Role of the anillin-like protein in growth of Cryptococcus neoformans at human host temperature.

Cryptococcus neoformans, a basidiomycete yeast, causes lethal meningitis in immunocompromised individuals. The ability of C. neoformans to proliferate at 37°C is essential for virulence. We identified anillin-like protein, CnBud4, as essential for proliferation of C. neoformans at 37°C and for virulence in a heterologous host Galleria mellonella at 25°C. C. neoformans cells lacking CnBud4 were inviable at 25°C in the absence of active calcineurin and were hypersensitive to membrane stress and an anti-fungal agent fluconazole, phenotypes previously described for C. neoformans mutants lacking septins. CnBud4 localized to the mother-bud neck during cytokinesis in a septin-dependent manner. In the absence of CnBud4, septin complex failed to transition from a collar-like single ring to the double ring during cytokinesis. In an ascomycete yeast, Saccharomyces cerevisiae, the anillin-like homologue ScBud4 participates in the organization of the septin ring at the mother-bud neck and plays an important role in specifying location for new bud emergence, known as axial budding pattern. In contrast to their role in S. cerevisiae, neither septins nor CnBud4 were needed to direct the position of the new bud in C. neoformans, suggesting that this function is not conserved in basidiomycetous yeasts. Our data suggest that the requirement of CnBud4 for growth at 37°C and pathogenicity in C. neoformans is based on its conserved role in septin complex organization.

Effects of environmental factors on sensitivity of Cryptococcus neoformans to fluconazole and amphotericin B.

Cryptococcus neoformans is a leading cause of fungal meningitis in immunocompromized populations. Amphotericin B (AMB) and fluconazole (FLC) are common anticryptococcal agents. AMB treatment leads to severe side-effects. In contrast, FLC-based therapy is relatively safe, although C. neoformans often develops resistance to this drug. C. neoformans must adapt to the challenging environment of the human host. Environmental effects on potency of AMB and FLC and development of drug resistance remain poorly characterized. Here, the effects of nutrients, temperature and antioxidants on susceptibility of C. neoformans towards FLC and AMB were investigated. Limited nutrients led to a decrease and an increase of sensitivity towards FLC and AMB, respectively. Co-treatment with various antioxidants also demonstrated reciprocal effects on susceptibility towards FLC and AMB. In contrast, elevated temperature increased the efficacy of both drugs, although the effect on FLC was more drastic as compared to that of AMB. In addition, temperatures of 37°C and above prevented development of FLC resistance. Our study pointed to a critical role of the environment on susceptibility towards AMB and FLC and revealed reciprocal effects towards these antifungal drugs, reflecting contrasting modes of action of AMB and FLC.

Genetic contribution to high temperature tolerance in Cryptococcus neoformans.

The human fungal pathogen Cryptococcus neoformans relies on a complex signaling network for the adaptation and survival at the host temperature. Protein phosphatase calcineurin is central to proliferation at 37°C but its exact contributions remain ill-defined. To better define genetic contributions to the C. neoformans temperature tolerance, 4031 gene knockouts were screened for genes essential at 37°C and under conditions that keep calcineurin inactive. Identified 83 candidate strains, potentially sensitive to 37°C, were subsequently subject to technologically simple yet robust assay, in which cells are exposed to a temperature gradient. This has resulted in identification of 46 genes contributing to the maximum temperature at which C. neoformans can proliferate (Tmax). The 46 mutants, characterized by a range of Tmax on drug-free media, were further assessed for Tmax under conditions that inhibit calcineurin, which led to identification of several previously uncharacterized knockouts exhibiting synthetic interaction with the inhibition of calcineurin. A mutant that lacked septin Cdc11 was among those with the lowest Tmax and failed to proliferate in the absence of calcineurin activity. To further define connections with calcineurin and the role for septins in high temperature growth, the 46 mutants were tested for cell morphology at 37°C and growth in the presence of agents disrupting cell wall and cell membrane. Mutants sensitive to calcineurin inhibition were tested for synthetic lethal interaction with deletion of the septin-encoding CDC12 and the localization of the septin Cdc3-mCherry. The analysis described here pointed to previously uncharacterized genes that were missed in standard growth assays indicating that the temperature gradient assay is a valuable complementary tool for elucidating the genetic basis of temperature range at which microorganisms proliferate.

Increase of reactive oxygen species contributes to growth inhibition by fluconazole in Cryptococcus neoformans.

BACKGROUND: Cryptococcus neoformans, a basidiomycetous yeast, is a fungal pathogen that can colonize the lungs of humans causing pneumonia and fungal meningitis in severely immunocompromised individuals. Recent studies have implied that the antifungal drug fluconazole (FLC) can induce oxidative stress in C. neoformans by increasing the production of reactive oxygen species (ROS), as presence of the antioxidant ascorbic acid (AA) could reverse the inhibitory effects of FLC on C. neoformans. However, in Candida albicans, AA has been shown to stimulate the expression of genes essential for ergosterol biosynthesis. Hence, the contribution of ROS in FLC-mediated growth inhibition remains unclear. RESULTS: In order to determine whether counteracting ROS generated by FLC in C. neoformans can contribute to diminishing inhibitory effects of FLC, we tested three other antioxidants in addition to AA, namely, pyrrolidine dithiocarbamate (PDTC), retinoic acid (RA), and glutathione (GSH). Our data confirm that there is an increase in ROS in the presence of FLC in C. neoformans. Importantly, all four antioxidants reversed FLC-mediated growth inhibition of C. neoformans to various extents. We further verified the involvement of increased ROS in FLC-mediated growth inhibition by determining that ROS-scavenging proteins, metallothioneins (CMT1 and CMT2), contribute to growth recovery by PDTC and AA during treatment with FLC. CONCLUSION: Our study suggests that ROS contributes to FLC-mediated growth inhibition and points to a complex nature of antioxidant-mediated growth rescue in the presence of FLC.

Mechanisms of Cytokinesis in Basidiomycetous Yeasts.

While mechanisms of cytokinesis exhibit considerable plasticity, it is difficult to precisely define the level of conservation of this essential part of cell division in fungi, as majority of our knowledge is based on ascomycetous yeasts. However, in the last decade more details have been uncovered regarding cytokinesis in the second largest fungal phylum, basidiomycetes, specifically in two yeasts, Cryptococcus neoformans and Ustilago maydis. Based on these findings, and current sequenced genomes, we summarize cytokinesis in basidiomycetous yeasts, indicating features that may be unique to this phylum, species-specific characteristics, as well as mechanisms that may be common to all eukaryotes.

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore-microtubule attachment.

Cdt1, a protein critical for replication origin licensing in G1 phase, is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase owing to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle-assembly-checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kMT attachment.

Stress-stimulated mitogen-activated protein kinases control the stability and activity of the Cdt1 DNA replication licensing factor.

DNA replication is tightly coordinated both with cell cycle cues and with responses to extracellular signals to maintain genome stability. We discovered that human Cdt1, an essential origin licensing protein whose activity must be restricted to G(1) phase, is a substrate of the stress-activated mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). These MAP kinases phosphorylate Cdt1 both during unperturbed G(2) phase and during an acute stress response. Phosphorylation renders Cdt1 resistant to ubiquitin-mediated degradation during S phase and after DNA damage by blocking Cdt1 binding to the Cul4 adaptor, Cdt2. Mutations that block normal cell cycle-regulated MAP kinase-mediated phosphorylation interfere with rapid Cdt1 reaccumulation at the end of S phase. Phosphomimetic mutations recapitulate the stabilizing effects of Cdt1 phosphorylation but also reduce the ability of Cdt1 to support origin licensing. Two other CRL4(Cdt2) targets, the cyclin-dependent kinase (CDK) inhibitor p21 and the methyltransferase PR-Set7/Set8, are similarly stabilized by MAP kinase activity. These findings support a model in which MAP kinase activity in G(2) promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete.

The emerging regulatory potential of SCFMet30 -mediated polyubiquitination and proteolysis of the Met4 transcriptional activator.

The yeast SCFMet30 ubiquitin ligase plays a critical role in cell division by regulating the Met4 transcriptional activator of genes that control the uptake and assimilation of sulfur into methionine and S-adenosyl-methionine. The initial view on how SCFMet30 performs its function has been driven by the assumption that SCFMet30 acts exclusively as Met4 inhibitor when high levels of methionine drive an accumulation of cysteine. We revisit this model in light of the growing evidence that SCFMet30 can also activate Met4. The notion that Met4 can be inhibited or activated depending on the sulfur metabolite context is not new, but for the first time both aspects have been linked to SCFMet30, creating an interesting regulatory paradigm in which polyubiquitination and proteolysis of a single transcriptional activator can play different roles depending on context. We discuss the emerging molecular basis and the implications of this new regulatory phenomenon.

Destabilization of binding to cofactors and SCFMet30 is the rate-limiting regulatory step in degradation of polyubiquitinated Met4.

The Met4 transcriptional activator of methionine biosynthesis is negatively regulated by the SCFMet30 ubiquitin ligase in response to accumulation of methionine. This mechanism requires polyubiquitination, but not proteolysis. We report that a previously unappreciated mechanism involving growth control regulates Met4. Unless methionine is present in the growth medium, polyubiquitinated Met4 is stabilized in late exponential cultures, correlating with transcriptional repression. Polyubiquitinated Met4 becomes destabilized in a proteasome-dependent manner upon reentry into exponential growth, correlating with transcriptional activation. Met4 stabilization is regulated at the level of SCFMet30 binding and requires transcriptional cofactors. These lock Met4 and SCFMet30 into a tight complex active in ubiquitination but incapable of binding the proteasome. Release of polyubiquitinated Met4 from SCFMet30 is sufficient for degradation, and specific sulfur amino acids can promote the degradation by destabilizing Met4 binding to cofactors and SCFMet30. Thus, destabilization of cofactors and SCFMet30 binding is the rate-limiting regulatory step in Met4 proteolysis.