SUMMARY In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (Smo) by a mechanism distinct from that of cyclopamine and other known Smo antagonists, and prevents the ciliary accumulation of Smo normally caused by Hh stimulation.

There have been increased reports of the isolation of unusual genotypic groups of Candida albicans (groups C and D) based on a well-defined genotypic method; this method uses cellular DNA digested with the EcoRI enzyme and the restriction fragment length polymorphisms (RFLPs) generated by agarose gel electrophoresis. The aim of the present study was to use additional molecular tools to characterize these unusual strains and to compare them with authentic strains of C. dubliniensis, a recently delineated species, and type I C. stellatoidea. The RFLPs of PCR products generated from the intergenic transcribed spacer (ITS) region did not differentiate among C. albicans genotypes A, B, and C and type I C. stellatoidea. However, this method did differentiate the C. albicans genotype D strains, which were identical to C. dubliniensis. The RFLPs generated by HaeIII digestion of the PCR products of the V3 region of the 25S rRNA gene (rDNA) could differentiate the same groups as RFLP analysis of the PCR amplicon of the ITS region.C. albicans genotype B isolates have been shown to have a transposable intron in the 25S rDNA, whereas genotype A isolates do not; C. dubliniensis strains also have an intron that is larger than that in genotype B C. albicansstrains but that is in the same location. PCR designed to span this region resulted in a single product for C. albicansgenotype A (450 bp), B (840 bp), type 1 C. stellatoidea (840 bp), and C. dubliniensis(1,080 bp), whereas the C. albicans genotype C isolates had two major products (450 and 840 bp). All C. albicans genotype D isolates gave a PCR product identical to that given by C. dubliniensis. These results indicate that those strains previously designated C. albicansgenotype D are in fact C. dubliniensis, that no differences were found between type 1 C. stellatoideaand C. albicans genotype B strains, and that theC. albicans genotype C strains appear to have the transposable intron incompletely inserted throughout the ribosomal repeats in their genomes. The results of the antifungal susceptibility testing of 105 of these strains showed that, for fluconazole, strains of C. dubliniensis were significantly more susceptible than strains of each of the C. albicans genotypes (genotypes A, B, and C). The flucytosine susceptibility results indicated that strains of C. albicans genotype A were significantly less susceptible than either C. albicansgenotype B or C. albicans genotype C strains. These results indicate that there is a correlation between theCandida groups and antifungal susceptibility.

Although considered nonpathogenic, Saccharomyces cerevisiae is being encountered more frequently in the clinical setting. To assess pathogenic potential, 13 clinical isolates, 10 nonclinical isolates, and 5 constructed strains of S. cerevisiae were analyzed. All were S. cerevisiae by biochemical profiles, sporulation, or genetic evidence. Intravenous inoculation of yeasts into CD-1 mice showed that some clinical isolates proliferated in the brain (5-fold) but nonclinical isolates were cleared (1000-fold) by day 7 after infection. Comparison of burdens with those of YJM128 (clinical) and Y55 (laboratory strain) revealed three virulence groupings: virulent, those greater than or equal to YJM128 (5 clinical and 2 genetic constructs); intermediate virulent, those less than YJM128 and greater than Y55 (5 clinical, 3 genetic constructs, and 4 nonclinical); and avirulent, those less than or equal to Y55 (1 clinical and 6 nonclinical). Genetic crosses indicated that virulence was a dominant trait. Growth of various isolates at 37 degrees C and 39 degrees C indicated that temperature is associated with but not solely responsible for differences in virulence. These data demonstrate that some clinical isolates of S. cerevisiae can proliferate and resist clearance in vivo and support the potential of S. cerevisiae as a cause of clinical disease.

The Candida parapsilosis family has emerged as a major opportunistic and nosocomial pathogen. It causes multifaceted pathology in immuno-compromised and normal hosts, notably low birth weight neonates. Its emergence may relate to an ability to colonize the skin, proliferate in glucose-containing solutions, and adhere to plastic. When clusters appear, determination of genetic relatedness among strains and identification of a common source are important. Its virulence appears associated with a capacity to produce biofilm and production of phospholipase and aspartyl protease. Further investigations of the host-pathogen interactions are needed. This review summarizes basic science, clinical and experimental information about C. parapsilosis.

Central nervous system (CNS) aspergillosis is

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents.We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.

No typing system exists for Aspergillus fumigatus, though isolates are distinguishable by phenotypic characteristics. DNA was prepared by lysis of protoplasts, followed by deproteination, phenolchloroform extraction, and dialysis. DNA prepared was of uniform size and exceeded 60 kb. After digestion with SalI and XhoI endonucleases, DNA was electrophoresed, stained, and photographed. Differences in the mobilities of 10- to 50-kb bands distinguished isolates. Reproducibility was shown by repeated preparations and animal passage. By use of a proposed notation system for describing restriction fragment length polymorphism patterns, 31 epidemiologically characterized isolates from three continents revealed 24 patterns (DNA types). Three DNA types were represented by 3 isolates each and 1 DNA type by 2 isolates; 20 types were unique. Two groups of 3 isolates of the same DNA type were from Stanford University Hospital. One patient isolate from Stanford was the same DNA type as a sewage isolate from New Jersey. Another Stanford isolate was the same as a German isolate. These observations indicate widespread dispersal of some clones and restricted locales for others. Paired isolates from airway fluids of three patients had two DNA types in each. Restriction endonuclease typing shows promise for investigating the epidemiology and ecology of A. fumigatus.

SUMMARY Paracoccidioidomycosis, one of the most important endemic and systemic mycoses in Latin America, presents several clinical pictures. Epidemiological studies indicate a striking rarity of disease (but not infection) in females, but only during the reproductive years. This suggested a hormonal interaction between female hormones and the etiologic dimorphic fungus Paracoccidioides brasiliensis . Many fungi have been shown to use hormonal (pheromonal) fungal molecules for intercellular communication, and there are increasing numbers of examples of interactions between mammalian hormones and fungi, including the specific binding of mammalian hormones by fungal proteins, and suggestions of mammalian hormonal modulation of fungal behavior. This suggests an evolutionary conservation of hormonal receptor systems. We recount studies showing the specific hormonal binding of mammalian estrogen to proteins in P. brasiliensis and an action of estrogen to specifically block the transition from the saprophytic form to the invasive form of the fungus in vitro . This block has been demonstrated to occur in vivo in animal studies. These unique observations are consistent with an estrogen-fungus receptor-mediated effect on pathogenesis. The fungal genes responsive to estrogen action are under study.