Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Parasites of wild howlers (Alouatta spp.)

A literature review of howler parasites provides the basis for an overview of the ecological significance of parasite surveys in primates. Within this framework, we have added insights into the interactions between primate hosts and their parasites from a long-term study in Costa Rica. We collected fecal samples from mantled howlers (Alouatta palliata) over a 9-year period (19861994 inclusive) and analyzed them for parasite eggs, larvae, cysts, and oocysts. We found many misperceptions inherent in the typical methodology of primate parasite surveys and in the reporting of the findings. Our work in Costa Rica suggests that a snapshot effect occurs with most surveys. A static view does not reflect the dynamic and changing ecological interaction between host and parasite. We describe some problems with parasite data analyses that emphasize the need for long-term longitudinal surveys in wild primate groups.

Working at the interface of phylogenetics and population genetics: a biogeographical analysis of Triaenops spp. (Chiroptera: Hipposideridae).

New applications of genetic data to questions of historical biogeography have revolutionized our understanding of how organisms have come to occupy their present distributions. Phylogenetic methods in combination with divergence time estimation can reveal biogeographical centres of origin, differentiate between hypotheses of vicariance and dispersal, and reveal the directionality of dispersal events. Despite their power, however, phylogenetic methods can sometimes yield patterns that are compatible with multiple, equally well-supported biogeographical hypotheses. In such cases, additional approaches must be integrated to differentiate among conflicting dispersal hypotheses. Here, we use a synthetic approach that draws upon the analytical strengths of coalescent and population genetic methods to augment phylogenetic analyses in order to assess the biogeographical history of Madagascar's Triaenops bats (Chiroptera: Hipposideridae). Phylogenetic analyses of mitochondrial DNA sequence data for Malagasy and east African Triaenops reveal a pattern that equally supports two competing hypotheses. While the phylogeny cannot determine whether Africa or Madagascar was the centre of origin for the species investigated, it serves as the essential backbone for the application of coalescent and population genetic methods. From the application of these methods, we conclude that a hypothesis of two independent but unidirectional dispersal events from Africa to Madagascar is best supported by the data.