, 2299–2309 11 Woods, J.P. (2002) Histoplasma capsulatum molecular genetics, pathogenesis, and responsiveness to its environment. Fungal Genet. Biol. 35, 81–97 12 Woods, J.P. (2003) Knocking on the right door and making a comfortable home: Histoplasma capsulatum intracellular pathogenesis. Curr. Opin. Microbiol. 6, 327–331 13 Magrini, V. et al. (2004) Fosmid-based physical mapping of the Histoplasma capsulatum genome. Genome Res. 14, 1603–1609 14 Retallack, D.M. et al. (1999) The URA5 gene is necessary for Histoplasma capsulatum growth during infection of mouse and human cells. Infect. Immun. 67, 624–629 15 Sebghati, T.S. et al. (2000) Intracellular parasitism by Histoplasma capsulatum: fungal virulence and calcium dependence. Science 290, 1368–1372 16 Tian, X. and Shearer, G., Jr. (2002) The mold-specific MS8 gene is required for normal hypha formation in the dimorphic pathogenic fungus Histoplasma capsulatum. Eukaryot. Cell 1, 249–256 17 Agrawal, N. et al. (2003) RNA interference: biology, mechanism, and applications. Microbiol. Mol. Biol. Rev. 67, 657–685 18 Klimpel, K.R. and Goldman, W.E. (1988) Cell walls from avirulent variants of Histoplasma capsulatum lack alpha-(1,3)-glucan. Infect. Immun. 56, 2997–3000 19 San-Blas, G. et al. (1977) Host-parasite relationships in the yeastlike form of Paracoccidiodes brasiliensis strain IVIC Pb9. Infect. Immun. 15, 343–346 20 Hogan, L.H. and Klein, B.S. (1994) Altered expression of surface a-1,3glucan in genetically related strains of Blastomyces dermatitidis that differ in virulence. Infect. Immun. 62, 3543–3546

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