The extent to which the transport of glucose across the plasma membrane of the yeast Saccharomyces bayanus controls the glycolytic flux was determined. The magnitude of control was quantified by measuring the effect of small changes in the activity of the glucose transport system on the rate of glucose consumption. Two effectors were used to modulate the activity of glucose transport : (i) maltose, a competitive inhibitor of the glucose transport system in S. bayanus (as well as in Saccharomyces cerevisiae) and (ii) extracellular glucose, the substrate of the glucose transport system. Two approaches were followed to derive from the experimental data the flux control coefficient of glucose transport on the glycolytic flux : (i) direct comparison of the steadystate glycolytic flux with the zero trans-influx of glucose and (ii) comparison of the change in glycolytic flux with the concomitant change in calculated glucose transport activity on variation of the extracellular glucose concentration. Both these approaches demonstrated that in cells of S. bayanus grown on glucose and harvested at the point of glucose exhaustion, a high proportion of the control of the glycolytic flux resides in the transport of glucose across the plasma membrane.

cThe present work reports the effects of caspofungin, a ␤-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting ␤-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the ␤-glucan synthase inhibitor against this fungus.

In the present report, we describe the first case of a phaeohyphomycotic brain abscess in a 5-year-old boy with chronic granulomatous disease (CGD) admitted to hospital with seizures. A computed tomography (CT) scan revealed a cerebral abscess and the microbiology study showed a dark, melanin-pigmented fungus, exhibiting only sterile hyphae. This fungus was identified by the amplification and sequencing of the 5.8S RNA gene and of the adjacent internal transcriber spacer domains, ITS1 and ITS2, as Alternaria infectoria. Due to the impossibility of a surgical excision, and although several therapeutic strategies were attempted, the patient died. Limitations in the routine identification procedures and therapeutic options of this emerging opportunistic agent are highlighted in this report.

The fungal cell wall polymer β-(1,3)-D-glucan is synthesized by the enzyme β-(1,3)- D-glucan synthase that is a complex composed of at least two proteins, Rho1p and Fks1p. Here, we report the nucleotide sequence of a single FKS gene and of the regulatory unit, RHO1 from the dematiaceous pathogenic fungus Alternaria infectoria. The predicted AiFks and AiRho share, respectively, 93% and 100% identity with that of Drechslera tritici-repentis. We also report that the sensitivity to caspofungin of eight different A. infectoria clinical strains is similar, with a MIC > 32 µg/ml and a MEC of 1 µg/ml, except for one strain which had a MEC of 1.4 µg/ml. This same strain exhibited one substitution at the hot spot 2, S1405A, compatible with less susceptible phenotypes, with the other seven strains having no mutations in either hot spot 1 or 2. The relative quantification of the expression of AiFKS and of AiRHO demonstrated a decrease in response to an exposure to caspofungin at 0.5 µg/ml.

In the model yeast Saccharomyces cerevisiae, hexose uptake is mediated exclusively by a family of facilitators (Hxt, hexose transporters). Some other Saccharomyces species (e.g. Saccharomyces bayanus and Saccharomyces pastorianus) possess, in addition, a specific fructose transporter (Fsy1, fructose symporter) that has been previously described to function as a proton symporter. In the present work, we compared growth of a yeast strain in which FSY1 occurs naturally in anaerobic, fructose- and glucose-limited chemostat cultures. Especially at low specific growth rates, fructose-proton symport was shown to have a strong impact on the biomass yield on sugar. We subsequently employed energized hybrid plasma membrane vesicles to confirm previous observations concerning the mode of operation and specificity of Fsy1 mediated transport. Surprisingly, these experiments suggested that the carrier exhibits an unusual fructose:H(+) stoichiometry of 1:2. This energetically expensive mode of operation was also found consistently in vivo, in shake flask and in chemostat cultures, and both when Fsy1 is the sole transporter and when the Hxt carriers are present. However, it is observed only when Fsy1 is operating at higher glycolytic fluxes, a situation that is normally prevented by downregulation of the gene. Taken together, our results suggest the possibility that fructose symport with more than one proton may constitute an energetically unfavorable mode of operation of the Fsy1 transporter that, in growing cultures, is prevented by transcriptional regulation.