Generation of phenotypic diversity in the fungal pathogen Candida Albicans

University of Minnesota Ph.D. dissertation. September 2010. Major: Microbiology, Immunology and Cancer Biology. Advisor: Dana Davis. 1 computer file (PDF); xiii, 303 pages.Microbial organisms have a diverse array of mechanisms to obtain phenotypic variation. Phenotypic variation not only enhances population fitness and competitiveness for a specific niche but it is also critical for the survival of a population to unexpected environmental changes. Further, in pathogenic organisms, phenotypic variation is directly associated with virulence. Therefore, besides of the contribution to our understanding of microbial evolution, dissecting the mechanisms that lead to phenotypic variation in pathogenic organisms is very clinically relevant. Candida albicans is the most successful opportunistic fungi that infect humans. C. albicans is an obligate diploid yeast with an almost exclusive clonal form of reproduction. In the absence of meiosis to introduce variation in the population, C. albicans needs alternative mechanisms to achieve variability, such as the colony morphology phenotypic switching (CMPS). CMPS is the formation of colonies that have an altered, heritable, and low frequency reversible morphology. CMPS is associated with pathogenesis in C. albicans: variant colony morphologies have been isolated during infections in humans and show an altered expression of diverse virulence factors, including the secretion of hydrolytic enzymes and resistance to antifungal drugs. Despite the potential role of CMPS in the pathogenesis of C. albicans, little is known about the mechanisms that regulate this phenomenon. In our lab, we serendipitously identified a negative regulator of CMPS in C. albicans: the Kelch protein Mds3. Mds3 had been previously associated with other morphogenetic processes in fungi, but the biological role of Mds3 in the cell was unknown. Therefore, my goals in this dissertation were to understand the function of Mds3 in the cell and to use this knowledge to gain insights into C. albican's CMPS mechanisms and regulation. Through a combination of bioinformatic, biochemical, and genetic analyses we found that Mds3 appears to be a large Kelch/BTB cytoplasmic scaffold protein that functions as a regulator of two major signaling cascades, the TOR and Ras pathways (Chapters 2, 3, and 5). With this information, I was able to identify more CMPS regulators that belong to these pathways and environmental signals that regulate CMPS and which are all strongly associated with signaling through these pathways (Chapters 4 and 5). Analyses of morphologically switched mds3delta/delta strains indicated that the phenotypic switch is accompanied by an increase in the sensitivity to the TOR inhibitor rapamycin, which suggests an increased dependence on TOR function in the switched strains. Further, the phenotypic switch was also accompanied by increased sensitivity to genotoxic agents and sometimes also by karyotypic rearrangements and aneuploidies (Chapter 4). Increased DNA damage and genomic instability are mechanisms associated with phenotypic variation in several highly diverse organisms, and could also be mechanisms leading to the phenotypic switch in C. albicans (Chapter 4). Taken together, I propose a model for CMPS in C. albicans in which defects in the signaling through the TOR and Ras signal transduction pathways as cells become nutrient limited and stressed lead to the accumulation of genetic and epigenetic alterations that eventually cause the phenotypic switch

Intracellular complexes of the early-onset torsion dystonia-associated AAA+ ATPase TorsinA

A single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer. Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein. We also discovered that the deletion of the N-terminal membrane-associating region of torsinA abolished oligomer formation. Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex

Prognostic Implications of the Complement Protein C1q in Gliomas

The contribution of the complement system in the pathophysiology of brain cancers has been recently considered in light of its well-known involvement in carcinogenesis. Complement system represents an important component of the inflammatory response, which acts as a functional bridge between the innate and adaptive immune response. C1q, the first recognition subcomponent of the complement classical pathway, has recently been shown to be involved in a range of pathophysiological functions that are not dependent on complement activation. C1q is expressed in the microenvironment of various types of human tumors, including melanoma, prostate, mesothelioma, and ovarian cancers, where it can exert a protective or a harmful effect on cancer progression. Despite local synthesis of C1q in the central nervous system, the involvement of C1q in glioma pathogenesis has been poorly investigated. We, therefore, performed a bioinformatics analysis, using Oncomine dataset and UALCAN database in order to assess whether the expression of the genes encoding for the three chains of C1q (C1qA, C1qB, and C1qC) could serve as a potential prognostic marker for gliomas. The obtained results were then validated using an independent glioma cohort from the Chinese Glioma Genome Atlas datasets. Our bioinformatics analysis, coupled with immunohistochemistry and fluorescence microscopy, appears to suggest a positive correlation between higher levels of C1q expression and unfavorable prognosis in a diverse grade of gliomas

THE CLINICOPATHOLOGICAL AND PROGNOSTIC SIGNIFICANCES OF C1Q EXPRESSION IN GLIOMAS: A BIOINFORMATICS ANALYSIS

Introduction. The complement system represents an important component of the inflammatory response and acts as a functional bridge between the innate and adaptive immune response. The contribution of the complement component C1q in the pathophysiology of brain cancers has been recently considered in light of its well-known involvement in carcinogenesis. Brain malignancies arise from cells of the CNS and are classified according to the tissue of phylogenetic origin. Gliomas represent the most common and aggressive form of brain tumours in adults. They derive from glial cells that help to support the functions of the other main brain cells type, the neurons (1). These are a heterogeneous group of diseases with multiple subtypes (1, 2). Glioblastoma multiforme (GBM) is the most common and fatal form of a primary brain tumour, accounting for approximately 60% of all glioma cases (3), whereas grade-II and -III gliomas are the second most common type of glioma in adults (~30%) (3). C1q molecule, together with other complement components, can be locally produced within the CNS by microglia and astrocytes, rendering it an attractive player in primary brain tumour development (4). The role of C1q in gliomas microenvironment is still poorly characterized and it is still quite puzzling whether it exerts a beneficial or a harmful activity for cancer progression. In the present study we performed a bioinformatics analysis aimed at investigating if C1q can serve as a potential prognostic marker for gliomas. Methods. The expression levels of C1qA, C1qB and C1qC genes in gliomas were analysed using Oncomine analysis. Available genomics data from The Cancer Genome Atlas project was used for Kaplan–Meier survival analysis to generate survival probability plots, using UALCAN analysis. Results. From the analysis performed on several data- sets using Oncomine, we showed a significantly higher mRNA expression levels for C1qA, C1qB and C1qC chains were detected in gliomas (different histotypes and grades) as compared to normal brain tissue (Fig. 1). We observed a positive correlation between the mRNA expression of C1qA, C1qB and C1qC mRNA poly- peptide chains and the unfavorable prognosis only in gliomas grade-II and -III, where the survival probability is indeed reduced (P <0.05) (Fig. 2). No correlation was observed in glioblastoma multiforme (Fig. 2). By immu- nohistochemical approaches we detected a high depo- sition of C1q in the tumor microenvironment of both in grade-II and -III gliomas and in GBMs examined (Fig. 3a glioma, 3b glioblastoma multiforme; 20x Magnification). Moreover, in double immunocytochemical experiments we demonstrated that CD68 positive infiltrating cells are actively synthesizing C1q in the tumor micro-envi- ronment. CD68 expression is characteristic of tumor- associated macrophages, whose enrichment in glioma has been associated with poor prognosis (5). Conclusion. In our study C1q expression was significantly correlated with poor survival probability in gliomas grade-II and -III while this is not the case for GBM. These data altogether underline how complex, multifaceted and still poorly understood is the role C1q can exert on tumor progression, and how the very same molecule can differentially affect the outcome depending on the biological context it comes to act

The VO: A Powerful Tool for Global Astronomy

Since its inception in the early 2000's, the Virtual Observatory (VO), developed as a collaboration of many national and international projects, has become a major factor in the discovery and dissemination of astronomical information worldwide. The International Virtual Observatory Alliance (IVOA) has been coordinating all these efforts worldwide to ensure a common VO framework that enables transparent access to and interoperability of astronomy resources (data and software) around the world. The VO is not a magic solution to all astronomy data management challenges but it does bring useful solutions in many areas borne out by the fact that VO interfaces are broadly found in astronomy's major data centres and projects worldwide. Astronomy data centres have been building VO services on top of their existing data services to increase interoperability with other VO-compliant data resources to take advantage of the continuous and increasing development of VO applications. VO applications have made multi-instrument and multi-wavelength science, a difficult and fruitful part of astronomy, somewhat easier. More recently, several major new astronomy projects have been directly adopting VO standards to build their data management infrastructure, giving birth to ‘VO built-in' archives. Embracing the VO framework from the beginning brings the double gain of not needing to reinvent the wheel and ensuring from the start interoperability with other astronomy VO resources. Some of the IVOA standards are also starting to be used by neighbour disciplines like planetary sciences. There is still quite a lot to be done on the VO, in particular tackling the upcoming big data challenge and how to find interoperable solutions to the new data analysis paradigm of bringing and running the software close to the data. We report on the current status and also desire to encourage others to adopt VO technology and engage them in the effort of developing the VO. Thus, we wish to ensure that the VO standards fit new astronomy projects requirements and needs

Prognostic implications of the complement protein C1q in gliomas

Associazione Italiana per la Ricerca sul Cancro (AIRC); Program Innovative Tools for Cancer Risk Assessment and Diagnosis

Pin1-dependent signaling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction

The cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis-trans isomerase Pin1. This signalling cascade negatively regulates NL2' s ability to interact with gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, gephyrin and GABA A receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1\ufffd/\ufffd) associated with an increase in amplitude of spontaneous GABA A -mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction. \ufffd 2014 Macmillan Publishers Limited. All rights reserved

Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin for Initiation and Maintenance

Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification