Control of the C. albicans Cell Wall Damage Response by Transcriptional Regulator Cas5

The fungal cell wall is vital for growth, development, and interaction of cells with their environment. The response to cell wall damage is well understood from studies in the budding yeast Saccharomyces cerevisiae, where numerous cell wall integrity (CWI) genes are activated by transcription factor ScRlm1. Prior evidence suggests the hypothesis that both response and regulation may be conserved in the major fungal pathogen Candida albicans. We have tested this hypothesis by using a new C. albicans genetic resource: we have screened mutants defective in putative transcription factor genes for sensitivity to the cell wall biosynthesis inhibitor caspofungin. We find that the zinc finger protein CaCas5, which lacks a unique ortholog in S. cerevisiae, governs expression of many CWI genes. CaRlm1 has a modest role in this response. The transcriptional coactivator CaAda2 is also required for expression of many CaCas5-dependent genes, as expected if CaCas5 recruits CaAda2 to activate target gene transcription. Many caspofungin-induced C. albicans genes specify endoplasmic reticulum and secretion functions. Such genes are not induced in S. cerevisiae, but promote its growth in caspofungin. We have used a new resource to identify a key C. albicans transcriptional regulator of CWI genes and antifungal sensitivity. Our gene expression findings indicate that both divergent and conserved response genes may have significant functional roles. Our strategy may be broadly useful for identification of pathogen-specific regulatory pathways and critical response genes

Function of Metallothionein-3 in Neuronal Cells: Do Metal Ions Alter Expression Levels of MT3?

A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochemistry. Gene expression of Mt2 and Mt3 was examined in tissues extracted from the dentate gyrus of mouse brains and in human neuronal cell cultures. The whole-genome gene expression analysis identified significant variations in the mRNA levels of genes associated with zinc homeostasis, including Mt2 and Mt3. Mt3 was found to be the most differentially expressed gene in the identified groups, pointing to the existence of a factor, not yet identified, that differentially controls Mt3 expression. To examine the expression of the human metallothioneins in neurons, mRNA levels of MT3 and MT2 were compared in BE(2)C and SH-SY5Y cell cultures treated with lead, zinc, cobalt, and lithium. MT2 was highly upregulated by Zn2+ in both cell cultures, while MT3 was not affected, and no other metal had an effect on either MT2 or MT3

Quantitative evaluation of all hexamers as exonic splicing elements

We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule. The effect of 6-mers on splicing often depended on their location; much of this context effect could be ascribed to the creation of different overlapping sequences at each site. Taking these overlaps into account, the splicing effect of each 6-mer could be quantified, and 6-mers could be designated as enhancers (ESEseqs) and silencers (ESSseqs), with an ESRseq score indicating their strength. Some 6-mers exhibited positional bias relative to the two splice sites. The distribution and conservation of these ESRseqs in and around human exons supported their classification. Predicted RNA secondary structure effects were also seen: Effective enhancers, silencers and 3′ splice sites tend to be single stranded, and effective 5′ splice sites tend to be double stranded. 6-mers that may form positive or negative synergy with another were also identified. Chromatin structure may also influence the splicing enhancement observed, as a good correspondence was found between splicing performance and the predicted nucleosome occupancy scores of 6-mers. This approach may prove of general use in defining nucleic acid regulatory motifs, substitute for functional SELEX in most cases, and provide insights about splicing mechanisms

Growth of Wild-Type, Mutant, and Complemented Strains on Caspofungin Medium

<p>Serial dilutions of an overnight culture were spotted on YPD medium with no addition (A–C), 25 ng/ml caspofungin (D and E), 125 ng/ml caspofungin (F), or 50 μg/ml Congo red (G–I). Growth was visualized after 1–2 d at 30 °C. The wild-type reference strain (DAY185), mutant (Δ/Δ), and complemented (Δ/Δ/+) strains are shown. All strains were prototrophic; detailed genotypes are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020021#ppat-0020021-st002" target="_blank">Table S2</a>.</p

Expression of Caspofungin-Responsive Genes

<p>Wild-type reference strain DAY185 was grown in YPD medium at 30 °C to midexponential phase, and aliquots of the culture received caspofungin at the indicated final concentration. After 1 h of further incubation, RNA was prepared for Northern blot analysis and probed for the indicated transcripts. Ca<i>TEF1</i> was used as a loading control.</p

Dependence of Caspofungin-Responsive Genes on CaCas5

<p>Reference strain DAY185 and prototrophic Ca<i>cas5</i>Δ/Δ mutant VIC1186 were grown and treated with 125 ng/ml caspofungin as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020021#ppat-0020021-g002" target="_blank">Figure 2</a>. RNA was prepared for Northern blot analysis and probed for the indicated transcripts. The rRNA bands visualized with ethidium bromide staining were used as a loading control.</p

Combining Small-Volume Metabolomic and Transcriptomic Approaches for Assessing Brain Chemistry

The integration of disparate data types provides a more complete picture of complex biological systems. Here we combine small-volume metabolomic and transcriptomic platforms to determine subtle chemical changes and to link metabolites and genes to biochemical pathways. Capillary electrophoresis–mass spectrometry (CE–MS) and whole-genome gene expression arrays, aided by integrative pathway analysis, were utilized to survey metabolomic/transcriptomic hippocampal neurochemistry. We measured changes in individual hippocampi from the mast cell mutant mouse strain, C57BL/6 <i>Kit</i>^{<i>W‑sh/W‑sh</i>}. These mice have a naturally occurring mutation in the white spotting locus that causes reduced c-Kit receptor expression and an inability of mast cells to differentiate from their hematopoietic progenitors. Compared with their littermates, the mast cell-deficient mice have profound deficits in spatial learning, memory, and neurogenesis. A total of 18 distinct metabolites were identified in the hippocampus that discriminated between the C57BL/6 <i>Kit</i>^{<i>W‑sh/W‑sh</i>} and control mice. The combined analysis of metabolite and gene expression changes revealed a number of altered pathways. Importantly, results from both platforms indicated that multiple pathways are impacted, including amino acid metabolism, increasing the confidence in each approach. Because the CE–MS and expression profiling are both amenable to small-volume analysis, this integrated analysis is applicable to a range of volume-limited biological systems