A Core Filamentation Response Network in <em>Candida albicans</em> Is Restricted to Eight Genes

<div><p>Although morphological plasticity is a central virulence trait of <i>Candida albicans</i>, the number of filament-associated genes and the interplay of mechanisms regulating their expression remain unknown. By correlation-based network modeling of the transcriptional response to different defined external stimuli for morphogenesis we identified a set of eight genes with highly correlated expression patterns, forming a core filamentation response. This group of genes included <i>ALS3</i>, <i>ECE1</i>, <i>HGT2</i>, <i>HWP1</i>, <i>IHD1</i> and <i>RBT1</i> which are known or supposed to encode for cell- wall associated proteins as well as the Rac1 guanine nucleotide exchange factor encoding gene <i>DCK1</i> and the unknown function open reading frame orf19.2457. The validity of network modeling was confirmed using a dataset of advanced complexity that describes the transcriptional response of <i>C. albicans</i> during epithelial invasion as well as comparing our results with other previously published transcriptome studies. Although the set of core filamentation response genes was quite small, several transcriptional regulators are involved in the control of their expression, depending on the environmental condition.</p> </div

A Virtual Infection Model Quantifies Innate Effector Mechanisms and <i>Candida albicans</i> Immune Escape in Human Blood

<div><p><i>Candida albicans</i> bloodstream infection is increasingly frequent and can result in disseminated candidiasis associated with high mortality rates. To analyze the innate immune response against <i>C. albicans</i>, fungal cells were added to human whole-blood samples. After inoculation, <i>C. albicans</i> started to filament and predominantly associate with neutrophils, whereas only a minority of fungal cells became attached to monocytes. While many parameters of host-pathogen interaction were accessible to direct experimental quantification in the whole-blood infection assay, others were not. To overcome these limitations, we generated a virtual infection model that allowed detailed and quantitative predictions on the dynamics of host-pathogen interaction. Experimental time-resolved data were simulated using a state-based modeling approach combined with the Monte Carlo method of simulated annealing to obtain quantitative predictions on <i>a priori</i> unknown transition rates and to identify the main axis of antifungal immunity. Results clearly demonstrated a predominant role of neutrophils, mediated by phagocytosis and intracellular killing as well as the release of antifungal effector molecules upon activation, resulting in extracellular fungicidal activity. Both mechanisms together account for almost of <i>C. albicans</i> killing, clearly proving that beside being present in larger numbers than other leukocytes, neutrophils functionally dominate the immune response against <i>C. albicans</i> in human blood. A fraction of <i>C. albicans</i> cells escaped phagocytosis and remained extracellular and viable for up to four hours. This immune escape was independent of filamentation and fungal activity and not linked to exhaustion or inactivation of innate immune cells. The occurrence of <i>C. albicans</i> cells being resistant against phagocytosis may account for the high proportion of dissemination in <i>C. albicans</i> bloodstream infection. Taken together, iterative experiment–model–experiment cycles allowed quantitative analyses of the interplay between host and pathogen in a complex environment like human blood.</p></div

Simulation <i>versus</i> experimental results of reinoculation of alive <i>C. albicans</i> cells.

<p>Results of inoculation of <i>C. albicans</i> into human whole blood at and (blue bars). At both time points, <i>C. albicans</i>/ml were inoculated in human whole blood and the FACS analysis was performed at . This analysis provides the relative number of <i>C. albicans</i> cells that were phagocytosed by PMN () or by monocytes () or those who remained in extracellular space (). For the comparison with primary inoculation of <i>C. albicans</i>, <i>C. albicans</i>/ml were inoculated and analyzed by FACS at (green bars). The experimental conditions were also applied for the simulation with estimated parameters. Filled bars refer to the simulation results and striped bars indicate data obtained by FACS analysis.</p

Transition rates of the state based model.

<p>The transition rates of the state-based model are given by the phagocytosis rate of PMN that phagocytose for their first time, the phagocytosis rate of PMN that phagocytose for at least the second time, the phagocytosis rate of monocytes, the intracellular killing rate of monocytes, the intracellular killing rate of PMN, the resistance rate and the rates that determine the extracellular killing and .</p

<i>C. albicans</i> predominantly associates with PMN and is killed rapidly.

<p>(A) Time-dependent increase of <i>C. albicans</i> association with blood cells as determined by flow cytometry. The majority of <i>C. albicans</i> cells associated to PMN whereas only low interactions could be observed for monocytes and no association to lymphocytes was detectable. The percentages of <i>C. albicans</i> associated with PMN (striped bars) or monocytes (black bars) were calculated relative to total <i>C. albicans</i> cells in blood (set to ). All values correspond to the means of five independent experiments with whole blood from five different donors. (B) Representative blood smears of <i>C. albicans</i>-infected blood after (a), (b), (c) and (d) demonstrate continuous filamentation of extracellular fungi (I). Ingested <i>C. albicans</i> (black arrows) were mainly found in PMN and showed different morphotypes. (C) Survival assay of <i>C. albicans</i> exposed to human whole blood shows a rapid killing of the fungus within of infection. Each dot represents <i>C. albicans</i> colony forming units (<i>C. a.</i> CFU/ml blood) of independent experiments with blood from different donors. The mean standard deviation is given for each time point.</p

Expression pattern of the core filamentation response genes in selected transcriptome studies.

<p>Transcriptional data from selected studies were analyzed for information about the eight core filamentation response network genes. As different technologies and normalization pattern were used, it is only possible to provide information about up (red)- or down- regulation (blue) or a no change of expression (yellow) of the indicated genes. The open reading frame orf19.2457 was partially not part of the microarray design in some studies and were marked absent (black). (A) Expression dynamics for the genes of the early and late filamentation networks as well as of the germ tube formation network during the invasion of human oral epithelial cells (TR146 cell line) with <i>C. albicans</i> wild type SC5314. The dataset were taken from the study of Wächtler and coworkers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058613#pone.0058613-Wachtler1" target="_blank">[44]</a>. (B) Expression dynamics for the core filamentation response genes in different transcriptome studies covering more hyphae- inducing conditions thant those which were used in this study.</p

Schematic representation of the state-based model.

<p>Circular symbols depict different states of the model, <i>i.e.</i> the green circle represents extracellular alive <i>C. albicans</i> (), the red circle indicates extracellularly killed <i>C. albicans</i> (), the black circles symbolize resistant <i>C. albicans</i> that are alive () or killed (), orange circles represent states of monocytes () with alive and killed <i>C. albicans</i> and the blue circles depict different states of PMN (). The model is not restricted by the number of immune cell states, as indicated by the dots, but is extended to account for all required states. The arrows represent allowed transitions between states, where their different colors correspond to the state of <i>C. albicans</i> (alive or dead) and the type of transition that they can perform (phagocytosis, killing or resistance). Alive <i>C. albicans</i> can be phagocytosed (green arrows), killed (purple arrows) or can became resistant (black arrow). <i>C. albicans</i> that are already killed can be phagocytosed (red arrows).</p

Result of the state-based model simulation generated by estimated transition rates.

<p>Time course of different combinations of simulated data (red solid lines) were fitted to associated experimental data from whole-blood infection assays (red dotted lines as guide for the eye) with corresponding standard deviations. The thickness of the solid lines represents the mean standard deviation of the simulation results that was obtained from 100 simulations for the normally distributed transition rates. Colored symbols refer to different <i>C. albicans</i> states, where their time courses are indicated by continuous lines with the same color. (A) Time-dependent relative number of killed <i>C. albicans</i> cells () that were experimentally measured by survival plates. The experimental results were compared with the combination of simulated data representing all killed <i>C. albicans</i> of the model, <i>i.e.</i> extracellularly killed <i>C. albicans</i> (), killed resistant <i>C. albicans</i> (), killed <i>C. albicans</i> that are in monocytes () or PMN (). (B) Alive <i>C. albicans</i> () that were measured by survival plates and simulated by the combination of alive <i>C. albicans</i> that are in extracellular space (), in monocytes (), in PMN () or became resistant against phagocytosis (). (C) Time course of <i>C. albicans</i> cells that are in extracellular space of blood (). Experimental data was obtained by FACS analysis and simulated data is represented by the combination of <i>C. albicans</i> cells that are extracellular alive (), extracellularly killed () and resistant against phagocytosis (). (D) The simulated resistant <i>C. albicans</i> () are the sum of alive and dead resistant <i>C. albicans</i> cells at each time point of the simulation time. (E) Time course of <i>C. albicans</i> cells that were phagocytosed by monocytes (). This is defined as sum of alive and killed <i>C. albicans</i> cells in monocytes, <i>i.e. </i> and , respectively. The corresponding experimental data was obtained by FACS analysis. (F) Relative number of <i>C. albicans</i> cells in PMN () during the whole-blood infection, where internalized <i>C. albicans</i> cells can be alive () or dead (). (G) Simulation result of killed <i>C. albicans</i> cells within monocytes (), that is defined as the sum of internalized <i>C. albicans</i> that were intracellularly killed() and those who were extracellularly killed (). (H) Simulated time course of killed <i>C. albicans</i> cells in PMN (), that is composed of intracellularly killed <i>C. albicans</i> cells () and extracellularly killed <i>C. albicans</i> cells () in PMN.</p

<i>In silico</i> promoter analysis of core filamentation response genes.

<p>in brackets: starting nucleotide of motif within the intergenic region.</p

Shift- specific gene expression patterns in <i>Candida albicans</i> hyphae.

<p>A summary of the data from the whole genome DNA microarrays used for this study. Genes showing fold changes of at least 1.5 were evaluated for significance (p≤0.05) and illustrated in blue for down- regulation and red for up- regulation. Genes were marked in yellow as not differentially expressed. (A). Differentially expressed genes for all three shifts at 1 h. (B–D) The expression dynamics of genes closely linked to the pH shift(B), the serum shift (C) or the change of the carbon source from glucose to N- acetylglucosamine (D) are shown. The presented data were taken from the whole genome DNA microarrays used for this study. The fold changes of at least 1.5 were evaluated for significance (p≤0.05) and illustrated in blue for down- regulation and red for up- regulation. Genes were marked in yellow as not differentially expressed.</p