Sugar Phosphorylation Controls Carbon Source Utilization and Virulence ofCandida albicans

Candida albicans is an opportunistic human fungal pathogen that relies upon different virulence traits, including morphogenesis, invasion, biofilm formation, and nutrient acquisition from host sources as well as metabolic adaptations during host invasion. In this study, we show how sugar kinases at the start of glycolysis modulate virulence of C. albicans. Sequence comparison with Saccharomyces cerevisiae identified four enzymes (Hxk1, Hxk2, Glk1, and Glk4) in C. albicans with putative roles in sugar phosphorylation. Hxk2, Glk1, and Glk4 demonstrate a critical role in glucose metabolism, while Hxk2 is the only kinase important for fructose metabolism. Additionally, we show that Hxk1 controls HXK2, GLK1, and GLK4 expression in the presence of fermentable as well as non-fermentable carbon sources, thereby indirectly controlling glycolysis. Moreover, these sugar kinases are important during virulence. Disabling the glycolytic pathway reduces adhesion capacity, while deletion of HXK1 decreases biofilm formation. Finally, we demonstrate that hxk2Δ/Δ glk1Δ/Δ glk4Δ/Δ and hxk1Δ/Δ hxk2Δ/Δ glk1Δ/Δ glk4Δ/Δ have attenuated virulence upon systemic infections in mice. These results indicate a regulatory role for Hxk1 during sugar phosphorylation. Furthermore, these kinases are essential during growth on glucose or fructose, and C. albicans relies on a functional glycolytic pathway for maximal virulence.status: publishe

Methionine is required for cAMP-PKA mediated morphogenesis and virulence of Candida albicans (vol 108, pg 258, 2018)

© 2018 John Wiley & Sons Ltd This article was published in Mol Microbiol (2018) 108(3), 258-275. This article corrects: Figure 1C We have made a mistake in the compiled figure 1C by introducing the same image for the re-integrant MUP1 strain on medium containing SLD + met as for the heterozygous mutant. We have now placed the correct image for the re-integrant strain. We sincerely apologize for this error and emphasize that they do not affect the conclusions reported in the paper. The figure legend remains the same. (Figure presented.).status: publishe

The histone chaperone HIR maintains chromatin states to control nitrogen assimilation and fungal virulence

Adaptation to changing environments and immune evasion is pivotal for fitness of pathogens. Yet, the underlying mechanisms remain largely unknown. Adaptation is governed by dynamic transcriptional re-programming, which is tightly connected to chromatin architecture. Here, we report a pivotal role for the HIR histone chaperone complex in modulating virulence of the human fungal pathogen Candida albicans. Genetic ablation of HIR function alters chromatin accessibility linked to aberrant transcriptional responses to protein as nitrogen source. This accelerates metabolic adaptation and increases the release of extracellular proteases, which enables scavenging of alternative nitrogen sources. Furthermore, HIR controls fungal virulence, as HIR1 deletion leads to differential recognition by immune cells and hypervirulence in a mouse model of systemic infection. This work provides mechanistic insights into chromatin-coupled regulatory mechanisms that fine-tune pathogen gene expression and virulence. Furthermore, the data point toward the requirement of refined screening approaches to exploit chromatin modifications as antifungal strategies

The Non-receptor Tyrosine Kinase Tec Controls Assembly and Activity of the Noncanonical Caspase-8 Inflammasome

<div><p>Tec family kinases are intracellular non-receptor tyrosine kinases implicated in numerous functions, including T cell and B cell regulation. However, a role in microbial pathogenesis has not been described. Here, we identified Tec kinase as a novel key mediator of the inflammatory immune response in macrophages invaded by the human fungal pathogen <i>C. albicans</i>. Tec is required for both activation and assembly of the noncanonical caspase-8, but not of the caspase-1 inflammasome, during infections with fungal but not bacterial pathogens, triggering the antifungal response through IL-1β. Furthermore, we identify dectin-1 as the pathogen recognition receptor being required for Syk-dependent Tec activation. Hence, Tec is a novel innate-specific inflammatory kinase, whose genetic ablation or inhibition by small molecule drugs strongly protects mice from fungal sepsis. These data demonstrate a therapeutic potential for Tec kinase inhibition to combat invasive microbial infections by attenuating the host inflammatory response.</p></div

Tec is required for the assembly of the caspase-8 inflammasome.

<p>(<b>a</b>) Caspase-8 activity after 60 Min of stimulation with <i>C. albicans</i> of cells left untreated, knockdown of a non-target (nTG; 25 nM) or respective siRNA knock down (25 nM) after 72 hrs of incubation; chemiluminenscence of unstimulated cells and <i>C. albicans</i> only was subtracted. (<b>b</b>–<b>e</b>) Immunoblot analysis of CARD9, Bcl-10, MALT1, ASC and caspase-8 (Casp8) after immunoprecipitation (IP) with antibodies against CARD9 (<b>b</b>), MALT1 (<b>c</b>), ASC (<b>d</b>) and caspase-8 (<b>e</b>) from whole-cell lysates of BMMs left unstimulated (-) or stimulated with <i>C. albicans</i> for 60 Min. Data are representative of two independent experiments for each IP. (<b>f</b>) Immunoblot analysis of p-Src, p-Syk, p-PLCγ2, p-PKCδ and p-RAF1 during the course of BMM infection with <i>C. albicans</i>. (<b>g</b>) <i>In vitro</i> kinase assay; Tec was immunoprecipitated from unstimulated BMMs and incubated with recombinant active Syk, BSA (70 ng each) and adenosine triphosphate (ATP, 200 nM) for 30 Min at 30°C; active phosphorylated Tec was detected with α-p-Tyr antibodies. (<b>h</b>) <i>In vitro</i> kinase assay; PLCγ2 was immunoprecipitated from unstimulated BMMs and incubated with active Tec, BSA (70 ng each) and adenosine triphosphate (ATP, 200 nM) for 30 Min at 30°C; active PLCγ2 was detected with α-p-PLCγ2 antibody. (<b>i</b>) Immunoblot of activated Tec and p-Src/p-Syk in cell lysates after stimulation with <i>C. albicans</i> and parallel Syk inhibition with R406 (3 µM); lysates were enriched for phospho-proteins. Data are representative of at least two (<b>b</b>–<b>i</b>) or three (<b>a</b>) independent experiments. Mean and SD are shown (<b>a</b>).</p

Lack of Tec impairs the inflammatory response to fungal pathogens.

<p>(<b>a</b>) Immunoblot analysis of Tec and (<b>b</b>) qPCR analysis of Tec expression after stimulating BMMs with <i>C. albicans</i> for 120 min; results are normalized to GAPDH (glyceraldehyde phosphate dehydrogenase). (<b>a</b>) Immunoblot of activated Tec in cell lysates after stimulation with <i>C. albicans</i>; lysates were enriched for phospho-proteins. (<b>b</b>) Detection of reactive oxygen species from BMMs after <i>C. albicans</i> challenge for 120 Min using luminol (ROS from unstimulated cells was subtracted). (<b>c</b>) qPCR analysis of cytokine response after 120 Min without (-) or with stimulation with <i>C. albicans</i> (Ca); results are normalized to GAPDH. (<b>d</b>) ELISA for cytokines in supernatants of BMMs with or without (-) <i>C. albicans</i> (Ca) stimulation. (<b>e</b>) Rate of phagocytosis after 45 Min of incubation with <i>C. albicans</i> (Ca). (<b>f</b>) Immunoblotting of p-IκBα and p-NF-κB p65 activation over the time course of <i>C. albicans</i> infection in BMMs. Data are representative of at least two (<b>a</b>–<b>c</b>, <b>g</b>) or three (<b>d</b>–<b>f</b>) independent experiments. Mean and SD are shown.</p

Caspase-8 activity in response to <i>C. albicans</i> requires Tec in BMMs.

<p>(<b>a</b>) Caspase-1 activity over the course of infection with <i>C. albicans</i>; absorbence of unstimulated cells and <i>C. albicans</i> only was subtracted. (<b>b</b>) Caspase-8 activity over the course of infection with <i>C. albicans</i>; chemiluminenscence of unstimulated cells and <i>C. albicans</i> only was subtracted. (<b>c</b>) Immunoblot analysis of full-length or active (p20) caspase-1 and full-length and active (p10) caspase-8 during the course of BMM infection with <i>C. albicans</i>. (<b>d</b>) ELISA of IL-1β in supernatants of BMMs after stimulation with <i>C. albicans</i> only (Ca) or with dimethylsulfoxide (DMSO), Casp1 inhibitor (Casp1 Inh; 5 mM) or Casp8 inhibitor (Casp8 Inh; 5 mM) and Ca or left unstimulated (-). (<b>e</b>) ELISA of pro-IL-1β in supernatants of BMMs after stimulation with <i>C. albicans</i> (Ca) or left unstimulated (-). Data are representative of at least two (<b>c</b>), three (<b>d,e</b>) or five (<b>a</b>,<b>b</b>) independent experiments. Mean and SD are shown.</p

Tec regulates fungal virulence <i>in vivo</i>.

<p>(<b>a</b>) Caspase-8 activity of total murine peritoneal lavage cells obtained after intraperitoneal infection (<i>i.p.</i>) with 5.10⁶ CfUs of <i>C. albicans</i> after indicated times of infection or control lavage cells from uninfected mice; n = 3 per genotype and time point (<b>b</b>) Caspase-1 activity of total murine peritoneal cells upon <i>i.p.</i> infection with 5.10⁶ CfUs of <i>C. albicans</i> after indicated times of infection or cells from uninfected mice; n = 3 per genotype and time point (<b>c</b>) Intracellular staining of active caspase-8 in neutrophils (CD11b⁺Ly6C⁺Ly6G⁺) and macrophages (CD11b⁺F4/80⁺) in peritoneal lavage cells obtained after i.p. infection with 5.10⁶ CfUs of <i>C. albicans</i> after 24 h. (<b>d</b>) Mice were infected <i>i.p.</i> with 3% brewer thioglykolate medium for 4 days; lavage cells were collected; total caspase-8 activity was measured after 60 Min of stimulation with <i>C. albicans</i> (Ca); chemiluminenscence of unstimulated cells or <i>C. albicans</i> only was subtracted; n = 4 per genotype and time point (<b>e</b>) Polymorph-nuclear neutrophils were isolated from bone marrow of respective mice using a Percoll-gradient; Caspase-8 activity was measured after 60 Min of stimulation with <i>C. albicans</i> (Ca); chemiluminenscence of unstimulated cells or <i>C. albicans</i> only was subtracted. (<b>f</b>) qPCR analysis of TNFα and IL-1β of total murine peritoneal cells upon intraperitoneal infection (<i>i.p.</i>) with 5.10⁶ CfUs of <i>C. albicans</i> after indicated time of infection or cells from uninfected mice; results are normalized to those of GAPDH. n = 3 per genotype and time point (<b>g</b>) ELISA of TNFα and IL-1β in lavage of mice upon intraperitoneal infection (<i>i.p.</i>) with 5.10⁶ CfUs of <i>C. albicans</i> after indicated time of infection or from uninfected mice. n = 3 per genotype and time point. Data are representative of at least two (<b>c</b>) three (<b>a,b, e–g</b>) or four (<b>d</b>) independent experiments. Mean and SD are shown.</p

Tec-deficient mice are highly resistant to candidiasis.

<p>(<b>a</b>) Survival of mice after intraperitoneal infection (<i>i.p.</i>) with 5.10⁷ CfUs of <i>C. albicans</i>; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 12 per genotype. (<b>b</b>) Survival of mice after intravenous infection (<i>i.v.</i>) with 1.10⁵ CfUs of <i>C. albicans</i>; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 11 per genotype. (<b>c</b>) Histopathological analysis of mice after i.v. infection with 1.10⁵ CfUs of <i>C. albicans</i>; HE-Staining. (<b>d</b>) Intracellular staining of active caspase-8 in neutrophils (CD11b⁺Ly6C⁺Ly6G⁺), inflammatory monocytes (CD11b⁺Ly6C⁺Ly6G⁻) and inflammatory DCs (CD11b⁺CD11c⁺Ly6C⁺) in leukocytes isolated from kidneys after <i>i.v.</i> infection with 5.10⁶ CfUs of <i>C. albicans</i> for 24 h. (<b>e</b>) ELISA of respective cytokines in sera of mice after <i>i.v.</i> infection with 5.10⁶ CfUs of <i>C. albicans</i> for 24 h. Data are representative of two (<b>d,e</b>) independent experiments.</p

Chemical-genetic inhibition of Tec rescues mice from fatal fungal sepsis.

<p>(<b>a</b>) Survival of mice after intraperitoneal infection with 5.10⁷ CfUs of <i>C. albicans</i> and oral treatment with PCI-32765 with daily doses as indicated; treatment was stopped after 5 days; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 9 per group. (<b>b</b>) Survival of mice after intraperitoneal infection with 5.10⁷ CfUs of <i>C. albicans</i> and oral treatment with 5 mg/kg bodyweight PCI-32765 with daily doses; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 6 per group. (<b>c</b>) Survival of mice after intraperitoneal infection with 5.10⁷ CfUs of <i>C. albicans</i> and oral treatment with 5 mg/kg bodyweight PCI-32765 with daily doses; treatment was stopped after 9 days; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 6 per group.</p