Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1⌬, yvh1⌬, sit4⌬, and ptc1⌬) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1⌬, yvh1⌬, and sit4⌬ mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1⌬ revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.The opportunistic fungal pathogen Candida albicans, a member of the normal human microflora, can cause superficial or life-threatening systemic infections, particularly in immunocompromised patients. Virulence determinants of C. albicans include the yeast-to-hypha transition, adherence to host receptors, and the ability to produce a variety of secreted hydrolytic enzymes, such as aspartic proteinases, phospholipases, and lipases. In addition, genes encoding metabolism and stress response proteins of this pathogen are important for pathogenicity (9,16,22).It would be of interest to determine whether other, previously uncharacterized C. albicans genes have a role in virulence. However, screening of whole families of C. albicans genes for virulence-related properties using a mammalian model of infection, such as the mouse model, would pose issues both of ethics and of practical costs. A number of substitute, invertebrate models of microbial virulence more suitable for screening experiments have been developed, including the use of Caenorhabditis elegans, Drosophila melanogaster, and Galleria mellonella (10, 18, 35, 41), silkworms (20), and locusts (32). The silkworm model of C. albicans infection has been used for the quantitative evaluation of antifungal agents, with results equivalent to those in a mouse model. In addition, the silkworm infection model has been used successfully to identify and evaluate uncharacterized genes required for the virulence of Staphylococcus aureus (28,29).The fungus must adapt to stresses encountered in vivo, such as various host defense mechanisms and/or microenvironmental changes in...

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One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation.

In response to stimulants, such as serum, the yeast cells of the opportunistic fungal pathogen Candida albicans form germ tubes, which develop into hyphae. Yvh1p, one of the 29 protein phosphatases encoded in the C. albicans genome, has 45 % identity with the dual-specific phosphatase Yvh1p of the model yeast Saccharomyces cerevisiae. In this study, Yvh1p expression was not observed during the initial step of germ tube formation, although Yvh1p was expressed constitutively in cell cycle progression of yeast or hyphal cells. In an attempt to analyse the function of Yvh1p phosphatase, the complete ORFs of both alleles were deleted by replacement with hph200-URA3-hph200 and ARG4. Although YVH1 has nine single-nucleotide polymorphisms in its coding sequence, both YVH1 alleles were able to complement the YVH1 gene disruptant. The vegetative growth of Dyvh1 was significantly slower than the wild-type. The hyphal growth of Dyvh1 on agar, or in a liquid medium, was also slower than the wild-type because of the delay in nuclear division and septum formation, although germ tube formation was similar between the wild-type and the disruptant. Despite the slow hyphal growth, the expression of several hypha-specific genes in Dyvh1 was not delayed or repressed compared with that of the wild-type. Infection studies using mouse models revealed that the virulence of Dyvh1 was less than that of the wild-type. Thus, YVH1 contributes to normal vegetative yeast or hyphal cell cycle progression and pathogenicity, but not to germ tube formation.

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