"Under Pressure" - How fungi evade, exploit, and modulate cells of the innate immune system.

The human immune system uses an arsenal of effector mechanisms to prevent and counteract infections. Yet, some fungal species are extremely successful as human pathogens, which can be attributed to a wide variety of strategies by which these fungi evade, exploit, and modulate the immune system. These fungal pathogens normally are either harmless commensals or environmental fungi. In this review we discuss how commensalism, but also life in an environmental niche without human contact, can drive the evolution of diverse and specialized immune evasion mechanisms. Correspondingly, we discuss the mechanisms contributing to the ability of these fungi to cause superficial to life-threatening infections.

Candida albicans translocation through the intestinal epithelial barrier is promoted by fungal zinc acquisition and limited by NFκB-mediated barrier protection.

The opportunistic fungal pathogen Candida albicans thrives on human mucosal surfaces as a harmless commensal, but frequently causes infections under certain predisposing conditions. Translocation across the intestinal barrier into the bloodstream by intestine-colonizing C. albicans cells serves as the main source of disseminated candidiasis. However, the host and microbial mechanisms behind this process remain unclear. In this study we identified fungal and host factors specifically involved in infection of intestinal epithelial cells (IECs) using dual-RNA sequencing. Our data suggest that host-cell damage mediated by the peptide toxin candidalysin-encoding gene ECE1 facilitates fungal zinc acquisition. This in turn is crucial for the full virulence potential of C. albicans during infection. IECs in turn exhibit a filamentation- and damage-specific response to C. albicans infection, including NFκB, MAPK, and TNF signaling. NFκB activation by IECs limits candidalysin-mediated host-cell damage and mediates maintenance of the intestinal barrier and cell-cell junctions to further restrict fungal translocation. This is the first study to show that candidalysin-mediated damage is necessary for C. albicans nutrient acquisition during infection and to explain how IECs counteract damage and limit fungal translocation via NFκB-mediated maintenance of the intestinal barrier.

"We've got to get out"-Strategies of human pathogenic fungi to escape from phagocytes.

Human fungal pathogens are a deadly and underappreciated risk to global health that most severely affect immunocompromised individuals. A virulence attribute shared by some of the most clinically relevant fungal species is their ability to survive inside macrophages and escape from these immune cells. In this review, we discuss the mechanisms behind intracellular survival and elaborate how escape is mediated by lytic and non-lytic pathways as well as strategies to induce programmed host cell death. We also discuss persistence as an alternative to rapid host cell exit. In the end, we address the consequences of fungal escape for the host immune response and provide future perspectives for research and development of targeted therapies.

The fungivorous amoeba Protostelium aurantium targets redox homeostasis and cell wall integrity during intracellular killing of Candida parapsilosis.

Predatory interactions among microbes are major evolutionary driving forces for biodiversity. The fungivorous amoeba Protostelium aurantium has a wide fungal food spectrum including foremost pathogenic members of the genus Candida. Here we show that upon phagocytic ingestion by the amoeba, Candida parapsilosis is confronted with an oxidative burst and undergoes lysis within minutes of processing in acidified phagolysosomes. On the fungal side, a functional genomic approach identified copper and redox homeostasis as primary targets of amoeba predation, with the highly expressed copper exporter gene CRP1 and the peroxiredoxin gene PRX1 contributing to survival when encountered with P. aurantium. The fungicidal activity was largely retained in intracellular vesicles of the amoebae. Following their isolation, the content of these vesicles induced immediate killing and lysis of C. parapsilosis in vitro. Proteomic analysis identified 56 vesicular proteins from P. aurantium. Although completely unknown proteins were dominant, many of them could be categorised as hydrolytic enzymes targeting the fungal cell wall, indicating that fungal cell wall structures are under selection pressure by predatory phagocytes in natural environments. TAKE AWAY: The amoeba Protostelium aurantium feeds on fungi, such as Candida parapsilosis. Ingested yeast cells are exposed to reactive oxygen species. A copper exporter and a peroxiredoxin contribute to fungal defence. Yeast cells undergo intracellular lysis. Lysis occurs via a cocktail of hydrolytic enzymes from intracellular vesicles.

Candidalysin is a fungal peptide toxin critical for mucosal infection.

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

A TRP1-marker-based system for gene complementation, overexpression, reporter gene expression and gene modification in Candida glabrata.

Although less prevalent than its relative Candida albicans, the yeast Candida glabrata is a successful pathogen of humans, which causes life-threatening candidiasis. It is thus vital to understand the pathogenicity mechanisms and contributing genes in C. glabrata. However, gene complementation as a tool for restoring the function of a previously deleted gene is not standardized in C. glabrata, and it is less frequently used than in C. albicans. In this study, we established a gene complementation strategy using genomic integration at the TRP1 locus. We prove that our approach can not only be used for integration of complementation cassettes, but also for overexpression of markers like fluorescent proteins and the antigen ovalbumin, or of potential pathogenicity-related factors like the biotin transporter gene VHT1. With urea amidolyase Dur1,2 as an example, we demonstrate the application of the gene complementation approach for the expression of sequence-modified genes. With this approach, we found that a lysine-to-arginine mutation in the biotinylation motif of Dur1,2 impairs urea-dependent growth of C. glabrata and C. albicans. Taken together, the TRP1-based gene complementation approach is a valuable tool for investigating novel gene functions and for elucidating their role in the pathobiology of C. glabrata.

Fungal biotin homeostasis is essential for immune evasion after macrophage phagocytosis and virulence.

Biotin is an important cofactor for multiple enzymes in central metabolic processes. While many bacteria and most fungi are able to synthesise biotin de novo, Candida spp. are auxotrophic for this vitamin and thus require efficient uptake systems to facilitate biotin acquisition during infection. Here we show that Candida glabrata and Candida albicans use a largely conserved system for biotin uptake and regulation, consisting of the high-affinity biotin transporter Vht1 and the transcription factor Vhr1. Both species induce expression of biotin-metabolic genes upon in vitro biotin depletion and following phagocytosis by macrophages, indicating low biotin levels in the Candida-containing phagosome. In line with this, we observed reduced intracellular proliferation of both Candida cells pre-starved of biotin and deletion mutants lacking VHR1 or VHT1 genes. VHT1 was essential for the full virulence of C. albicans during systemic mouse infections, and the lack of VHT1 led to reduced fungal burden in C. glabrata-infected brains and C. albicans-infected brains and kidneys. Together, our data suggest a critical role of Vht1-mediated biotin acquisition for C. glabrata and C. albicans during intracellular growth in macrophages and systemic infections.

Metabolic adaptation of intracellular bacteria and fungi to macrophages.

The mature phagosome of macrophages is a hostile environment for the vast majority of phagocytosed microbes. In addition to active destruction of the engulfed microbes by antimicrobial compounds, restriction of essential nutrients in the phagosomal compartment contributes to microbial growth inhibition and killing. However, some pathogenic microorganisms have not only developed various strategies to efficiently withstand or counteract antimicrobial activities, but also to acquire nutrients within macrophages for intracellular replication. Successful intracellular pathogens are able to utilize host-derived amino acids, carbohydrates and lipids as well as trace metals and vitamins during intracellular growth. This requires sophisticated strategies such as phagosome modification or escape, efficient nutrient transporters and metabolic adaptation. In this review, we discuss the metabolic adaptation of facultative intracellular bacteria and fungi to the intracellular lifestyle inside macrophages.

Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera.

One small step for a yeast--microevolution within macrophages renders Candida glabrata hypervirulent due to a single point mutation.

Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.

Induction of caspase-11 by aspartyl proteinases of Candida albicans and implication in promoting inflammatory response.

We recently demonstrated that the secreted aspartyl proteinases (Saps), Sap2 and Sap6, of Candida albicans have the potential to induce the canonical activation of the NLRP3 inflammasome, leading to the secretion of interleukin-1ß (IL-1ß) and IL-18 via caspase-1 activation. We also observed that the activation of caspase-1 is partially independent from the NLRP3 activation pathway. In this study, we examined whether Sap2 and Sap6 are also able to activate the noncanonical inflammasome pathway in murine macrophages. Our data show that both Sap2 and Sap6 can activate caspase-11 through type I interferon (IFN) production. Caspase-11 cooperates to activate caspase-1, with a subsequent increase of IL-1ß secretion. Endocytosis and internalization of Saps are required for the induction of type I IFN production, which is essential for induction of noncanonical inflammasome activation. Our study indicates a sophisticated interplay between caspase-1 and caspase-11 that connects the canonical and noncanonical pathways of inflammasome activation in response to C. albicans Saps.

Intracellular survival of Candida glabrata in macrophages: immune evasion and persistence.

Candida glabrata is a successful human opportunistic pathogen which causes superficial but also life-threatening systemic infections. During infection, C. glabrata has to cope with cells of the innate immune system such as macrophages, which belong to the first line of defense against invading pathogens. Candida glabrata is able to survive and even replicate inside macrophages while causing surprisingly low damage and cytokine release. Here, we present an overview of recent studies dealing with the interaction of C. glabrata with macrophages, from phagocytosis to intracellular growth and escape. We review the strategies of C. glabrata that permit intracellular survival and replication, including poor host cell activation, modification of phagosome maturation and phagosome pH, adaptation to antimicrobial activities, and mechanisms to overcome the nutrient limitations within the phagosome. In summary, these studies suggest that survival within macrophages may be an immune evasion and persistence strategy of C. glabrata during infection.

Immune evasion, stress resistance, and efficient nutrient acquisition are crucial for intracellular survival of Candida glabrata within macrophages.

Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes' functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.

Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome.

In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL-1ß, TNF-α, and IL-6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL-1ß and IL-18 production through inflammasome activation. This occurs via NLRP3 and caspase-1 activation, which cleaves pro-IL-1ß into secreted bioactive IL-1ß, a cytokine that was induced by Saps in monocytes, in monocyte-derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL-1ß. Inflammasome activation required Sap internalization via a clathrin-dependent mechanism, intracellular induction of K(+) efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS-ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.

A family of glutathione peroxidases contributes to oxidative stress resistance in Candida albicans.

Candida albicans is a well-adapted human commensal but is also a facultative pathogen that can cause superficial and systemic infections. Its remarkable capacity to thrive within the human host relies on its ability to adapt and respond to the local environment of different niches. C. albicans is able to cope with oxidative stress in a coordinated fashion via upregulation of different protective mechanisms. Here, we unravel the role of a family of glutathione peroxidase (GPx), designated Gpx31, Gpx32, and Gpx33, in oxidative stress resistance. We show that GPx activity in C. albicans is induced upon exposure to peroxides and that this enzymatic activity is required for full resistance to oxidative stress. The GPx activity relies on the presence of GPX31, with no apparent contribution from GPX32 and GPX33 during in vitro short-term (3 h) exposure to peroxides. However, a triple gpx31-33Δ/Δ mutant exhibited a more pronounced sensitivity than a single gpx31Δ/Δ mutant on solid media in the presence of oxidants, suggesting that GPX32 and GPX33 may be involved in long-term adaptation to oxidative stress. Interestingly, reintegration of a single allele of GPX31 was sufficient to restore the wild-type phenotype in both the single and triple mutants. We found that mutants lacking GPX31-33 were more susceptible to killing by phagocytic cells, suggesting that GPxs are required for full resistance to innate immune effector cells. Despite the sensitivity to oxidative stress and phagocytes, these mutants were not affected in their virulence in the chicken embryo model of candidiasis.

Secretion of the fungal toxin candidalysin is dependent on conserved precursor peptide sequences.

The opportunistic fungal pathogen Candida albicans damages host cells via its peptide toxin, candidalysin. Before secretion, candidalysin is embedded in a precursor protein, Ece1, which consists of a signal peptide, the precursor of candidalysin and seven non-candidalysin Ece1 peptides (NCEPs), and is found to be conserved in clinical isolates. Here we show that the Ece1 polyprotein does not resemble the usual precursor structure of peptide toxins. C. albicans cells are not susceptible to their own toxin, and single NCEPs adjacent to candidalysin are sufficient to prevent host cell toxicity. Using a series of Ece1 mutants, mass spectrometry and anti-candidalysin nanobodies, we show that NCEPs play a role in intracellular Ece1 folding and candidalysin secretion. Removal of single NCEPs or modifications of peptide sequences cause an unfolded protein response (UPR), which in turn inhibits hypha formation and pathogenicity in vitro. Our data indicate that the Ece1 precursor is not required to block premature pore-forming toxicity, but rather to prevent intracellular auto-aggregation of candidalysin sequences.

Thriving within the host: Candida spp. interactions with phagocytic cells.

Certain Candida spp. (e.g. C. albicans, C. tropicalis, C. parapsilosis and C. glabrata) are not only well-adapted fungal commensals of humans, but are also able to cause superficial mucosal infections or even systemic disease. Professional phagocytes (neutrophils, macrophages and dendritic cells) constitute the first line of defence against Candida spp. Here, we review the interactions of phagocytes with pathogenic Candida spp., focusing on macrophages and neutrophils. We discuss the mechanisms involved in recognition, uptake and killing of these fungi. We go on to analyse the cellular responses of these yeasts towards phagocyte-imposed stresses, including metabolic flexibility, robust oxidative stress response and ability to cope with nitrosative stress. Finally, we address strategies that allow these opportunistic pathogens to thrive within the host, evading and escaping from the phagocyte attack.

Toll-like receptor 4 and CD11b expressed on microglia coordinate eradication of Candida albicans cerebral mycosis.

The fungal pathogen Candida albicans is linked to chronic brain diseases such as Alzheimer's disease (AD), but the molecular basis of brain anti-Candida immunity remains unknown. We show that C. albicans enters the mouse brain from the blood and induces two neuroimmune sensing mechanisms involving secreted aspartic proteinases (Saps) and candidalysin. Saps disrupt tight junction proteins of the blood-brain barrier (BBB) to permit fungal brain invasion. Saps also hydrolyze amyloid precursor protein (APP) into amyloid ß (Aß)-like peptides that bind to Toll-like receptor 4 (TLR4) and promote fungal killing in vitro while candidalysin engages the integrin CD11b (Mac-1) on microglia. Recognition of Aß-like peptides and candidalysin promotes fungal clearance from the brain, and disruption of candidalysin recognition through CD11b markedly prolongs C. albicans cerebral mycosis. Thus, C. albicans is cleared from the brain through innate immune mechanisms involving Saps, Aß, candidalysin, and CD11b.

From intestinal colonization to systemic infections: Candida albicans translocation and dissemination.

Candida species are the most prevalent cause of invasive fungal infections, of which Candida albicans is the most common. Translocation across the epithelial barrier into the bloodstream by intestinal-colonizing C. albicans cells serves as the main source for systemic infections. Understanding the fungal mechanisms behind this process will give valuable insights on how to prevent such infections and keep C. albicans in the commensal state in patients with predisposing conditions. This review will focus on recent developments in characterizing fungal translocation mechanisms, compare what we know about enteric bacterial pathogens with C. albicans, and discuss the different proposed hypotheses for how C. albicans enters and disseminates through the bloodstream immediately following translocation.

The Dual Function of the Fungal Toxin Candidalysin during Candida albicans-Macrophage Interaction and Virulence.

The dimorphic fungus Candida albicans is both a harmless commensal organism on mucosal surfaces and an opportunistic pathogen. Under certain predisposing conditions, the fungus can overgrow the mucosal microbiome and cause both superficial and life-threatening systemic infections after gaining access to the bloodstream. As the first line of defense of the innate immune response, infecting C. albicans cells face macrophages, which mediate the clearance of invading fungi by intracellular killing. However, the fungus has evolved sophisticated strategies to counteract macrophage antimicrobial activities and thus evade immune surveillance. The cytolytic peptide toxin, candidalysin, contributes to this fungal defense machinery by damaging immune cell membranes, providing an escape route from the hostile phagosome environment. Nevertheless, candidalysin also induces NLRP3 inflammasome activation, leading to an increased host-protective pro-inflammatory response in mononuclear phagocytes. Therefore, candidalysin facilitates immune evasion by acting as a classical virulence factor but also contributes to an antifungal immune response, serving as an avirulence factor. In this review, we discuss the role of candidalysin during C. albicans infections, focusing on its implications during C. albicans-macrophage interactions.