The CRZ1/SP1-like gene links survival under limited aeration, cell integrity and biofilm formation in the pathogenic yeast Cryptococcus neoformans.

AIMS: Limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2-arrest in the obligate aerobic pathogenic yeast Cryptococcus neoformans. Also, the ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. The aim of this study was to identify and characterize genes that are necessary for the proliferation slowdown and G2-arrest caused by limited aeration. METHODS: Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The CNAG_00156.2 gene coding for a zinc-finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1-dependent (SP1-like) transcription factors. RESULTS: We have confirmed the role of the cryptococcal homologue of CRZ1/SP1-like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole. CONCLUSIONS: Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.

Towards understanding cell cycle control in Cryptococcus neoformans: structure-function relationship of G1 and G1/S cyclins homologue CnCln1.

We have previously reported that only a single Cdk1-related G1 and G1/S cyclin homologue was found in the genome sequence of the pathogenic basidiomycetous yeast Cryptococcus neoformans (C. neoformans) and designated it CnCln1. Surprisingly, CnCln1 was not only able to complement the function of the G1 cyclins of the ascomycetous budding yeast Saccharomyces cerevisiae (S. cerevisiae), such as ScCln3, but also the G1/S cyclins of S. cerevisiae, such as ScCln1 and ScCln2. In this study, we investigated how CnCln1 cooperates with the cyclin-dependent kinases of S. cerevisiae (ScCdk1) and substitutes the function of G1 and G1/S cyclins of S. cerevisia from a point of view of their structure-function relationship. Our in silico analysis demonstrated that the CnCln1/ScCdk1 complex was more stable than any of the yeast cyclin and ScCdk1complexes. Thus, these results are consistent with in vitro analysis that has revealed the flexible functional capacity of CnCln1 as a Cdk1-related G1 and G1/S cyclins of S. cerevisiae.

Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata.

Protoplast regeneration of a wild-type and two mutant strains of Candida glabrata defective in CHS3 homologues encoding class IV chitin synthase in Saccharomyces cerevisiae was examined by scanning and negative-staining electron microscopy. In the wild-type strain, small particles and short filaments appeared on the protoplast surface at 10 min, filamentous materials covered the entire surface of the protoplast at 1 h, granular materials started filling interspaces of filamentous materials at 2 h and regeneration was completed at 6 h. The filamentous materials consisted of microfibrils of various widths ranging from ≤5 to 40 nm, and composed of ß-glucan. Protoplasts of the two chitin synthase mutant strains of Δchs3A and Δchs3B completed regeneration essentially by the same process as wild-type strain, although it took more time. These results suggest that CHS3A and CHS3B genes may have important roles in cell wall formation during protoplast regeneration, but can be compensated by other cell wall enzymes.

The single Cdk1-G1 cyclin of Cryptococcus neoformans is not essential for cell cycle progression, but plays important roles in the proper commitment to DNA synthesis and bud emergence in this yeast.

The cell cycle pattern of the pathogenic basidiomycetous yeast Cryptococcus neoformans differs from that of the ascomycetous budding yeast Saccharomyces cerevisiae. To clarify the cell cycle control mechanisms at the molecular level, homologues of cell cycle control genes in C. neoformans were cloned and analyzed. Here, we report on the cloning and characterization of genes coding for CDK1 cyclin homologues, in particular, the C. neoformans G1 cyclin. We have identified three putative CDK1 cyclin homologues and two putative CDK5 (PHO85) cyclin homologues from the genome. Complementation tests in an S. cerevisiae G1 cyclin triple mutant confirmed that C. neoformans CLN1 is able to complement S. cerevisiae G1 cyclin deficiency, demonstrating that it is a G1 cyclin homologue. Interestingly, cells deleted of the single Cdk1-G1 cyclin were viable, demonstrating that this gene is not essential. However, it exhibited aberrant budding and cell division and a clear delay in the initiation of DNA synthesis as well as an extensive delay in budding. The fact that the mutant managed to traverse the G1 to M phase may be due to the activities of Pho85-related G1 cyclins. Also, that C. neoformans had only a single Cdk1-G1 cyclin highlighted the importance of keeping in order the commitment to the initiation of DNA synthesis first and then that of budding, as discussed.

[Cell cycle control and CDC28/Cdc2 homologue and related gene cloning of Cryptococcus neoformans].

In Cryptococcus neoformans the DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in the early exponential phase. However, bud emergence was delayed to G2 during transition to the stationary phase, and in the early stationary phase budding scarcely occurred, although roughly half of the cells completed DNA synthesis. The timing of budding in C. neoformans was shifted to later cell cycle points with progression of the growth phase of the culture. Similarly, a deficit in oxygen was demonstrated to delay the timing of budding, prolong the G2 phase and cause accumulation of cells after DNA synthesis, but before commitment to budding. The C. neoformans homologue of the main cell cycle control gene CDC28/Cdc2 was isolated using degenerate RT-PCR. The full-length coding region was then amplified using primers to target the regions around the start and stop codons. The gene was called CnCdk1 and was found to have high homologies to S. cerevisiae CDC28 and S. pombe cdc2. To determine its function, its ability to rescue S. cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2-CnCdk1 construct exhibited growth at the restrictive temperature. Results of the sequence analysis and the ability of CnCdk1 to complement the S. cerevisiae cdc28-ts mutations support its assumed role as the CDC28/cdc2 homologue in C. neoformans.

Regulatory role of hepatocyte nuclear factor-4alpha on gastric choriocarcinoma function.

Gastric choriocarcinoma is a highly aggressive carcinoma, most probably originating from somatic cells in the gastric mucosal layer. We herein investigated the regulatory role of hepatocyte nuclear factor (HNF)-4alpha, a transcriptional regulator expressed in non-neoplastic and neoplastic gastric tissues, on functions of gastric choriocarcinoma cells. HNF-4alpha cDNA was stably transfected to a gastric choriocarcinoma cell line, SCH. Alterations in SCH cell functions such as histology, ultrastructure, proliferation, production of trophoblast-specific proteins, and chemosensitivity to methotrexate (MTX) were examined. Neither in vitro and in vivo proliferations nor HLA-G expression differed significantly between the mock-transfected and HNF-4alpha-transfected SCH cells, while suppressed human chorionic gonadotropin (hCG) secretions, increased human placental lactogen (hPL) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) immunoreactivity, and decreased chemosensitivity to MTX were seen in HNF-4alpha-transfected SCH cells. General histologic features in xenograft nodules were unaltered, but, ultrastructurally, fascicles of paranuclear filaments were significantly more numerous in HNF-4alpha-transfected SCH cells. These results indicated an HNF-4alpha-rendered functional regulation in SCH cells, suggesting a role of transcriptional factors abundant in gastric but not in trophoblastic tissues/cells on the functional modulation of gastric choriocarcinoma cells.

Fluconazole-resistant pathogens Candida inconspicua and C. norvegensis: DNA sequence diversity of the rRNA intergenic spacer region, antifungal drug susceptibility, and extracellular enzyme production.

The opportunistic fungal pathogens Candida inconspicua and C. norvegensis are very rarely isolated from patients and are resistant to fluconazole. We collected 38 strains of the two microorganisms isolated from Europe and Japan, and compared the polymorphism of the rRNA intergenic spacer (IGS) and internal transcribed spacer (ITS) regions, antifungal drug susceptibility, and extracellular enzyme production as a potential virulence factor. While the IGS sequences of C. norvegensis were not very divergent (more than 96.7% sequence similarity among the strains), those of C. inconspicua showed remarkable diversity, and were divided into four genotypes with three subtypes. In the ITS region, no variation was found in either species. Since the sequence similarity of the two species is approximately 70% at the ITS region, they are closely related phylogenetically. Fluconazole resistance was reconfirmed for the two microorganisms but they were susceptible to micafungin and amphotericin B. No strain of either species secreted aspartyl proteinase or phospholipase B. These results provide basal information for accurate identification, which is of benefit to global molecular epidemiological studies and facilitates our understanding of the medical mycological characteristics of C. inconspicua and C. norvegensis.

Isolation of a CDC28 homologue from Cryptococcus neoformans that is able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae.

A partial cDNA fragment of the Cryptococcus neoformans homologue of the main cell cycle control gene CDC28/cdc2 was isolated using degenerate primer RT-PCR. A subsequent search in the C. neoformans genome database identified several sequences similar to CDC28/cdc2. A part of the sequence which showed the highest similarity to CDC28/cdc2 turned out to be identical to the partial cyclin-dependent kinase (Cdk) cDNA fragment isolated by degenerate RT-PCR. The full-length coding region of this Cdk homologue was amplified by RT-PCR using primers designed to target regions around start and stop codons, and the gene was named CnCdk1. To determine its function, an analysis of deduced amino acid sequence of the CnCdk1 was performed and its ability to rescue Saccharomyces cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2- CnCdk1 construct exhibited growth at 36.5 degrees C in galactose-raffinose medium, but not in glucose medium. Results of the sequence analysis and the fact that CnCdk1 is able to complement the S. cerevisiae cdc28-ts mutation support its assumed role as the CDC28/cdc2 homologue in C. neoformans.