Zebrafish genes analysis procedure diagram.

<p>The analysis of zebrafish was summarized in this figure, which includes the method used, key findings and the related Tables and Figures. First significant genes were first singled out by ANOVA and Bonferroni correction. GO analysis of these genes revealed that the key function is related to iron-related function. PCA and K-mean clustering suggested that the genes profile can be cluster into two main groups: progressively expressed and progressively suppressed. Finally, binomial tests were used to identify the specific function, iron-related and immune-related. Iron-related genes demonstrated a significant bias toward Group 1 p-value 0.0011. Immune-related genes demonstrated a significant association bias toward Group 2 with p-value 0.0215.</p

Expression profiles of the zebrafish immune-related and iron-related genes.

<p>The immune response is a key process for zebrafish to defend themselves against <i>C. albicans</i> Of the 576 significant genes identified, 13 genes had GO related to the immune response and immune system development. PCA divided these into two main groups. (A) shows the immune-related gene expression profile. Analysis of the <i>C. albicans</i> expression profiles identified iron transport and scavenging as important factors during infection. GO analysis also revealed that iron homeostasis was an important biological process among the significant genes. There were 24 genes in the 576 significant zebrafish genes that had GO associated with iron binding and iron transport. PCA divided these into two main groups. (B) shows the iron-related gene expression profile.</p

Validation of expression patterns by real-time polymerase chain reaction.

<p>(A) The <i>C</i>. <i>albicans</i> gene expression patterns (<i>RBT5</i>, <i>CHT2</i>, <i>MNT1</i>, and <i>PMT1</i>) revealed by the DNA microarray are shown on the top. Real-time PCR of <i>C</i>. <i>albicans</i> genes (RNA isolated from three infected zebrafish per group) are shown in the bottom. <i>C. albicans</i> expressed <i>RBT5</i> to use iron in the invasion and damage phase (after 6 hpi), expressed <i>CHT2</i> in the late stage (after 12 hpi), and activated <i>MNT1</i> and <i>PMT1</i> in the adhesion phase (0.5 and 2 hpi). (B) The zebrafish gene expression patterns (<i>TNFA</i>, <i>SLC25A37</i>, <i>VTNB</i>, and <i>CYP4V2</i>) revealed by the DNA microarray are shown on the top. Real-time PCR of the zebrafish genes (RNA isolated from three infected zebrafish per group) are shown on the bottom. The zebrafish progressively expressed <i>TNFA</i> and <i>SLC25A37</i> during the infection and turned on <i>VTNB</i> and <i>CYP4V2</i> in the adhesion phase (0.5, 1 hpi). The profiles of the real time PCR were identical and could validate the present genome microarray in both the host and the pathogen. The asterisks above the bar indicate that the level of target gene expression was significantly different from the levels of expression in the other time points: * = P<0.2; ** = P<0.05; *** = P<0.01.</p

Expression profiles of significant <i>C.</i><i>albicans</i> genes on well-known iron uptake pathway.

<p>In reductive system, <i>ALS3</i>, needed to acquire iron from the host's ferritin, transiently expressed in the early invasion phase. <i>FTR1</i>, a high-affinity iron permease, was progressively induced during the invasion phase, and expression was then sustained in the damage phase. In heme-iron uptake system, <i>C. albicans</i> up-regulated <i>RBT5</i> during the invasion and damage phases and expressed <i>HMX1</i> in the damage phase. These data indicated that the uptake of iron had already started in the early invasion phase and the possible uptake of iron from the host's hemoglobin as the zebrafish suffered massive hemorrhaging during the damage phase.</p

Comparison of gene expressions of mouse and zebrafish models during <i>C.</i><i>albicans</i> infection.

<p>The present work was compared with the <i>C. albicans</i> genome wide data set in Thewes et al. (2007) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072483#pone.0072483-Thewes1" target="_blank">[18]</a>. (A) shows that the intersection of this work and that of Thewes et al. had 130 genes. The proposed method was used to re-analyze the gene expression profiles and cluster them into two groups that contained288 up-regulated genes and 185 down-regulated genes. (B) and (C) show that 39 up-regulated genes and 45 down-regulated genes had the same expression profile in the zebrafish model.</p

<i>C.</i><i>albicans</i> genes analysis procedure diagram.

<p>The analysis of <i>C. albicans</i> was summarized in this figure, which includes the method used, key findings and the related Tables and Figures. First significant genes were first singled out by ANOVA and Bonferroni correction. GO analysis of these genes revealed that the key function is related to membrane transport. PCA showed that the infection can be classified into three stages according to gene expression profiles. K-mean clustering suggested that the genes profile can be cluster into two main groups: progressively expressed and progressively suppressed. Binomial tests were used to identify important GO-terms in the two main groups. For both total significant genes and cell surface-related genes, hyphal formation and cell wall reorganization demonstrated a significant association bias toward Group 1. Cellular homeostasis was found to demonstrate a significant bias toward Group 2, and most cell surface-realted genes in cellular homeostasis are iron related.</p

Significant <i>C. albicans</i> genes in signaling pathway of yeast-to-hyphae transition.

<p>Map significant filamentous related genes onto well-known controlling morphological transition pathways.</p

PC load bar plot and histology of zebrafish infected by <i>C.</i><i>albicans.</i>

<p>(A) The pathogen's load contributions to the first principal component indicated that the differences in expression between 0.5 and 1 and 12, 16 and 18 hpi represented the axes of largest variation. The load contributions to the second principal components featured the gene expression differences between time points 2, 4, 6 and 8 hpi and 0.5, 1, 12, 16 and 18 hpi. Two principal components sufficiently explained more than 93% of the variations of the zebrafish expression profiles. (B) The host's load contributions to the first principal component indicated that the differences in expression between time points 0.5, 1, 2 and 4 hpi and 12, 16 and 18 hpi represented the axes of largest variation. The load contributions to the second principal components featured the gene expression differences between time points 6, 8 and 12 hpi and 0.5, 1, 2, 4, 16 and 18 hpi. (C) The histology of zebrafish liver indicated that <i>C. albicans</i> mainly grew in yeast form at 1 hpi and began to attach to the zebrafish liver. 0.5 and 1 hpi were thus defined as the adhesion phase. <i>C. albicans</i> began to transit to the hyphal form at 2 hpi. Invasion to the liver was evident at 4 to 8 hpi. Hence, time points 4 to 8 hpi were defined as the invasion phase, with 2 hpi defined as a transition point for morphogenesis. Beyond 12 hpi, tissue damage, more extensive penetration, and fish death occurred. Time points 12, 16 and 18 hpi were defined as the damage phase.</p

Profile of iron-related genes during inection and reference.

<p>Compare profile of iron-related genes with those previous researches, which link these genes' induction to the surrounding iron concentration.</p

<i>C. albicans</i> genes cluster by PCA subspace.

<p>This table lists the key biological processes that demonstrated an association bias toward either Group 1 or Group 2 with a <i>p</i>-value of less than 0.05 (A complete list was shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072483#pone.0072483.s005" target="_blank">Table S3</a>).</p