Receptor-kinase EGFR-MAPK adaptor proteins mediate the epithelial response to Candida albicans via the cytolytic peptide toxin, candidalysin.

Candida albicans (C. albicans) is a dimorphic commensal human fungal pathogen that can cause severe oropharyngeal candidiasis (oral thrush) in susceptible hosts. During invasive infection, C. albicans hyphae invade oral epithelial cells (OECs) and secrete candidalysin, a pore-forming cytolytic peptide that is required for C. albicans pathogenesis at mucosal surfaces. Candidalysin is produced in the hyphal invasion pocket and triggers cell damage responses in OECs. Candidalysin also activates multiple MAPK-based signaling events that collectively drive the production of downstream inflammatory mediators that coordinate downstream innate and adaptive immune responses. The activities of candidalysin are dependent on signaling through the epidermal growth factor receptor (EGFR). Here, we interrogated known EGFR-MAPK signaling intermediates for their roles mediating the OEC response to C. albicans infection. Using RNA silencing and pharmacological inhibition, we identified five key adaptors, including growth factor receptor-bound protein 2 (Grb2), Grb2-associated binding protein 1 (Gab1), Src homology and collagen (Shc), SH2-containing protein tyrosine phosphatase-2 (Shp2), and casitas B-lineage lymphoma (c-Cbl). We determined that all of these signaling effectors were inducibly phosphorylated in response to C. albicans. These phosphorylation events occurred in a candidalysin-dependent manner and additionally required EGFR phosphorylation, matrix metalloproteinases (MMPs), and cellular calcium flux to activate a complete OEC response to fungal infection. Of these, Gab1, Grb2, and Shp2 were the dominant drivers of ERK1/2 activation and the subsequent production of downstream innate-acting cytokines. Together, these results identify the key adaptor proteins that drive the EGFR signaling mechanisms that underlie oral epithelial responses to C. albicans.

Candidalysins Are a New Family of Cytolytic Fungal Peptide Toxins.

Candidalysin is the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is secreted by Candida albicans and is critical for driving infection and host immune responses in several model systems. However, Candida infections are also caused by non-C. albicans species. Here, we identify and characterize orthologs of C. albicans candidalysin in C. dubliniensis and C. tropicalis. The candidalysins have different amino acid sequences, are amphipathic, and adopt a predominantly α-helical secondary structure in solution. Comparative functional analysis demonstrates that each candidalysin causes epithelial damage and calcium influx and activates intracellular signaling pathways and cytokine secretion. Importantly, C. dubliniensis and C. tropicalis candidalysins have higher damaging and activation potential than C. albicans candidalysin and exhibit more rapid membrane binding and disruption, although both fungal species cause less damage to epithelial cells than C. albicans. This study identifies the first family of peptide cytolysins in human-pathogenic fungi. IMPORTANCE Pathogenic fungi kill an estimated 1.5 million people every year. Recently, we discovered that the fungal pathogen Candida albicans secretes a peptide toxin called candidalysin during mucosal infection. Candidalysin causes damage to host cells, a process that supports disease progression. However, fungal infections are also caused by Candida species other than C. albicans. In this work, we identify and characterize two additional candidalysin toxins present in the related fungal pathogens C. dubliniensis and C. tropicalis. While the three candidalysins have different amino acid sequences, all three toxins are α-helical and amphipathic. Notably, the candidalysins from C. dubliniensis and C. tropicalis are more potent at inducing cell damage, calcium influx, mitogen-activated protein kinase signaling, and cytokine responses than C. albicans candidalysin, with the C. dubliniensis candidalysin having the most rapid membrane binding kinetics. These observations identify the candidalysins as the first family of peptide toxins in human-pathogenic fungi.

Albumin Neutralizes Hydrophobic Toxins and Modulates Candida albicans Pathogenicity.

Albumin is abundant in serum but is also excreted at mucosal surfaces and enters tissues when inflammation increases vascular permeability. Host-associated opportunistic pathogens encounter albumin during commensalism and when causing infections. Considering the ubiquitous presence of albumin, we investigated its role in the pathogenesis of infections with the model human fungal pathogen, Candida albicans. Albumin was introduced in various in vitro models that mimic different stages of systemic or mucosal candidiasis, where it reduced the ability of C. albicans to damage host cells. The amphipathic toxin candidalysin mediates necrotic host cell damage induced by C. albicans. Using cellular and biophysical assays, we determined that albumin functions by neutralizing candidalysin through hydrophobic interactions. We discovered that albumin, similarly, can neutralize a variety of fungal (α-amanitin), bacterial (streptolysin O and staurosporin), and insect (melittin) hydrophobic toxins. These data suggest albumin as a defense mechanism against toxins, which can play a role in the pathogenesis of microbial infections. IMPORTANCE Albumin is the most abundant serum protein in humans. During inflammation, serum albumin levels decrease drastically, and low albumin levels are associated with poor patient outcome. Thus, albumin may have specific functions during infection. Here, we describe the ability of albumin to neutralize hydrophobic microbial toxins. We show that albumin can protect against damage induced by the pathogenic yeast C. albicans by neutralizing its cytolytic toxin candidalysin. These findings suggest that albumin is a toxin-neutralizing protein that may play a role during infections with toxin-producing microorganisms.

Role for IL-1 Family Cytokines in Fungal Infections.

Fungal pathogens kill approximately 1.5 million individuals per year and represent a severe disease burden worldwide. It is estimated over 150 million people have serious fungal disease such as recurrent mucosal infections or life-threatening systemic infections. Disease can ensue from commensal fungi or new infection and involves different fungal morphologies and the expression of virulence factors. Therefore, anti-fungal immunity is complex and requires coordination between multiple facets of the immune system. IL-1 family cytokines are associated with acute and chronic inflammation and are essential for the innate response to infection. Recent research indicates IL-1 cytokines play a key role mediating immunity against different fungal infections. During mucosal disease, IL-1R and IL-36R are required for neutrophil recruitment and protective Th17 responses, but function through different mechanisms. During systemic disease, IL-18 drives protective Th1 responses, while IL-33 promotes Th2 and suppresses Th1 immunity. The IL-1 family represents an attractive anti-fungal immunotherapy target. There is a need for novel anti-fungal therapeutics, as current therapies are ineffective, toxic and encounter resistance, and no anti-fungal vaccine exists. Furthering our understanding of the IL-1 family cytokines and their complex role during fungal infection may aid the development of novel therapies. As such, this review will discuss the role for IL-1 family cytokines in fungal infections.

Analysis of Epithelial Cell Responses to Microbial Pathogens.

The epithelial cell is usually the first host cell that interacts with the microbiota present at mucosal surfaces. Although initially thought of as "bystander" cells with barrier function, the epithelial cell is now known to be a sentinel cell in the recognition and discrimination of commensal and pathogenic microorganisms and a key cell in initiating subsequent innate and adaptive immune responses. Here, we describe the main assays utilized in analyzing the activation of epithelial cell signaling (western blotting), transcription factors (TransAm), gene expression (quantitative reverse transcription PCR (qRT-PCR)), cytokine responses (ELISA, Luminex), and damage induction (lactate dehydrogenase (LDH) release). While our laboratory focuses on the epithelial response to Candida pathogens, these assays can be applied universally to analyze the activation of epithelial cells in response to any microbial pathogen.

Candida albicans Enhances the Progression of Oral Squamous Cell Carcinoma In Vitro and In Vivo.

Oral squamous cell carcinoma (OSCC) is associated with oral Candida albicans infection, although it is unclear whether the fungus promotes the genesis and progression of OSCC or whether cancer facilitates fungal growth. In this study, we investigated whether C. albicans can potentiate OSCC tumor development and progression. In vitro, the presence of live C. albicans, but not Candida parapsilosis, enhanced the progression of OSCC by stimulating the production of matrix metalloproteinases, oncometabolites, protumor signaling pathways, and overexpression of prognostic marker genes associated with metastatic events. C. albicans also upregulated oncogenes in nonmalignant cells. Using a newly established xenograft in vivo mouse model to investigate OSCC-C. albicans interactions, oral candidiasis enhanced the progression of OSCC through inflammation and induced the overexpression of metastatic genes and significant changes in markers of the epithelial-mesenchymal transition. Finally, using the 4-nitroquinoline 1-oxide (4NQO) murine model, we directly correlate these in vitro and short-term in vivo findings with the progression of oncogenesis over the long term. Taken together, these data indicate that C. albicans upregulates oncogenes, potentiates a premalignant phenotype, and is involved in early and late stages of malignant promotion and progression of oral cancer. IMPORTANCE Oral squamous cell carcinoma (OSCC) is a serious health issue worldwide that accounts for 2% to 4% of all cancer cases. Previous studies have revealed a higher yeast carriage and diversity in oral cancer patients than in healthy individuals. Furthermore, fungal colonization in the oral cavity bearing OSCC is higher on the neoplastic epithelial surface than on adjacent healthy surfaces, indicating a positive association between oral yeast carriage and epithelial carcinoma. In addition to this, there is strong evidence supporting the idea that Candida contributes to carcinogenesis events in the oral cavity. Here, we show that an increase in Candida albicans burden promotes an oncogenic phenotype in the oral cavity.

Candida albicans and candidalysin in inflammatory disorders and cancer.

As our understanding of mycology progresses, the impact of fungal microbes on human health has become increasingly evident. Candida albicans is a common commensal fungus that gives rise to local and systemic infections, particularly in immunocompromised patients where it can result in mortality. However, C. albicans has also been quietly linked with a variety of inflammatory disorders, to which it has traditionally been considered incidental; recent studies may now provide new aspects of these relationships for further consideration. This review provides a novel perspective on the impact of C. albicans and its peptide toxin, candidalysin, on human health, exploring their contributions to pathology within a variety of diseases.

Candidalysin Is a Potent Trigger of Alarmin and Antimicrobial Peptide Release in Epithelial Cells.

Host released alarmins and antimicrobial peptides (AMPs) are highly effective as antifungal agents and inducers. Whilst some are expressed constitutively at mucosal tissues, the primary site of many infections, others are elicited in response to pathogens. In the context of Candida albicans, the fungal factors inducing the release of these innate immune molecules are poorly defined. Herein, we identify candidalysin as a potent trigger of several key alarmins and AMPs known to possess potent anti-Candida functions. We also find extracellular ATP to be an important activator of candidalysin-induced epithelial signalling responses, namely epidermal growth factor receptor (EGFR) and MAPK signalling, which mediate downstream innate immunity during oral epithelial infection. The data provide novel mechanistic insight into the induction of multiple key alarmins and AMPs, important for antifungal defences against C. albicans.

Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.

Candida albicans is a fungal pathobiont, able to cause epithelial cell damage and immune activation. These functions have been attributed to its secreted toxin, candidalysin, though the molecular mechanisms are poorly understood. Here, we identify epidermal growth factor receptor (EGFR) as a critical component of candidalysin-triggered immune responses. We find that both C. albicans and candidalysin activate human epithelial EGFR receptors and candidalysin-deficient fungal mutants poorly induce EGFR phosphorylation during murine oropharyngeal candidiasis. Furthermore, inhibition of EGFR impairs candidalysin-triggered MAPK signalling and release of neutrophil activating chemokines in vitro, and diminishes neutrophil recruitment, causing significant mortality in an EGFR-inhibited zebrafish swimbladder model of infection. Investigation into the mechanism of EGFR activation revealed the requirement of matrix metalloproteinases (MMPs), EGFR ligands and calcium. We thus identify a PAMP-independent mechanism of immune stimulation and highlight candidalysin and EGFR signalling components as potential targets for prophylactic and therapeutic intervention of mucosal candidiasis.

Candida albicans Interactions with Mucosal Surfaces during Health and Disease.

Flexible adaptation to the host environment is a critical trait that underpins the success of numerous microbes. The polymorphic fungus Candida albicans has evolved to persist in the numerous challenging niches of the human body. The interaction of C. albicans with a mucosal surface is an essential prerequisite for fungal colonisation and epitomises the complex interface between microbe and host. C. albicans exhibits numerous adaptations to a healthy host that permit commensal colonisation of mucosal surfaces without provoking an overt immune response that may lead to clearance. Conversely, fungal adaptation to impaired immune fitness at mucosal surfaces enables pathogenic infiltration into underlying tissues, often with devastating consequences. This review will summarise our current understanding of the complex interactions that occur between C. albicans and the mucosal surfaces of the human body.

Candida innate immunity at the mucosa.

The tremendous diversity in microbial species that colonise the mucosal surfaces of the human body is only now beginning to be fully appreciated. Distinguishing between the behaviour of commensal microbes and harmful pathogens that reside at mucosal sites in the body is a complex, and exquisitely fine-tuned process central to mucosal health. The fungal pathobiont Candida albicans is frequently isolated from mucosal surfaces with an asymptomatic carriage rate of approximately 60% in the human population. While normally a benign member of the microbiota, overgrowth of C. albicans often results in localised mucosal infection causing morbidity in otherwise healthy individuals, and invasive infection that often causes death in the absence of effective immune defence. C. albicans triggers numerous innate immune responses at mucosal surfaces, and detection of C. albicans hyphae in particular, stimulates the production of antimicrobial peptides, danger-associated molecular patterns and cytokines that function to reduce fungal burdens during infection. This review will summarise our current understanding of innate immune responses to C. albicans at mucosal surfaces.

IL-36 and IL-1/IL-17 Drive Immunity to Oral Candidiasis via Parallel Mechanisms.

Protection against microbial infection by the induction of inflammation is a key function of the IL-1 superfamily, including both classical IL-1 and the new IL-36 cytokine families. Candida albicans is a frequent human fungal pathogen causing mucosal infections. Although the initiators and effectors important in protective host responses to C. albicans are well described, the key players in driving these responses remain poorly defined. Recent work has identified a central role played by IL-1 in inducing innate Type-17 immune responses to clear C. albicans infections. Despite this, lack of IL-1 signaling does not result in complete loss of immunity, indicating that there are other factors involved in mediating protection to this fungus. In this study, we identify IL-36 cytokines as a new player in these responses. We show that C. albicans infection of the oral mucosa induces the production of IL-36. As with IL-1α/ß, induction of epithelial IL-36 depends on the hypha-associated peptide toxin Candidalysin. Epithelial IL-36 gene expression requires p38-MAPK/c-Fos, NF-κB, and PI3K signaling and is regulated by the MAPK phosphatase MKP1. Oral candidiasis in IL-36R-/- mice shows increased fungal burdens and reduced IL-23 gene expression, indicating a key role played by IL-36 and IL-23 in innate protective responses to this fungus. Strikingly, we observed no impact on gene expression of IL-17 or IL-17-dependent genes, indicating that this protection occurs via an alternative pathway to IL-1-driven immunity. Thus, IL-1 and IL-36 represent parallel epithelial cell-driven protective pathways in immunity to oral C. albicans infection.

Candida-Epithelial Interactions.

A plethora of intricate and dynamic molecular interactions occur between microbes and the epithelial cells that form the mucosal surfaces of the human body. Fungi, particularly species of Candida, are commensal members of our microbiota, continuously interacting with epithelial cells. Transient and localised perturbations to the mucosal environment can facilitate the overgrowth of fungi, causing infection. This minireview will examine the direct and indirect mechanisms by which Candida species and epithelial cells interact with each other, and explore the factors involved in the central processes of adhesion, invasion, and destruction of host mucosal surfaces.

Processing of Candida albicans Ece1p Is Critical for Candidalysin Maturation and Fungal Virulence.

Candida albicans is an opportunistic fungal pathogen responsible for superficial and life-threatening infections in humans. During mucosal infection, C. albicans undergoes a morphological transition from yeast to invasive filamentous hyphae that secrete candidalysin, a 31-amino-acid peptide toxin required for virulence. Candidalysin damages epithelial cell plasma membranes and stimulates the activating protein 1 (AP-1) transcription factor c-Fos (via p38-mitogen-activated protein kinase [MAPK]), and the MAPK phosphatase MKP1 (via extracellular signal-regulated kinases 1 and 2 [ERK1/2]-MAPK), which trigger and regulate proinflammatory cytokine responses, respectively. The candidalysin toxin resides as a discrete cryptic sequence within a larger 271-amino-acid parental preproprotein, Ece1p. Here, we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two-step posttranslational processing of Ece1p to produce candidalysin. Kex2p-mediated proteolysis of Ece1p after Arg61 and Arg93, but not after other processing sites within Ece1p, is required to generate immature candidalysin from Ece1p, followed by Kex1p-mediated removal of a carboxyl arginine residue to generate mature candidalysin. C. albicans strains harboring mutations of Arg61 and/or Arg93 did not secrete candidalysin, were unable to induce epithelial damage and inflammatory responses in vitro, and showed attenuated virulence in vivo in a murine model of oropharyngeal candidiasis. These observations identify enzymatic processing of C. albicans Ece1p by kexin-like proteinases as crucial steps required for candidalysin production and fungal pathogenicity.IMPORTANCECandida albicans is an opportunistic fungal pathogen that causes mucosal infection in millions of individuals worldwide. Successful infection requires the secretion of candidalysin, the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is derived from its parent protein Ece1p. Here, we identify two key amino acids within Ece1p vital for processing and production of candidalysin. Mutations of these residues render C. albicans incapable of causing epithelial damage and markedly reduce mucosal infection in vivo Importantly, candidalysin production requires two individual enzymatic events. The first involves processing of Ece1p by Kex2p, yielding immature candidalysin, which is then further processed by Kex1p to produce the mature toxin. These observations identify important steps for C. albicans pathogenicity at mucosal surfaces.

Oral epithelial cells orchestrate innate type 17 responses to Candida albicans through the virulence factor candidalysin.

Candida albicans is a dimorphic commensal fungus that causes severe oral infections in immunodeficient patients. Invasion of C. albicans hyphae into oral epithelium is an essential virulence trait. Interleukin-17 (IL-17) signaling is required for both innate and adaptive immunity to C. albicans During the innate response, IL-17 is produced by γδ T cells and a poorly understood population of innate-acting CD4+ αß T cell receptor (TCRαß)+ cells, but only the TCRαß+ cells expand during acute infection. Confirming the innate nature of these cells, the TCR was not detectably activated during the primary response, as evidenced by Nur77eGFP mice that report antigen-specific signaling through the TCR. Rather, the expansion of innate TCRαß+ cells was driven by both intrinsic and extrinsic IL-1R signaling. Unexpectedly, there was no requirement for CCR6/CCL20-dependent recruitment or prototypical fungal pattern recognition receptors. However, C. albicans mutants that cannot switch from yeast to hyphae showed impaired TCRαß+ cell proliferation and Il17a expression. This prompted us to assess the role of candidalysin, a hyphal-associated peptide that damages oral epithelial cells and triggers production of inflammatory cytokines including IL-1. Candidalysin-deficient strains failed to up-regulate Il17a or drive the proliferation of innate TCRαß+ cells. Moreover, candidalysin signaled synergistically with IL-17, which further augmented the expression of IL-1α/ß and other cytokines. Thus, IL-17 and C. albicans, via secreted candidalysin, amplify inflammation in a self-reinforcing feed-forward loop. These findings challenge the paradigm that hyphal formation per se is required for the oral innate response and demonstrate that establishment of IL-1- and IL-17-dependent innate immunity is induced by tissue-damaging hyphae.

The Role of ErbB Receptors in Infection.

Members of the epidermal growth factor receptor family (ErbB family) possess a wide distribution and diverse functions ranging from cellular growth to migration and apoptosis. Though highly implicated in a variety of cancers, their involvement in infectious disease is less recognised. A growing body of evidence now highlights the importance of the ErbB family in a variety of infections. Their role as growth factor receptors, along with other characteristics, such as surface expression and continuous intracellular trafficking, make this receptor family ideally placed for exploitation by pathogens. Herein, we review our current understanding of the role of the ErbB family in the context of infectious disease, exploring the mechanisms that govern pathogen exploitation of this system.

Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection.

UNLABELLED: Skeletal muscle, at 30 to 40% of body mass, is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and particularly avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors and were readily susceptible to low-pathogenicity (H2N3 A/mallard duck/England/7277/06) and high-pathogenicity (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) avian and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notably, the rapid accumulation of viral nucleoprotein and matrix (M) gene RNA in chicken and duck myotubes was accompanied by extensive cytopathic damage with marked myotube apoptosis (widespread microscopic blebs, caspase 3/7 activation, and annexin V binding at the plasma membrane). Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells. Additionally, in chicken myotubes infected with H5N1 viruses, the induction of interferon beta (IFN-ß) and IFN-inducible genes, including the melanoma differentiation-associated protein 5 (MDA-5) gene, was relatively weak compared to infection with the corresponding H2N3 virus. Our findings highlight that avian skeletal muscle fibers are capable of productive influenza virus replication and are a potential tissue source of infection. IMPORTANCE: Infection with high-pathogenicity H5N1 viruses in ducks is often asymptomatic, and skeletal muscle from such birds could be a source of infection of humans and animals. Little is known about the ability of influenza A viruses to replicate in avian skeletal muscle fibers. We show here that cultured chicken and duck myotubes were highly susceptible to infection with both low- and high-pathogenicity avian influenza viruses. Infected myotubes of both avian species displayed rapid virus accumulation, apoptosis, and extensive cellular damage. Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.