Nanoscale chemical imaging of phagocytosis : a battle for metals between host and microbe

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The Functions of Mediator in Candida albicans Support a Role in Shaping Species-Specific Gene Expression

The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species

The mediator of transcription in Candida albicans: roles in cell wall biogenesis, morphogenesis and host-pathogen interactions

The fungal pathogen Candida albicans is the fourth leading cause of nosocomial infections and continues to cause life-threatening candidiasis. C. albicans virulence results from the intricate gene regulatory pathways that dictate morphogenesis, particularly cell wall remodelling, dimorphism and biofilm formation that allows the organism to evade the host. Studies in non pathogenic yeasts have demonstrated that gene regulatory networks rely on multi-subunit transcriptional complexes such as the Mediator of transcription to modulate gene activation and repression in association with RNA polymerase II. While Mediator is well-studied in non-pathogenic model yeasts, before the commencement of this project its roles in C. albicans were unknown. In this thesis work, I characterised the roles of three Mediator subunits in C. albicans, Med20 from the Head domain of the complex, Med31 from the Middle domain and Srb9 from the Kinase module. I identified global roles for Mediator in the genetic programs that regulate morphogenesis, including conserved roles with non-pathogenic yeasts in the regulation of components of the RAM (regulation of Ace2 and morphogenesis) cell wall remodelling pathway in association with the Ace2 transcription factor. Phenotypic and gene expression comparison using deletion mutants of the most evolutionarily conserved subunit Med31, provided evidence for both conserved and divergent roles, with differences observed in the regulation of cellular adhesion. Med31 controls the yeast to hyphal transition, and it affects the expression of genes coding for key morphogenesis regulators including transcription factors required in the nutrient-sensing cAMP filamentation signalling pathway, (e.g. EFG1, TEC1, CPH2 and RIM101). The downstream targets of the Mediator complex in C. albicans also include the adhesion genes ALS1, ALS3 and HWP1, as well as other cell surface molecules such as the immunologically relevant cell wall component 1,3 β-glucan. Collectively, the above mentioned effects of Mediator on cell wall regulation and cellular morphogenesis translate into cellular roles in biofilm formation by C. albicans, which is a key virulence attribute, as well as essential functions for Mediator in the ability of C. albicans to escape the innate immune response by evading macrophages. Overall, the Mediator complex was essential for C. albicans pathogenesis and my published work with collaborators showed the Mediator is also required for C. albicans virulence in vivo. I further used C. albicans Mediator mutants and a novel assay that I developed, which monitors in real time the killing of macrophages by C. albicans, to discover that macrophages are killed in a biphasic fashion by C. albicans hyphal cells. I demonstrated for the first time that C. albicans hyphal cells trigger the inflammatory suicide response in macrophages – pyroptosis. This work further demonstrated that the long-standing view that C. albicans hyphal cells mechanically damage macrophages has to be revised – instead, hyphal cells activate pyroptosis, which is lytic, and then hijack this processes to escape. I showed that hyphal morphogenesis is necessary for pyroptotic macrophage death, and have data that implicate hyphal cell wall 1,3 β-glucan in the mechanism. The pathogen and host factors and mechanisms of C. albicans morphogenesis and immune evasion identified and characterised in my thesis work add to the knowledge base and provide a platform for the identification of future strategies against human fungal infections

The mediator of transcription in Candida albicans: roles in cell wall biogenesis, morphogenesis and host-pathogen interactions

The fungal pathogen Candida albicans is the fourth leading cause of nosocomial infections and continues to cause life-threatening candidiasis. C. albicans virulence results from the intricate gene regulatory pathways that dictate morphogenesis, particularly cell wall remodelling, dimorphism and biofilm formation that allows the organism to evade the host. Studies in non pathogenic yeasts have demonstrated that gene regulatory networks rely on multi-subunit transcriptional complexes such as the Mediator of transcription to modulate gene activation and repression in association with RNA polymerase II. While Mediator is well-studied in non-pathogenic model yeasts, before the commencement of this project its roles in C. albicans were unknown. In this thesis work, I characterised the roles of three Mediator subunits in C. albicans, Med20 from the Head domain of the complex, Med31 from the Middle domain and Srb9 from the Kinase module. I identified global roles for Mediator in the genetic programs that regulate morphogenesis, including conserved roles with non-pathogenic yeasts in the regulation of components of the RAM (regulation of Ace2 and morphogenesis) cell wall remodelling pathway in association with the Ace2 transcription factor. Phenotypic and gene expression comparison using deletion mutants of the most evolutionarily conserved subunit Med31, provided evidence for both conserved and divergent roles, with differences observed in the regulation of cellular adhesion. Med31 controls the yeast to hyphal transition, and it affects the expression of genes coding for key morphogenesis regulators including transcription factors required in the nutrient-sensing cAMP filamentation signalling pathway, (e.g. EFG1, TEC1, CPH2 and RIM101). The downstream targets of the Mediator complex in C. albicans also include the adhesion genes ALS1, ALS3 and HWP1, as well as other cell surface molecules such as the immunologically relevant cell wall component 1,3 β-glucan. Collectively, the above mentioned effects of Mediator on cell wall regulation and cellular morphogenesis translate into cellular roles in biofilm formation by C. albicans, which is a key virulence attribute, as well as essential functions for Mediator in the ability of C. albicans to escape the innate immune response by evading macrophages. Overall, the Mediator complex was essential for C. albicans pathogenesis and my published work with collaborators showed the Mediator is also required for C. albicans virulence in vivo. I further used C. albicans Mediator mutants and a novel assay that I developed, which monitors in real time the killing of macrophages by C. albicans, to discover that macrophages are killed in a biphasic fashion by C. albicans hyphal cells. I demonstrated for the first time that C. albicans hyphal cells trigger the inflammatory suicide response in macrophages – pyroptosis. This work further demonstrated that the long-standing view that C. albicans hyphal cells mechanically damage macrophages has to be revised – instead, hyphal cells activate pyroptosis, which is lytic, and then hijack this processes to escape. I showed that hyphal morphogenesis is necessary for pyroptotic macrophage death, and have data that implicate hyphal cell wall 1,3 β-glucan in the mechanism. The pathogen and host factors and mechanisms of C. albicans morphogenesis and immune evasion identified and characterised in my thesis work add to the knowledge base and provide a platform for the identification of future strategies against human fungal infections

Immune Resolution Dilemma: Host Antimicrobial Factor S100A8/A9 Modulates Inflammatory Collateral Tissue Damage During Disseminated Fungal Peritonitis

Intra-abdominal infection (peritonitis) is a leading cause of severe disease in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial and pro-inflammatory protein complex used as a biomarker for diagnosis of numerous inflammatory disorders. Here we describe the role of S100A8/A9 in inflammatory collateral tissue damage (ICTD). Using a mouse model of disseminated intra-abdominal candidiasis (IAC) in wild-type and S100A8/A9-deficient mice in the presence or absence of S100A9 inhibitor paquinimod, the role of S100A8/A9 during ICTD and fungal clearance were investigated. S100A8/A9-deficient mice developed less ICTD than wild-type mice. Restoration of S100A8/A9 in knockout mice by injection of recombinant protein resulted in increased ICTD and fungal clearance comparable to wild-type levels. Treatment with paquinimod abolished ICTD and S100A9-deficient mice showed increased survival compared to wild-type littermates. The data indicates that S100A8/A9 controls ICTD levels and antimicrobial activity during IAC and that targeting of S100A8/A9 could serve as promising adjunct therapy against this challenging disease

The pathogen Candida albicans hijacks pyroptosis for escape from macrophages

The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection. IMPORTANCE Upon phagocytosis by macrophages, Candida albicans can transition to the hyphal form, which causes macrophage death and enables fungal escape. The current model is that the highly polarized growth of hyphae results in macrophage piercing. This model is challenged by recent reports of C. albicans mutants that form hyphae of wild-type morphology but are defective in killing macrophages. We show that C. albicans causes macrophage cell death by at least two mechanisms. Phase 1 killing (first 6 to 8 h) depends on the activation of the pyroptotic programmed host cell death by fungal hyphae. Phase 2 (up to 24 h) is rapid and depends on robust hyphal formation but is independent of pyroptosis. Our data provide a new model for how the interplay between fungal morphogenesis and activation of a host cell death pathway mediates macrophage killing by C. albicans hyphae

Cell wall integrity is linked to mitochondria and phospholipid homeostasis in Candida albicans through the activity of the post-transcriptional regulator Ccr4-Pop2

Peer reviewed: YesNRC publication: Ye

Proline catabolism is a key factor facilitating Candida albicans pathogenicity

Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens