The composition and organization of the cell walls from Saccharomyces cerevisiae, Candida albicans, Aspergillus fumigatus, Schizosaccharomyces pombe, Neurospora crassa, and Cryptococcus neoformans are compared and contrasted. These cell walls contain chitin, chitosan, β-1,3-glucan, β-1,6-glucan, mixed β-1,3-/β-1,4-glucan, α-1,3-glucan, melanin, and glycoproteins as major constituents. A comparison of these cell walls shows that there is a great deal of variability in fungal cell wall composition and organization. However, in all cases, the cell wall components are cross-linked together to generate a cell wall matrix. The biosynthesis and properties of each of the major cell wall components are discussed. The chitin and glucans are synthesized and extruded into the cell wall space by plasma membrane-associated chitin synthases and glucan synthases. The glycoproteins are synthesized by ER-associated ribosomes and pass through the canonical secretory pathway. Over half of the major cell wall proteins are modified by the addition of a glycosylphosphatidylinositol anchor. The cell wall glycoproteins are also modified by the addition of O-linked oligosaccharides, and their N-linked oligosaccharides are extensively modified during their passage through the secretory pathway. These cell wall glycoprotein posttranslational modifications are essential for cross-linking the proteins into the cell wall matrix. Cross-linking the cell wall components together is essential for cell wall integrity. The activities of four groups of cross-linking enzymes are discussed. Cell wall proteins function as cross-linking enzymes, structural elements, adhesins, and environmental stress sensors and protect the cell from environmental changes.

Fungal Cell Wall Organization and Biosynthesis

The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms re- mained 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 trig- gers 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 thefirst 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 mor- phogenesis 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 re- duced exposed 1,3-glucan in hyphae. Our report shows how Candidauses 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 py- roptosis 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 macro- phage 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. albi- canshyphae.

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The Pathogen Candida albicans Hijacks Pyroptosis for Escape from Macrophages

Candida albicans is a major fungal pathogen of humans, causing approximately 400,000 life-threatening systemic infections world-wide each year in severely immunocompromised patients. An important fungicidal mechanism employed by innate immune cells involves the generation of toxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. Consequently, there is much interest in the strategies employed by C. albicans to evade the oxidative killing by macrophages and neutrophils. Our understanding of how C. albicans senses and responds to ROS has significantly increased in recent years. Key findings include the observations that hydrogen peroxide triggers the filamentation of this polymorphic fungus and that a superoxide dismutase enzyme with a novel mode of action is expressed at the cell surface of C. albicans. Furthermore, recent studies have indicated that combinations of the chemical stresses generated by phagocytes can actively prevent C. albicans oxidative stress responses through a mechanism termed the stress pathway interference. In this review, we present an up-date of our current understanding of the role and regulation of oxidative stress responses in this important human fungal pathogen.

Oxidative Stress Responses in the Human Fungal Pathogen, Candida albicans

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose-grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate-grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose-grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate-grown cells. We identified mating and pheromone-regulated proteins that were exclusive to lactate-grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa-specific and other niche-specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.

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Carbon source‐induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans

In this era of efficacious antiretroviral therapy and consequent immune reconstitution, oropharyngeal and esophageal candidiasis (OPC and OEC) still remain two clinically relevant presentations in the global HIV setting. Both diseases are predominantly caused by Candida albicans, a polymorphic fungus which is a commensal microbe in the healthy individual but can become an aggressive pathogen in a debilitated host. Actually, C. albicans commensalism is not the result of a benign behavior of one of the many components of human microbiota, but rather the result of host's potent innate and adaptive immune responses that restrict the growth of a potentially dangerous microrganism on the epithelia. An important asset guarding against the fungus is the Th17 functional subset of T helper cells. The selective loss of these cells with the progression of HIV infection causes the decay of fungal containment on the oral epithelium and allows C. albicans to express its pathogenic potential. An important part of this potential is represented by mechanisms to evade host immunity and enhance inflammation and immunoactivation. In C. albicans, these mechanisms are mostly incorporated into and expressed by characteristic morphogenic transitions such as the yeast-to-hyphal growth and the white-to-opaque switch. In addition, HIV infection generates an 'environment' selecting for overexpression of the virulence potential by the fungus, particularly concerning the secreted aspartyl proteinases (Saps). These enzymes can degrade critical host defense components such as complement and epithelial defensive proteins such as histatin-5 and E-cadherin. It appears that part of this enhanced Candida virulence could be induced by the binding of the fungus to HIV and/or induced by HIV proteins such as GP160 and tat. Both OPC and OEC can be controlled by old and new antimycotics, but in the absence of host collaboration, anticandidal therapy may become ineffective in the long run. For these reasons, new therapeutics targeting virulence factors and specific immune interventions are being addressed. Among these new approaches, vaccination is a promising one. Two subunit vaccines based on antigens dominantly expressed by C. albicans in vivo, that is the Als3 adhesin and Sap2, have recently undergone phase 1 clinical trials. Overall, studies of Candida and candidiasis in the HIV-positive patient while certainly contributing to a more effective control of the microorganism may also provide useful information on HIV-host relationship itself that can assist the fight against the virus.

Candida and candidiasis in HIV-infected patients: where commensalism, opportunistic behavior and frank pathogenicity lose their borders.

The ability of Candida albicans to switch from yeast to hyphal growth is essential for its virulence. The walls and especially the covalently attached wall proteins are involved in the primary host–pathogen interactions. Three hyphal induction methods were compared, based on fetal calf serum, the amino sugar N-acetylglucosamine (GlcNAc) and the mammalian cell culture medium Iscove’s modified Dulbecco’s medium (IMDM). GlcNAc and IMDM were preferred, allowing stable hyphal growth over a prolonged period without significant reversion to yeast growth and with high biomass yields. We employed Fourier transform-MS combined with a 15N-metabolically labelled reference culture as internal standard for relative quantification of changes in the wall proteome upon hyphal induction. A total of 21 wall proteins were quantified. Our induction methods triggered a similar response characterized by (i) a category of wall proteins showing strongly increased incorporation levels (Als3, Hwp2, Hyr1, Plb5 and Sod5), (ii) another category with strongly decreased levels (Rhd3, Sod4 and Ywp1) and (iii) a third one enriched for carbohydrate-active enzymes (including Cht2, Crh11, Mp65, Pga4, Phr1, Phr2 and Utr2) and showing only a limited response. This is, to our knowledge, the first systematic, quantitative analysis of the changes in the wall proteome of C. albicans upon hyphal induction. Finally, we propose new wall-protein-derived candidates for vaccine development.

Hyphal induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein profile

Introduction: Bioartificial livers (BALs) were originally developed to treat patients suffering from severe liver failure and relied on primary hepatocytes or on hepatoblastoma-derived cell lines. Currently, new in vitro BAL applications are emerging, including drug toxicity testing, disease modeling and basic clinical research, and in recent years, advances in the field of stem cell biology have resulted in potential alternative cell sources.Areas covered: This review identifies the demands of clinical and in vitro BAL applications to their biocomponent and summarizes the functionality and developmental state of BAL technology and cell types currently available. Relevant studies identified by searching the MEDLINE database until April 2014 were reviewed, supplemented with some of our own unpublished data.Expert opinion: BALs have the potential to meet demands currently left unmet in both clinical and in vitro applications. All the reviewed biocomponents show limitations towards one or more BAL applicatio...

Adriaan R. Siliakus

Papers

Hyphal induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein profile

Bioartificial livers in vitro and in vivo: tailoring biocomponents to the expanding variety of applications.