On how a transcription factor can avoid its proteolytic activation in the absence of signal transduction

In response to alkaline ambient pH, the Aspergillus nidulans PacC transcription factor mediating pH regulation of gene expression is activated by proteolytic removal of a negative-acting C–terminal domain. We demonstrate interactions involving the ∼150 C–terminal PacC residues and two regions located immediately downstream of the DNA binding domain. Our data indicate two full-length PacC conformations whose relative amounts depend upon ambient pH: one ‘open’ and accessible for processing, the other ‘closed’ and inaccessible. The location of essential determinants for proteolytic processing within the two more upstream interacting regions probably explains why the interactions prevent processing, whereas the direct involvement of the C–terminal region in processing-preventing interactions explains why C–terminal truncating mutations result in alkalinity mimicry and pH-independent processing. A mutant PacC deficient in pH signal response and consequent processing behaves as though locked in the ‘closed’ form. Single-residue substitutions, obtained as mutations bypassing the need for pH signal transduction, identify crucial residues in each of the three interactive regions and overcome the processing deficiency in the ‘permanently closed’ mutant

Glycosylphosphatidylinositols synthesized by Trichophyton rubrum in a cell-free system. Nachweis von Glykosylphosphatidylinositolen von Trichophyton rubrum synthetisiert im zellfreien System

Pusch U, Effendy I, Schwarz RT, Azzouz N. Glycosylphosphatidylinositols synthesized by Trichophyton rubrum in a cell-free system. Mycoses. 2003;46(3-4):104-113.The opportunistic fungi Trichophyton rubrum and T. mentagrophytes , are responsible for relatively non-inflammatory chronic dermatophytes infections in immunocompromised patients but also in healthy individuals. This chronic infection is associated with immunosuppressive effects of the cell wall components particularly the polysaccharides secreted by these organisms. We have studied glycosylphosphatidylinositol (GPI) anchor biosynthesis in the pathogenic fungus T. rubrum and could demonstrate that T. rubrum is able to synthesize GPI structures. Glycolipids synthesized in a cell-free system prepared from the dermatophyte T. rubrum and labeled with [(3) H]mannose, and [(3) H]galactose using GDP-[(3) H]mannose and UDP-[(3) H]galactose, respectively, were identified and structurally characterized as GPIs. The evolutionary conserved backbone of T. rubrum GPIs incorporates galactose. Further, all glycolipids lack the acyl group on the inositol which was shown for Saccharomyces cerevisiae and mammalian GPIs. Our data suggest significant differences in the GPI biosynthetic pathway between mammalian and T. rubrum cells that could perhaps be exploited for the development of an antimycotic for Trichophyton infection

Targeted changes of the cell wall proteome influence [i]Candida albican[/i]s ability to form single- and multi-strain biofilms

International audienceBiofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (similar to 10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence

From Genes to Networks: The Regulatory Circuitry Controlling Candida albicans Morphogenesis

Online first chapterInternational audienceCandida albicans is a commensal yeast of most healthy individuals, but also one of the most prevalent human fungal pathogens. During adaptation to the mammalian host, C. albicans encounters different niches where it is exposed to several types of stress, including oxidative, nitrosative (e.g., immune system), osmotic (e.g., kidney and oral cavity) stresses and pH variation (e.g., gastrointestinal (GI) tract and vagina). C. albicans has developed the capacity to respond to the environmental changes by modifying its morphology, which comprises the yeast-to-hypha transition, white-opaque switching, and chlamydospore formation. The yeast-to-hypha transition has been very well characterized and was shown to be modulated by several external stimuli that mimic the host environment. For instance, temperature above 37 ℃, serum, alkaline pH, and CO2 concentration are all reported to enhance filamentation. The transition is characterized by the activation of an intricate regulatory network of signaling pathways, involving many transcription factors. The regulatory pathways that control either the stress response or morphogenesis are required for full virulence and promote survival of C. albicans in the host. Many of these transcriptional circuitries have been characterized, highlighting the complexity and the interconnections between the different pathways. Here, we present the major signaling pathways and the main transcription factors involved in the yeast-to-hypha transition. Furthermore, we describe the role of heat shock transcription factors in the morphogenetic transition, providing an edifying example of the complex cross talk between pathways involved in morphogenesis and stress response