- Volume 148, Issue 11, 2002

Copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is suspected to be one of the anti-oxidant enzymes and virulence determinants active in some pathogenic micro-organisms. To elucidate the role of Cu/ZnSOD in the major human fungal pathogen Candida albicans, its gene, designated SOD1, was disrupted by the URA-blaster technique. The resulting sod1/sod1 mutant showed delayed hyphal growth on Spider medium but could still form hyphae on other solid media or in liquid media, particularly in response to serum. Moreover, the sod1/sod1 mutant was more sensitive to menadione, a redox-cycling agent, than the isogenic wild-type strain, although it still showed an adaptive oxidative stress response. Furthermore, the sod1/sod1 mutant cells exhibited slow growth in minimal medium when compared to the wild-type cells, but their growth was restored by the addition of lysine to the medium. Interestingly, C. albicans cells lacking Cu/ZnSOD showed increased susceptibility to macrophage attack and had attenuated virulence in mice. Thus, these results suggest that Cu/ZnSOD is required for the protection of C. albicans against oxidative stresses and for the full virulence of the organism to be expressed.

A continuous culture of Saccharomyces cerevisiae IFO 0233, growing with glucose as the major carbon and energy source, shows oscillations of respiration with a period of 48 min. Samples taken at maxima and minima indicate that (i) periodic changes do not occur as a result of carbon depletion, (ii) intrinsic differences in respiratory activity occur in washed organisms and (iii) a respiratory inhibitor accumulates during respiratory oscillations. Plasma membrane and inner mitochondrial membranes generate transmembrane electrochemical potentials; changes in these can be respectively assessed using anionic or cationic fluorophores. Thus flow cytometric analyses indicated that an oxonol dye [DiBAC4(3); bis(1,3-dibutylbarbituric acid)trimethine oxonol] was excluded from yeasts to a similar extent (in >98% of the population) at all stages, showing that the plasma membrane potential was maintained at a steady value. However, uptake of Rhodamine 123 was greatest at that phase characterized by a low respiratory rate. Addition of uncouplers of energy conservation [CCCP (m-chlorocarbonylcyanide phenylhydrazone) or S-13(5-chloro-3-t-butyl-2-chloro-41-nitrosalicylanilide)] to the continuous cultures increased the respiration, but had only a transient effect on the period of the oscillation. Electron microscopy showed changes in mitochondrial ultrastructure during the respiratory oscillation. At low respiration the cristae were more clearly defined due to swelling of the matrix; this corresponds to the ‘orthodox’ conformation. When respiration was high the mitochondrial configuration was ‘condensed’. It has been shown previously that a temperature-compensated ultradian clock operates in S. cerevisiae. It is proposed that mitochondria undergo cycles of energization in response to energetic demands driven by this ultradian clock output.

Most pathogenic fungi have the ability to alternate between a unicellular yeast form and different filamentous forms (hyphae and pseudohyphae). This attribute is generally regarded as an important virulence factor and has also attracted attention because of its implications in the study of eukaryotic cell differentiation. To identify genes that are involved in the regulation of these events, chemical mutagenesis of the dimorphic yeast Yarrowia lipolytica was performed and morphological mutants that were unable to form hyphal cells were isolated. Screening of a Y. lipolytica genomic DNA library for genes able to complement this defect led to the isolation of YlBMH1, a gene encoding a 14-3-3 protein and whose transcription levels are increased during the yeast-to-hypha transition. Remarkably, overexpression of YlBMH1 was able to enhance pseudohyphae formation in a strain lacking functional YlRAC1 but caused no visible effects in Δmhy1 and Δbem1 cells, thus suggesting that YlBMH1 is involved in the regulation of both hyphal and pseudohyphal growth in Y. lipolytica. The identification of YlBMH2, a gene encoding a second 14-3-3 protein (YlBmh2p) that contains a 19 aa insertion absent in all other members of the 14-3-3 family, is also reported. Differently from YlBMH1, the transcription levels of YlBMH2 do not show any apparent variation during the induction of hyphal growth, and its overexpression has no effects on cells lacking functional MHY1, YlRAC1 or YlBEM1. Taken together, these observations suggest that, in spite of their high conservation, YlBmh1p and YlBmh2p have different cellular functions.

Muscodor vitigenus is a recently described endophytic fungus of Paullinia paullinioides, a liana growing in the understorey of the rainforests of the Peruvian Amazon. This fungus produces naphthalene under certain cultural conditions. Naphthalene produced by M. vitigenus was identified by gas chromatography/mass spectrometry. Its chromatographic and mass spectral properties were identical to authentic naphthalene. Agar plugs supporting growth of the fungus and producing known amounts of naphthalene effectively repelled the adult stage of the wheat stem sawfly, Cephus cinctus, in Y-tube bioassay tests. Authentic naphthalene, at comparable concentrations to those in tests involving the fungus itself, mimicked the insect repellency of the fungus. Although other Muscodor spp. produce volatile antimicrobials, M. vitigenus is unique in its ability to produce naphthalene almost exclusively. This report also describes the potential practical implications of M. vitigenus.

Secreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multi-gene family and are considered to be putative virulence factors for candidiasis. SAP4–6 mRNAs were first detected during hyphae formation and were assumed to play roles in the development of disseminated candidiasis. Recombinant Sap proteins (Sap2–6) were prepared and specific antibodies were generated against Sap2–6. The presence of Sap4, Sap5 and Sap6, but not Sap2 or Sap3, was demonstrated in culture supernatants of C. albicans after induction of hyphae formation. In parallel to hyphae formation, Sap5 (∼40 kDa) was detected as early as ∼6 h after induction at neutral pH, and Sap4/6 (∼43 kDa) were detected after ∼24 h when the culture medium became acidic. The differential secretion of Sap5 and Sap4/6 was affected when the culture medium pH was buffered at pH 6·5 or pH 4·5. In addition, intracellular pools of Sap4–6 seem to exist, and protein is not necessary for Sap4–6 induction. This study provides the first evidence that Sap4–6 proteins in C. albicans are differentially produced and secreted during hyphae formation.