Iron Source Preference and Regulation of Iron Uptake in Cryptococcus neoformans

The level of available iron in the mammalian host is extremely low, and pathogenic microbes must compete with host proteins such as transferrin for iron. Iron regulation of gene expression, including genes encoding iron uptake functions and virulence factors, is critical for the pathogenesis of the fungus Cryptococcus neoformans. In this study, we characterized the roles of the CFT1 and CFT2 genes that encode C. neoformans orthologs of the Saccharomyces cerevisiae high-affinity iron permease FTR1. Deletion of CFT1 reduced growth and iron uptake with ferric chloride and holo-transferrin as the in vitro iron sources, and the cft1 mutant was attenuated for virulence in a mouse model of infection. A reduction in the fungal burden in the brains of mice infected with the cft1 mutant was observed, thus suggesting a requirement for reductive iron acquisition during cryptococcal meningitis. CFT2 played no apparent role in iron acquisition but did influence virulence. The expression of both CFT1 and CFT2 was influenced by cAMP-dependent protein kinase, and the iron-regulatory transcription factor Cir1 positively regulated CFT1 and negatively regulated CFT2. Overall, these results indicate that C. neoformans utilizes iron sources within the host (e.g., holo-transferrin) that require Cft1 and a reductive iron uptake system

Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for anti-cryptococcal therapy

Gene Regulatory Mechanisms Underlying the Spatial and Temporal Regulation of Target-Dependent Gene Expression in Drosophila Neurons

Neuronal differentiation often requires target-derived signals from the cells they innervate. These signals typically activate neural subtype-specific genes, but the gene regulatory mechanisms remain largely unknown. Highly restricted expression of the FMRFa neuropeptide in Drosophila Tv4 neurons requires target-derived BMP signaling and a transcription factor code that includes Apterous. Using integrase transgenesis of enhancer reporters, we functionally dissected the Tv4-enhancer of FMRFa within its native cellular context. We identified two essential but discrete cis-elements, a BMP-response element (BMP-RE) that binds BMP-activated pMad, and a homeodomain-response element (HD-RE) that binds Apterous. These cis-elements have low activity and must be combined for Tv4-enhancer activity. Such combinatorial activity is often a mechanism for restricting expression to the intersection of cis-element spatiotemporal activities. However, concatemers of the HD-RE and BMP-RE cis-elements were found to independently generate the same spatiotemporal expression as the Tv4-enhancer. Thus, the Tv4-enhancer atypically combines two low-activity cis-elements that confer the same output from distinct inputs. The activation of target-dependent genes is assumed to 'wait' for target contact. We tested this directly, and unexpectedly found that premature BMP activity could not induce early FMRFa expression; also, we show that the BMP-insensitive HD-RE cis-element is activated at the time of target contact. This led us to uncover a role for the nuclear receptor, seven up (svp), as a repressor of FMRFa induction prior to target contact. Svp is normally downregulated immediately prior to target contact, and we found that maintaining Svp expression prevents cis-element activation, whereas reducing svp gene dosage prematurely activates cis-element activity. We conclude that the target-dependent FMRFa gene is repressed prior to target contact, and that target-derived BMP signaling directly activates FMRFa gene expression through an atypical gene regulatory mechanism

Mutant sequence analysis of the Tv4-enhancer.

<p>(<b>A</b>) Conservation track from UCSC Browser showing conservation islands between 12 sequenced <i>Drosophila</i> species. (<b>B1-B9</b>) Graphic representation of the position of putative HD (green), Mad (red and magenta) and Med motifs (yellow) as the coloured vertical bars within the Tv4-enhancer. These overlay the depicted deletion series of the Tv4-enhancer B1-B9. We generated a series of deletion transgenes (B1-B9; dotted region deleted). <b>B1</b> is the full length wild-type Tv4-enhancer, denoted by the complete horizontal solid black line. <b>B2-B9</b> show the region present in the reporter transgene (horizontal black bar) after removal of certain sequences (dotted regions). <b>B4,5,7</b> show a deletion between two regions; the points of fusion are shown by vertical bars, between which the intervening sequence is removed. <b>B8,9</b> show the type of sequence conversion performed within the region shown. (<b>C</b>) Reporter expression driven from control or deletion mutants B1-B9, or the empty reporter vector, expressed in the bar graph as the % nEYFP fluorescence intensity relative to mean of the B1 control. The right-most panel provides the number of Tv4 neurons (out of six) expressing significant nEYFP above the 99% confidence interval of the empty vector control. Removal of the HD-A motif (green, B3,B4) or the Mad-D motif (magenta, B3,B5) severely reduced expression. The enhancer fragment spanning the HD-A to Mad-D motifs (B6) expressed at moderate levels, but alteration of the intervening sequence severely reduced reporter expression, whether by sequence elimination (B7), conversion to complementary sequence (B8) or non-canonical sequence conversion (B9). (<b>D</b>) Point substitution mutants of Mad, HD and Med motifs, showing (bar graph) expression levels as % nEYFP fluorescence intensity relative to mean of the control, or (bottom-most panel) the number of Tv4 neurons (out of six) expressing significant nEYFP above the 99% confidence interval of the empty vector control. Only substitution mutants that alter the HD-A or Mad-D motifs essentially eliminate reporter expression. n = 10–20 animals per genotype. All data represented as mean±SEM. Data compared using one-way ANOVA with Tukey HSD <i>post-hoc</i> test. * = p<0.05 compared to controls.</p

Sensitivity of PKA Mutants to Caffeine

<p>Ten-fold serial dilutions of the WT and the <i>pka1</i> and <i>pkr1</i> mutants were grown on YPD medium containing caffeine at 30 °C and 37 °C. The strains used for all treatments are listed on the left side of the panel. The plates were incubated for 5 d before being photographed.</p

Genetic and biochemical analysis of the HD-RE and BMP-RE concatemers.

<p>(<b>A,B</b>) HD-RE (<i>6xHD-A-nEYFP</i>) and (<b>C,D</b>) BMP-RE (<i>4xMad-D-nEYFP</i>) reporter expression in the genotypes shown. We show the relative intensity of reporter expression as the % of the mean of the genetic control. (<b>A</b>) Expression of HD-RE is reduced in <i>ap</i> mutants and eliminated in <i>eya</i> mutants, but not altered in <i>wit</i> or <i>sqz</i> mutants. Expression of <i>UAS-Glued</i>^<i>DN</i> (retrograde traffic blocker) using <i>ap</i>^<i>GAL4</i> had no effect on HD-RE. (<b>B</b>) <i>UAS-dac</i> overexpression (by <i>OK6-GAL4</i>) strongly increased HD-RE expression in Tv4 neurons. (<b>C</b>) Expression of BMP-RE is reduced in <i>ap</i>, <i>wit</i> and <i>eya</i> mutants. <i>BMP-RE</i> expression was also eliminated by overexpression of <i>UAS-Mad</i>^<i>1</i> (DNA-binding defective Mad) or <i>UAS-Glued</i>^<i>DN</i> (retrograde trafficking blocker) using <i>ap</i>^<i>GAL4</i>. (<b>D</b>) <i>UAS-dac</i> overexpression increased <i>BMP-RE</i> expression in Tv4 neurons. (<b>E-G</b>) EMSA studies show sequence-specific binding of Apterous to the HD-A motif of HD-RE and of Mad to the Mad-D motif of BMP-RE. (<b>E</b>) HD-RE labeled probes (sequences shown below as WT HD-RE) are shifted in the presence of recombinant GST-CtermAp (C-terminal half of Apterous containing the Homeodomain) and efficiently out-competed by wildtype unlabeled probe (WT comp). The binding is not outcompeted by unlabeled HD-RE with a mutated HD-A motif (MutHD HD-RE). The number under each lane indicates the stoichiometric ratio between unlabeled and labeled probe. (<b>F</b>) The BMP-RE is not shifted in the presence of GST-CtermAp, indicating a lack of Ap binding. (<b>G</b>) The BMP-RE is shifted when presented with GST-MH1-Mad (that contains the DNA-binding domain). The band shift is out-competed by increasing amounts of unlabeled wild-type probe (WT comp). Competition is not observed when unlabelled probe with a mutated putative Mad-binding site (MutMad comp) is added. Data in A-C represented as mean±SEM. n = 10–20 animals per genotype and compared using one-way ANOVA with Tukey HSD <i>post-hoc</i> test. * = p<0.05 compared to control. <b>Genotypes.</b><i>[cis-element]</i> in the following refers to either <i>6xHD-A-nEYFP</i> or <i>4xMad-D-nEYFP</i>. <b>(A,C) <i>ctrl</i></b><i>(Tv</i>^{<i>[cis-element]</i>}<i>/+)</i>. <b><i>ap</i></b>, <i>(ap</i>^<i>GAL4</i><i>/ap</i>^<i>P44</i><i>; Tv</i>^{<i>[cis-element]</i>}<i>/+)</i>. <b><i>wit</i></b><i>(Tv</i>^{<i>[cis-element]</i>},<i>wit</i>^<i>A12</i><i>/ wit</i>^<i>B11</i><i>)</i>. <b><i>sqz</i></b><i>(Tv</i>^{<i>[cis-element]</i>},<i>sqz</i>^<i>ie</i><i>/sqz</i>^<i>ie</i><i>)</i>. <b><i>eya</i></b><i>(eya</i>^{<i>Cli-IID</i>}<i>/eya</i>^<i>D1</i><i>; Tv</i>^{<i>[cis-element]</i>}<i>/+)</i>. <b><i>UAS-Mad</i></b>^{<b><i>1</i></b>} (<i>ap</i>^<i>GAL4</i><i>/UAS-Mad</i>^<i>1</i><i>; Tv</i>^{<i>[cis-element]</i>}/<i>/UAS-Mad</i>^<i>1</i>). <b><i>UAS-Glued</i></b>^{<b><i>DN</i></b>} (<i>ap</i>^<i>GAL4</i><i>/UAS-Glued</i>^<i>DN</i><i>; Tv</i>^{<i>[cis-element]</i>}/<i>+</i>) <b>(B,D) <i>Ctrl</i></b> (<i>OK6-GAL4</i><b><i>/+;</i></b><i>Tv</i>^{<i>[cis-element]</i>}<i>/</i><b><i>+</i>). <i>UAS-dac</i></b> (<i>OK6-GAL4</i><b><i>/+;</i></b><i>Tv</i>^{<i>[cis-element]</i>}<i>/UAS-dac</i>).</p

The 6xHD-A and 4xMad-D <i>cis</i>-elements encode sufficient information for Tv4-specific expression.

<p>(<b>A</b>) Relative position of putative HD, Mad and Medea motifs. We concatemerized fragments from the Tv4-enhancer (shown in A). The number of concatemeric direct sequence repeats is shown, as is the number of nucleotides within each direct repeat. (<b>B-F</b>) Reporter expression for each concatemer, shown above, in the early L1 VNC. Two regions generate reporter expression in Tv4 neurons, the 25 bp HD-A-containing conserved region (<b>B</b>) and the 39 bp Mad-D containing conserved region (<b>D</b>). All other regions generate weak widespread neuronal expression (<b>C</b>), ectopic expression (<b>E</b>), or fail to express (<b>F</b>). Scale bars are 30 μm in all images.</p

Svp represses <i>FMRF</i> via the HD-RE and BMP-RE prior to target contact.

<p>(<b>A-D</b>) The HD-RE (<i>6xHD-A-nEYFP)</i> and BMP-RE (<i>4xMad-D-nEYFP</i>) reporters are not expressed at late Stg. 16 (during Svp expression) but become expressed by late Stg. 17 (after mouth hook appearance). (<b>E-H</b>) Precocious, robust activation of BMP (shown by pMad immunoreactivity, red) is seen by Stg. 16 in Tv neurons (green), using <i>ap</i>^<i>GAL4</i> to drive <i>UAS-tkv</i>^<i>Act</i>,<i>UAS-sax</i>^<i>Act</i> and <i>UAS-myc</i>::<i>mad</i> transgenes (G). However, even when BMP is precociously activated, FMRFa expression (blue) is not detected until late Stg. 17, its normal initiation time (F,H). Tv4 neurons are indicated by dotted circles. (<b>I,J</b>) Maintaining <i>UAS-svp</i> expression using <i>ap</i>^<i>GAL4</i> results in total loss of FMRFa immunoreactivity in all Tv4 neurons, here shown at early L1. Also, pMad accumulation is unaffected in Tv4 neurons in these animals; numbers under insets represent fraction of Tv clusters with pMad or FMRFa expressing cells (n = 30 and 48 for controls and <i>UAS-svp</i>, respectively.) (<b>K-N</b>) Expression of HD-RE and BMP-RE reporters is strongly reduced by early L1 larvae when <i>UAS-svp</i> is overexpressed. Numbers in lower right corner indicate the mean number of nEYFP cells per animal ± SEM (n = 10–16 animals per group, p<10⁻³ two-tailed <i>t</i>-test between experimental and control for each reporter). (<b>O,P</b>) Svp expression is not detectable in early L1 larvae in control or <i>wit</i> mutant animals. Thus, BMP signaling is not required to downregulate Svp expression. The <i>HD-RE</i> reporter is used to identify the Tv4 neuron in these <i>wit</i> mutants. Tv4 neurons are indicated by dotted circles. Svp immunoreactivity can be detected in Tv2 and Tv3 neurons. <b>Genotypes</b>: (<b>A-D</b>) Concatemerized <i>cis</i>-elements <i>[cis-element]</i> (<i>6xHD-A-nEYFP</i> or <i>4xMad-D-nEYFP</i>) were analyzed as homozygotes. (<b>E-F</b>) <b>BMP gain of function</b> (<i>ap</i>^<i>GAL4</i><i>/+;+/+</i> vs. <i>ap</i>^<i>GAL4</i><i>/UAS-tkv</i>^<i>Act</i>,<i>UAS-sax</i>^<i>Act</i><i>;+/UAS-myc</i>::<i>mad</i>). (<b>I-N</b>) <b><i>Svp</i> gain of function</b> (<i>ap</i>^<i>GAL4</i><i>/+;+/+</i> vs. <i>ap</i>^<i>GAL4</i><i>/+;+/UAS-svp)</i> (<i>ap</i>^<i>GAL4</i><i>/+; Tv</i>^{<i>[cis-element]</i>}<i>/+</i> vs. <i>ap</i>^<i>GAL4</i><i>/+; Tv</i>^{<i>[cis-element]</i>}<i>/UAS-svp)</i>. (<b>O,P</b>) <b>BMP loss of function</b> (<i>ap</i>^<i>lacZ</i><i>/+;wit</i>^<i>A12</i><i>/ Tv</i>^{<i>6xHD-A-nEYFP</i>}<i>+</i> vs. <i>ap</i>^<i>lacZ</i><i>/+;wit</i>^<i>A12</i><i>/ wit</i>^<i>A12</i>,<i>Tv</i>^{<i>6xHD-A-nEYFP</i>}).</p