Coccidioides immitis, cause of a recent epidemic of "Valley fever" in California, is typical of many eukaryotic microbes in that mating and meiosis have yet to be reported, but it is not clear whether sex is truly absent or just cryptic. To find out, we have undertaken a population genetic study using PCR amplification, screening for single-strand conformation polymorphisms, and direct DNA sequencing to find molecular markers with nucleotide-level resolution. Both population genetic and phylogenetic analyses indicate that C. immitis is almost completely recombining. To our knowledge, this study is the first to find molecular evidence for recombination in a fungus for which no sexual stage has yet been described. These results motivate a directed search for mating and meiosis and illustrate the utility of single-strand conformation polymorphism and sequencing with arbitrary primer pairs in molecular population genetics.Unlike most plants and animals, the vast majority of eukaryotic microorganisms can reproduce asexually, and this together with their small size can make it very difficult to determine the relative importance of sexual reproduction in nature by direct observation alone. Rather, molecular markers must be used to test for the clonal population structure expected if sex is absent and the recombinant genotypes expected if sex is present (1-3). It is important in such studies that marker identity reflect common descent and that identities due to convergences, parallelisms, and reversals be minimized; in this respect, the most informative markers are DNA sequences (4,5). However, most studies of human pathogens have used allozymes or random amplified polymorphic DNAs (RAPDs) (1-3, 6). Neither approach is completely satisfactory, as allozyme patterns can be misleading because of natural selection (7), and RAPD patterns can be difficult both to repeat and to interpret in terms of Mendelian loci (8)(9)(10) (coccidioidomycosis) in California, with case reports 10 times more frequent than normal (13,14). Like some 20% of fungi (15), no sexual stage has ever been reported in C. immitis (16). Unfortunately, the inability to cross strains has hindered basic and applied research on this species.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. MATERIALS AND METHODSFor our study we have analyzed 30 clinical isolates from 25 patients at a single hospital in Tucson, Arizona. Three patients contributed multiple samples, collected up to 3 weeks, 16 months, and 8 years apart, respectively (Table 1). ¶ All isolates were collected in [1979][1980][1981][1982][1983][1984][1985][1986][1987][1988][1989][1990], before the epidemic.Our strategy for finding molecular markers begins with low-stringency PCR amplification from genomic DNA using arbitrary primers (-20-mers) in various pairwise combinations (11). Genomic DNA was isolated following heat treatment to k...

Many parasitic Apicomplexa, such as Plasmodium falciparum, contain an unpigmented chloroplast remnant termed the apicoplast, which is a target for malaria treatment. However, no close relative of apicomplexans with a functional photosynthetic plastid has yet been described. Here we describe a newly cultured organism that has ultrastructural features typical for alveolates, is phylogenetically related to apicomplexans, and contains a photosynthetic plastid. The plastid is surrounded by four membranes, is pigmented by chlorophyll a, and uses the codon UGA to encode tryptophan in the psbA gene. This genetic feature has been found only in coccidian apicoplasts and various mitochondria. The UGA-Trp codon and phylogenies of plastid and nuclear ribosomal RNA genes indicate that the organism is the closest known photosynthetic relative to apicomplexan parasites and that its plastid shares an origin with the apicoplasts. The discovery of this organism provides a powerful model with which to study the evolution of parasitism in Apicomplexa.

Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein-coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein-coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.