Mini-blaster-mediated targeted gene disruption and marker complementation in Candida albicans.

<p>Several gene disruption strategies have been described in Candida albicans to create homozygous mutants. We describe here a recyclable mini-blaster cassette containing C. albicans URA3 gene and 200-bp flanking repeats that is useful for disruption of C. albicans genes. The cassette can be used to create unmarked homozygous mutants which can be complemented at the HIS1 gene locus. This strategy of creating gene disruptions and subsequent complementation can be used to study gene function.</p

Fungal biofilms.

<p>Biofilms are a principal form of microbial growth and are critical to development of clinical infection. They are responsible for a broad spectrum of microbial infections in the human host. Many medically important fungi produce biofilms, including <em>Candida</em> <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002585#ppat.1002585-Finkel1">[1]</a>, <em>Aspergillus</em> <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002585#ppat.1002585-Beauvais1">[2]</a>,<em>Cryptococcus</em> <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002585#ppat.1002585-Martinez1">[3]</a>, <em>Trichosporon</em> <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002585#ppat.1002585-DiBonaventura1">[4]</a>, <em>Coccidioides</em> <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002585#ppat.1002585-Davis1">[5]</a>, and <em>Pneumocystis</em> <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002585#ppat.1002585-Cushion1">[6]</a>. In this review we emphasize common features among fungal biofilms, and point toward genes and pathways that may have conserved roles.</p

Genetic control of Candida albicans biofilm development.

Candida species cause frequent infections owing to their ability to form biofilms - surface-associated microbial communities - primarily on implanted medical devices. Increasingly, mechanistic studies have identified the gene products that participate directly in the development of Candida albicans biofilms, as well as the regulatory circuitry and networks that control their expression and activity. These studies have uncovered new mechanisms and signals that govern C. albicans biofilm development and associated drug resistance, thus providing biological insight and therapeutic foresight.</p

Candida albicans adds more weight to iron regulation.

The pathogen Candida albicans can occupy both the bloodstream and gastrointesintal (GI) tract, niches that differ in iron availability. Chen et al. report that a distinct transcription factor, Sef1, alters the conserved fungal iron regulatory paradigm. Sef1 is pivotal for bloodstream infection, but contributes to GI tract colonization as well.</p

Fungal morphogenesis: in hot pursuit.

<p>Temperature affects diverse biological processes. In fungi such as the pathogen Candida albicans, temperature governs a morphogenetic switch between yeast and hyphal growth. A new report connects the thermosensor Hsp90 to a CDK-cyclin-transcription factor module that controls morphogenesis.</p

Fungal Biofilms, Drug Resistance, and Recurrent Infection.

<p>A biofilm is a surface-associated microbial community. Diverse fungi are capable of biofilm growth. The significance of this growth form for infection biology is that biofilm formation on implanted devices is a major cause of recurrent infection. Biofilms also have limited drug susceptibility, making device-associated infection extremely difficult to treat. Biofilm-like growth can occur during many kinds of infection, even when an implanted device is not present. Here we summarize the current understanding of fungal biofilm formation, its genetic control, and the basis for biofilm drug resistance.</p

An extensive circuitry for cell wall regulation in Candida albicans.

Protein kinases play key roles in signaling and response to changes in the external environment. The ability of Candida albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. Our guiding hypothesis was that creating and screening a set of protein kinase mutant strains would reveal signaling pathways that mediate stress response in C. albicans. A library of protein kinase mutant strains was created and screened for sensitivity to a variety of stresses. For the majority of stresses tested, stress response was largely conserved between C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. However, we identified eight protein kinases whose roles in cell wall regulation (CWR) were not expected from functions of their orthologs in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. Analysis of the conserved roles of these protein kinases indicates that establishment of cell polarity is critical for CWR. In addition, we found that septins, crucial to budding, are both important for surviving and are mislocalized by cell wall stress. Our study shows an expanded role for protein kinase signaling in C. albicans cell wall integrity. Our studies suggest that in some cases, this expansion represents a greater importance for certain pathways in cell wall biogenesis. In other cases, it appears that signaling pathways have been rewired for a cell wall integrity response.</p

Teach, then trust Elizabeth W. Jones (1939-2008): mentor to many.

Essa

cis- and trans-acting localization determinants of pH response regulator Rim13 in Saccharomyces cerevisiae.

<p>The Rim101/PacC pathway governs adaptation to alkaline pH in many fungi. Output of the pathway is mediated by transcription factors of the Rim101/PacC family, which are activated by proteolytic cleavage. The proteolytic complex includes scaffold protein Rim20 and endosome-associated subunits of the endosomal sorting complex required for transport (ESCRT). We provide here evidence that Saccharomyces cerevisiae Rim13, the protease that is implicated in Rim101 cleavage, is associated with the Rim20-ESCRT complex, and we investigate its regulation. Rim13-GFP is dispersed in cells grown in acidic medium but forms punctate foci when cells encounter alkaline conditions. A vps4Δ mutant, which accumulates elevated levels of endosomal ESCRT, also accumulates elevated levels of Rim13-GFP foci, independently of external pH. In the vps4Δ background, mutation of ESCRT subunit Snf7 or of Rim20 blocks the formation of Rim13 foci, and we found that Rim13 and Rim20 are colocalized. The Rim13 ortholog PalB of Aspergillus nidulans has been shown to undergo ESCRT and membrane association through an N-terminal MIT domain, but Rim13 orthologs in the Saccharomyces clade lack homology to this N-terminal region. Instead, there is a clade-limited C-terminal region, and we show that point mutations in this region prevent punctate localization and impair Rim13 function. We suggest that RIM13 arose from its ancestral gene through two genome rearrangements. The ancestor lost the coding region for its MIT domain through a 5' rearrangement and acquired the coding region for the Saccharomyces-specific functional equivalent through a 3' rearrangement.</p

Detection of protein-protein interactions through vesicle targeting.

The detection of protein-protein interactions through two-hybrid assays has revolutionized our understanding of biology. The remarkable impact of two-hybrid assay platforms derives from their speed, simplicity, and broad applicability. Yet for many organisms, the need to express test proteins in Saccharomyces cerevisiae or Escherichia coli presents a substantial barrier because variations in codon specificity or bias may result in aberrant protein expression. In particular, nonstandard genetic codes are characteristic of several eukaryotic pathogens, for which there are currently no genetically based systems for detection of protein-protein interactions. We have developed a protein-protein interaction assay that is carried out in native host cells by using GFP as the only foreign protein moiety, thus circumventing these problems. We show that interaction can be detected between two protein pairs in both the model yeast S. cerevisiae and the fungal pathogen Candida albicans. We use computational analysis of microscopic images to provide a quantitative and automated assessment of confidence.</p