Over the past two decades, several fungal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

Summary The fungal meningitis pathogen Cryptococcus neoformans is a central driver of mortality in HIV/AIDS. We report a genome-scale chemical genetic data map for this pathogen that quantifies the impact of 439 small molecule challenges on 1448 gene knockouts. We identified chemical phenotypes for 83% of mutants screened and at least one genetic response for each compound. C. neoformans chemical-genetic responses are largely distinct from orthologous published profiles of Saccharomyces cerevisiae, demonstrating the importance of pathogen-centered studies. We used the chemical-genetic matrix to predict novel pathogenicity genes, infer compound mode-of-action, and to develop an algorithm, O2M, that predicts antifungal synergies. These predictions were experimentally validated, thereby identifying virulence genes, a molecule that triggers G2/M arrest and inhibits the Cdc25 phosphatase, and many compounds that synergize with the antifungal drug fluconazole. Our work establishes a chemical-genetic foundation for approaching an infection responsible for greater than one-third of AIDS-related deaths.

The aminoglycoside-3-O-acetyltransferase-I gene (aacC1) from R plasmids of two incompatibility groups (R1033 [Tn1696], and R135) was cloned and sequenced. In the case of R1033, it was shown that the aacC gene is coded by a precise insertion of 833 bp between the aadA promoter and its structural gene in a Tn21 related transposon (Tn1696). This insertion occurs at the same target sequence as that of the OXA-1 beta-lactamase gene insertion in Tn2603. Upstream of the aacC gene, we found an open reading frame (ORF) which is probably implicated in the site-specific recombinational events involved in the evolution of this family of genetic elements. These results provide additional confirmation of the role of Tn21 elements as naturally occurring interspecific transposition and expression cassettes.

c Amphotericin B (AMB) is an effective antifungal agent. However, its therapeutic use is hampered by its toxicity, mainly due to channel formation across kidney cell membranes and the disruption of postendocytic trafficking. We previously described a safe injectable AMB-arabinogalactan (AG) conjugate with neutralized toxicity. Here we studied the mechanism of the toxicity of free AMB and its neutralization by conjugation with AG. AMB treatment of a kidney cell line modulated the trafficking of three receptors (C-X-C chemokine receptor type 4 [CXCR4], M1 receptor, and human transferrin receptor [hTfnR]) due to an increase in endosomal pH. Similar data were also obtained in yeast but with an increase in vacuolar pH and the perturbation of Hxt2-green fluorescent protein (GFP) trafficking. The conjugation of AMB with AG neutralized all elements of the toxic activity of AMB in mammalian but not in fungal cells. Based on these results, we provide an explanation of how the conjugation of AMB with AG neutralizes its toxicity in mammalian cells and add to the knowledge of the mechanism of action of free AMB in both fungal and mammalian cells. Opportunistic fungal infections have emerged as an important cause of morbidity and mortality in immunodeficient patients (34). Amphotericin B (AMB) is considered one of the most effective antifungal agents; it exhibits wide-spectrum activity against both filamentous and yeast-like fungi, its pharmacokinetic and pharmacodynamic profiles are superior to those of other antifungal agents, and it is fungicidal, in contrast to most azoles which are fungistatic (3,39,53). The fungicidal effect is important, since most patients suffering from invasive fungal infections are immunocompromised (34). However, the infusionrelated and cumulative toxicities, particularly nephrotoxicity (14,20,30), of AMB have resulted in reductions in the routine use of deoxycholate micellar AMB formulations and the development of less-toxic high-cost lipid AMB formulations (16,36). To develop a soluble, less-toxic, and less costly formulation, AMB has been conjugated with various soluble macromolecules (18,37,(47)(48)(49).We conjugated AMB with arabinogalactan (AG) (18), which significantly increased the water solubility of AMB, reduced its toxicity, and resulted in an efficacy similar to that of Fungizone (a deoxycholate micellar formulation) and AmBisome (a lipid-based formulation) (18). AMB-related toxicity is associated with the inductions of interleukin 1␤ (IL-1␤), tumor necrosis factor ␣ (TNF-␣), and apoptosis in organs. These effects were not observed with the AMB-AG conjugate (AMB-AGC), suggesting its potential as a safer formulation for therapeutic use (19). AG is an inexpensive natural product, and the conjugation reactions are performed at room temperature, revealing promise for a potentially low-cost drug. AMB penetrates the plasma membrane (PM) and interacts with its sterols to form transmembrane channels, resulting in the leakage of monovalent ions and metabolites, which leads to cell death (5,22,4...

We developed novel anti-biofilm agents that both inhibited and destroyed C. albicans biofilm. With some further development, these agents might be suitable for therapeutic purposes.