Candida spp. and Oxidative Stress Response in Innate Immune Cells

In dieser Arbeit wurde der „Oxidative Burst“ des angeborenen Immunsystems in der Interaktion mit Candida albicans untersucht. Das klinische Spektrum des opportunistischen Pathogens C. albicans reicht von mucokutanen Infektionen bis hin zu lebensbedrohlichen, systemischen Krankheiten in immunsupprimierten Patienten. Eine der ersten Reaktionen der Zellen des angeborenen Immunsystems, sogenannte Phagozyten, ist die Produktion von Reaktiven Oxygen Spezies (ROS) wenn sie auf Pathogene stoßen. ROS spielen eine wichtige Rolle bei Entzündungsreaktionen, zum Beispiel zerstören sie eindringende Krankheitserreger. Durch eine Überproduktion von ROS kann aber auch das Endothel beschädigt werden. Frühere Studien haben gezeigt, dass Zymosan, eine Zellwand Aufbereitung von Saccharomyces cerevisiae, und C. albicans die ROS Produktion in Makrophagen aktivieren. Das C. albicans Genom codiert sechs Superoxid Dismutasen (SOD1 bis SOD6), die an der Zersetzung von ROS beteiligt sind, SOD1 bis SOD3 sind intrazellular und SOD4 bis SOD6 sind wahrscheinlich an der Zellwand von C. albicans lokalisiert. Diese Arbeit zeigt, dass die Co-Kultur von Makrophagen oder myeloischen dendritischen Zellen mit C. albicans denen Sod5 genetisch entfernt wurde zu einer massiven extrazellulären Anhäufung von ROS in vitro führt. Diese ROS Akkumulierung ist in der Interaktion mit Makrophagen noch höher wenn C. albicans weder Sod4 noch Sod5 haben. Weiteres werden C. albicans Sod5 und Sod4 Mutanten von Makrophagen in vitro besser getötet als Wildtyp C. albicans. Makrophagen, die einen Defekt im Oxidativen Burst haben weil ihnen das gp91Phox Gen fehlt, können diese Mutanten nicht mehr töten, dies zeigt eine ROS-abhängige Eliminierung von pathogenen Pilzen durch Makrophagen. Diese Daten zeigen die physiologische Rolle der C. albicans Zellwand SODs bei der Entgiftung von ROS und weisen auf einen Mechanismus, mit dem C. albicans das Immunsystems in vivo überlistet, hin. Im zweiten Teil dieser Arbeit wurden potentielle Rezeptor(en) untersucht, durch die Makrophagen C. albicans erkennen, um den oxidative Burst zu induzieren. Die Toll Like Rezeptor-Familie und das intrazelluläre MyD88 Adapter-Protein sind nicht an der ROS-Produktion durch Zymosan oder C. albicans Stimulation beteiligt. Wenn der C-Typ-Lectin-Rezeptor Dectin-1 mit Zymosan oder Hitze-getöteter C. albicans stimuliert wird, induziert Dectin-1 die ROS Antwort indem die Src und Syk-Kinase aktiviert wird. Darüber hinaus aktiviert Zymosan auch die ERK1/2 MAP-Kinasen via Dectin-1. Im Gegensatz dazu ist Dectin-1 nur mäßig an der Aktivierung von ROS und ERK1 beteiligt wenn die Makrophagen mit lebenden C. albicans stimuliert werden. Interessanterweise ist die Aktivierung der Src und Syk-Kinasen auch wichtig für ROS Induktion durch Stimulierung mit lebender C. albicans. Dies führt zu dem Schluss, dass ein Rezeptor oder Adapter-Protein mit einem ITAM Motif an der Induktion von ROS beteiligt ist. Ein siRNA-basierendes knock-down-Experiment zeigt, dass das ITAM Adapter-Protein DAP12 für die ROS Produktion durch C. albicans und Zymosan mitverantwortlich ist.In this work the oxidative burst of the innate immune system in response to Candida albicans infection was investigated. The clinical spectrum of the human opportunistic pathogen C. albicans ranges from mucocutaneous infections to systemic life-threatening diseases in immunocompromised patients. One of the immediate early responses of cells of the innate immune system on encountering microbial pathogens is the production of reactive oxygen species (ROS) by phagocytes. ROS play important roles in inflammatory reactions by destroying invading pathogens. However, overproduction of ROS may also cause endothelial damage, and excessive inflammation. Previous studies have shown that zymosan, a cell wall preparation of Saccharomyces cerevisiae, as well as C. albicans in the yeast form, strongly induce ROS in macrophages. The C. albicans genome harbours six superoxide dismutases (SOD1-6) involved in ROS degradation; SOD1 to SOD3 are intracellular and SOD4 to SOD6 are located in the cell wall. This work demonstrates that co-culture of macrophages or myeloid dendritic cells with C. albicans cells lacking Sod5 leads to massive extracellular ROS accumulation in vitro. ROS accumulation was further increased in co-culture with fungal cells lacking both Sod4 and Sod5. Survival experiments show that C. albicans Sod5 and Sod4 double mutants exhibit a severe loss of viability in the presence of macrophages in vitro. The reduced viability of the mutants relative to wild type is not evident with macrophages from gp91phox-/- mice defective in the oxidative burst activity, demonstrating a ROS-dependent killing activity of macrophages targeting fungal pathogens. These data show a physiological role for cell surface SODs in detoxifying ROS, and suggest a mechanism whereby C. albicans can evade host immune surveillance in vivo. The second part of this thesis aims to identify putative receptor(s) by which macrophages recognise C. albicans and induce the oxidative burst. The Toll-like receptor family and its MyD88 adaptor protein are not involved in ROS production due to zymosan or C. albicans stimulation. The c-type lectin receptor Dectin-1 can induce the ROS response via activation of Syk kinase with its immunoreceptor tyrosine-based activation motif (ITAM)-like domain upon zymosan or heat-killed C. albicans stimulation. Furthermore, zymosan also activates extracellular signal related kinase ERK1/2 MAPK dependent on Dectin-1. In contrast, Dectin-1 is only moderately involved in activation of ROS and ERK1/2 when stimulated with live C. albicans. Interestingly, activation of Src and Syk kinases is essential to induce the ROS response by live C. albicans. This leads us to conclude that an ITAM-containing receptor or adaptor protein is involved in the recognition of live C. albicans. Using a siRNA-based knock-down assay, we found that one ITAM-containing adaptor protein, DAP12, may contribute to the ROS response upon fungal pathogens such as C. albicans

The Set3/Hos2 Histone Deacetylase Complex Attenuates cAMP/PKA Signaling to Regulate Morphogenesis and Virulence of Candida albicans

Candida albicans, like other pleiomorphic fungal pathogens, is able to undergo a reversible transition between single yeast-like cells and multicellular filaments. This morphogenetic process has long been considered as a key fungal virulence factor. Here, we identify the evolutionarily conserved Set3/Hos2 histone deacetylase complex (Set3C) as a crucial repressor of the yeast-to-filament transition. Cells lacking core components of the Set3C are able to maintain all developmental phases, but are hypersusceptible to filamentation-inducing signals, because of a hyperactive cAMP/Protein Kinase A signaling pathway. Strikingly, Set3C-mediated control of filamentation is required for virulence in vivo, since set3Δ/Δ cells display strongly attenuated virulence in a mouse model of systemic infection. Importantly, the inhibition of histone deacetylase activity by trichostatin A exclusively phenocopies the absence of a functional Set3C, but not of any other histone deacetylase gene. Hence, our work supports a paradigm for manipulating morphogenesis in C. albicans through alternative antifungal therapeutic strategies

Cells lacking Hat1 show reduced virulence but persist in mouse kidneys.

<p>(A) Reduced growth rate of the <i>hat1</i>Δ/Δ strain was determined by measuring the OD₆₀₀ of cells growing in YPD at 30°C. (B) Cells lacking Hat1 are not cleared efficiently from kidneys. At the indicated time points, fungal burdens in kidneys of mice infected with <i>C</i>. <i>albicans</i> strains were determined and expressed as CFUs per gram kidney. Groups of 5–10 mice were analyzed at each time point and statistical significance was determined using the non-parametric Mann-Whitney-test. n.s.: not significant, *P<0.05 and **P<0.01 relative to the corresponding wild-type. (C) <i>hat1</i>Δ/Δ cells are defective in killing the host. Survival of mice infected with the indicated strains was monitored over 32 days post infection (p.i.). The data are presented as Kaplan-Meier survival curves. Groups of 6 mice were infected per <i>C</i>. <i>albicans</i> strain. Statistical significance was determined using the Log-rank test. ns: not significant; (D) Fungal burdens in kidneys of surviving mice from panel C were determined and expressed as CFUs per gram organ. One mouse infected with the <i>hat1</i>Δ/Δ strain was able to clear <i>Candida</i>. (E) The <i>cac2</i>Δ/Δ strain is not cleared efficiently from kidneys. Experiment was performed as described in (B). Groups of 4–5 mice were analyzed at each time point. (F) Infection with <i>hat1</i>Δ/Δ cells causes reduced kidney damage. Urea levels were determined in sera of infected mice at day 3 and 7 post infection. n.s.: not significant, *P<0.05, **P<0.01 relative to the wild-type (Student's t-test).</p

Specific functional gene groups are upregulated in cells lacking Hat1.

<p>(A) GO terms enriched among 2-fold significantly upregulated genes in logarithmically growing <i>hat1</i>Δ/Δ cells are shown. (B) The plot shows GO terms found within genes significantly upregulated in the <i>hat1</i>Δ/Δ and <i>rtt109</i>Δ/Δ strains only. (C) GO terms enriched within genes significantly upregulated in the <i>hat1</i>Δ/Δ and <i>cac2</i>Δ/Δ strains only. (D) The panel shows GO terms found among genes significantly upregulated in the <i>hat1</i>Δ/Δ mutant only and not in the <i>rtt109</i>Δ/Δ and the <i>cac2</i>Δ/Δ strains. (E) GO terms enriched among significantly upregulated genes in <i>hat1</i>Δ/Δ cells after treatment with H₂O₂ are shown. (F) The plot shows GO terms found within genes significantly upregulated in the <i>hat1</i>Δ/Δ strain only and not in the <i>rtt109</i>Δ/Δ and the <i>cac2</i>Δ/Δ strains upon H₂O₂ treatment. (A-F) The corresponding p-values for the enrichment (empty bars) and the percentage of genes changed within the GO group (filled bars) are presented. The absolute number of regulated genes within a GO group is presented in brackets. Groups containing identical genes are depicted in the same color. Significantly regulated genes were defined by a p-value <0.05.</p

Deletion of <i>HAT1</i> and <i>HAT2</i> increases oxidative stress resistance and azole tolerance.

<p>(A) Cells lacking Hat1 or Hat2 show increased resistance to H₂O₂. Lack of both genes mimics the corresponding single deletion strains. (B) Deletion of <i>HAT1</i> increases resistance to <i>tert</i>-butyl hydroperoxide (tBOOH). Lack of Rtt109 does not affect tBOOH sensitivity. (C) Loss of Hat1 causes reduced susceptibility to voriconazole (Voric.) and itraconazole (Itrac.). Deletion of <i>HAT2</i> or <i>HAT1</i> and <i>HAT2</i> mimics loss of Hat1. (D) Deletion of <i>RTT109</i> or <i>RAD52</i> does not increase voriconazole tolerance. (A-D) Fivefold serial dilutions of the indicated strains were spotted on agar plates containing the indicated substances and pictures were taken after incubation at 30°C for 3 days.</p

Lack of histone chaperones mimics deletion of <i>HAT1</i>.

<p>(A) Loss of Cac2 increases H₂O₂ resistance. Deletion of <i>RTT106</i> or <i>HIR1</i> does not affect susceptibility to hydrogen peroxide. Fivefold serial dilutions of the indicated strains were spotted on agar plates containing the indicated substances and pictures were taken after incubation at 30°C for 3 days. (B) Deletion of <i>HAT1</i> or <i>CAC2</i> increases survival to transient hydrogen peroxide treatment. Exponentially growing cells were treated with the indicated concentrations of H₂O₂ for 2 hours. Cells were plated and colonies counted after 3 days of incubation on YPD plates at 30°C to determine viability. Data are shown as mean + SD from three independent experiments. (C) Deletion of <i>HIR1</i> reduces voriconazole (Voric.) susceptibility. The <i>hat1hir1</i>Δ/Δ double deletion strain mimics lack of Hat1. Loss of Cac2 has only a minor effect and deletion of <i>RTT106</i> does not alter azole susceptibility. Experiment was performed as described in (A). (D) Increased azole tolerance of <i>hat1</i>Δ/Δ, <i>hir1</i>Δ/Δ and <i>hat1hir1</i>Δ/Δ was confirmed using a liquid growth inhibition assay. Logarithmically growing cells were diluted into medium containing the indicated concentrations of voriconazole (Voric.) and incubated at 30°C for 18 hours. OD₆₀₀ was determined and growth inhibition relative to untreated samples was calculated. Data are shown as mean + SD from three independent experiments. (E) Lack of Spt6 reduces H₂O₂ susceptibility. Experiment was performed as described in (B). Cells were treated with 10 mM H₂O₂. Data are shown as mean + SD from two independent experiments. (F) Deletion of <i>SPT6</i> increases H₂O₂ resistance and azole tolerance. Fivefold serial dilutions of the indicated strains were spotted on agar plates containing the indicated substances and pictures were taken after incubation at 30°C for 5 days. (G) Reduction of histone gene dosage decreases H₂O₂ and azole susceptibility. Experiment was performed as described in (A). (B, D, E) *P<0.05, **P<0.01 and ***P<0.001 relative to the corresponding wild-type (Student's t-test).</p

Deletion of <i>HAT1</i> primarily leads to upregulation of genes.

<p>(A) Lack of Hat1 causes mainly induction of genes in logarithmically growing cells. Each dot corresponds to one protein-coding gene. The fold change in RNA expression between untreated wild-type and <i>hat1</i>Δ/Δ cells (y-axis) is plotted against the expression level of each gene in this dataset (x-axis). Differentially expressed genes in the <i>hat1</i>Δ/Δ mutant are depicted in red. logCPM: log2 counts per million reads; logFC: log2 fold change; (B+C) Loss of Cac2 or Rtt109 causes almost exclusively upregulation of genes in logarithmically growing cells. Plots were created as described in (A). (D) Venn diagram showing the overlaps of upregulated genes in the <i>hat1</i>Δ/Δ, <i>cac2</i>Δ/Δ and <i>rtt109</i>Δ/Δ mutants in the absence of H₂O₂. (E) Venn diagram showing the overlaps of upregulated genes in the <i>hat1</i>Δ/Δ, <i>cac2</i>Δ/Δ and <i>rtt109</i>Δ/Δ mutants upon treatment with H₂O₂. (F) H₂O₂ repressed genes are upregulated in the <i>hat1</i>Δ/Δ mutant upon peroxide treatment. Each dot corresponds to one protein-coding gene. The -fold change in RNA expression between H₂O₂ treated wild-type and <i>hat1</i>Δ/Δ strains (y-axis) is plotted against the fold change between the wild-type without and with treatment (x-axis). Differentially expressed genes in the <i>hat1</i>Δ/Δ mutant are depicted in red. logFC: log2 fold change; (A-F) Differentially regulated genes were defined by a fold change > = 2 and p-value <0.05.</p

Loss of Hat1 raises antioxidant enzyme activity and glutathione-mediated H₂O₂ resistance.

<p>(A) Faster <i>CAT1</i> induction increases catalase activity in <i>hat1</i>Δ/Δ cells. Catalase activity was determined in whole cell extracts isolated from cells before and after H₂O₂ treatment. Data are shown as mean + SD from three independent experiments. (B) Loss of Hat1 leads to increased glutathione peroxidase activity. GPx activity was determined in whole cell extracts isolated from cells before and after H₂O₂ treatment. Data are shown as mean + SD from two independent experiments. (C) Lack of <i>CAT1</i> does not abolish Hat1-mediated H₂O₂ resistance. Cells of the indicated strains were treated with 1 mM H₂O₂ for 2 hours, plated and colonies counted after 3 days of incubation on YPD plates at 30°C to determine viability. Data are shown as mean + SD from three independent experiments. (D) Depletion of glutathione biosynthesis abolishes Hat1-mediated H₂O₂ resistance. Cells of the indicated strains were treated with H₂O₂ for 2 hours and plated on YPD plates containing glutathione. Colonies were counted to determine viability after growth for 3 days at 30°C. Data are shown as mean + SD from three independent experiments. (A-D) n.s.: not significant, *P<0.05, **P<0.01 and ***P<0.001 relative to the corresponding control (Student's t-test).</p

Higher ROS detoxification capacity of <i>hat1</i>Δ/Δ cells causes resistance to neutrophil killing.

<p>(A) Superoxide dismutases Sod4 and Sod5 are induced in <i>hat1</i>Δ/Δ cells. Expression levels of <i>SOD4</i> and <i>SOD5</i> in logarithmically growing cells were detected by RT-qPCR. Transcript levels were normalized to the expression level of the reference gene (RG) <i>RIP1</i>. Data are shown as mean + SD from 3 independent experiments. (B) Infection of macrophages with <i>hat1</i>Δ/Δ cells causes reduced ROS accumulation. ROS levels were determined by measuring luminol-dependent chemiluminescence [relative luciferase units (RLU) min^-1 per 1000 immune cells] in 2.5 min intervals during interaction of the indicated <i>C</i>. <i>albicans</i> strains with bone marrow-derived murine macrophages (BMDMs). One representative experiment is shown. Data were reproduced in three independent experiments. (C) Quantification of total ROS release upon interaction with BMDMs. Experiment was performed as described in (B). The area under the curve within 90 min of interaction was calculated. Data are shown as mean + SD from three independent experiments. (D) Cells lacking Hat1 show increased survival to neutrophil killing. Survival of <i>C</i>. <i>albicans</i> cells upon one hour interaction with murine bone marrow neutrophils was determined by plating and CFU counting. Data are shown as mean + SD from three independent experiments. (A-D) *P<0.05, **P<0.01 relative to the wild-type (Student's t-test).</p