Competitive fungal commensalism mitigates candidiasis pathology.

The mycobiota are a critical part of the gut microbiome, but host-fungal interactions and specific functional contributions of commensal fungi to host fitness remain incompletely understood. Here, we report the identification of a new fungal commensal, Kazachstania heterogenica var. weizmannii, isolated from murine intestines. K. weizmannii exposure prevented Candida albicans colonization and significantly reduced the commensal C. albicans burden in colonized animals. Following immunosuppression of C. albicans colonized mice, competitive fungal commensalism thereby mitigated fatal candidiasis. Metagenome analysis revealed K. heterogenica or K. weizmannii presence among human commensals. Our results reveal competitive fungal commensalism within the intestinal microbiota, independent of bacteria and immune responses, that could bear potential therapeutic value for the management of C. albicans-mediated diseases.

Dectin-1/CARD9 induction of the TFEB and TFE3 gene network is dispensable for phagocyte anti-Aspergillus activity in the lung.

Myeloid phagocytes of the respiratory immune system, such as neutrophils, monocytes, and alveolar macrophages, are essential for immunity to Aspergillus fumigatus, the most common etiologic agent of mold pneumonia worldwide. Following the engulfment of A. fumigatus conidia, fusion of the phagosome with the lysosome is a critical process for killing conidia. TFEB and TFE3 are transcription factors that regulate lysosomal biogenesis under stress and are activated by inflammatory stimuli in macrophages, but it is unknown whether TFEB and TFE3 contribute to anti-Aspergillus immunity during infection. We found that lung neutrophils express TFEB and TFE3, and their target genes were upregulated during A. fumigatus lung infection. In addition, A. fumigatus infection induced nuclear accumulation of TFEB and TFE3 in macrophages in a process regulated by Dectin-1 and CARD9. Genetic deletion of Tfeb and Tfe3 impaired macrophage killing of A. fumigatus conidia. However, in a murine immune-competent Aspergillus infection model with genetic deficiency of Tfeb and Tfe3 in hematopoietic cells, we surprisingly found that lung myeloid phagocytes had no defects in conidial phagocytosis or killing. Loss of TFEB and TFE3 did not impact murine survival or clearance of A. fumigatus from the lungs. Our findings indicate that myeloid phagocytes activate TFEB and TFE3 in response to A. fumigatus, and while this pathway promotes macrophage fungicidal activity in vitro, genetic loss can be functionally compensated in the lung, resulting in no measurable defect in fungal control and host survival.

The quorum-sensing peptidic inhibitor rescues host immune system eradication: A novel infectivity mechanism.

Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell-cell signaling peptide PapR7 regulate virulence gene expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to affect the host immune system. Here, we designed a peptide-based QS inhibitor that suppresses PlcR-dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system-mediated eradication by reducing the expression of PlcR-regulated major toxins similarly to the profile that was observed for isogenic strains. Our findings provide evidence that Bc infectivity is regulated by QS circuit-mediated destruction of host immunity, thus reveal a interesting strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes a readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics.

Dectin-1/CARD9-induction of the TFEB and TFE3 gene network is dispensable for phagocyte anti-Aspergillus activity in the lung.

Myeloid phagocytes of the respiratory immune system, such as neutrophils, monocytes, and alveolar macrophages, are essential for immunity to Aspergillus fumigatus, the most common etiologic agent of mold pneumonia worldwide. Following engulfment of A. fumigatus conidia, fusion of the phagosome with the lysosome, is a critical process for killing conidia. TFEB and TFE3 are transcription factors that regulate lysosomal biogenesis under stress and are activated by inflammatory stimuli in macrophages, but it is unknown whether TFEB and TFE3 contribute to anti-Aspergillus immunity during infection. We found that lung neutrophils express TFEB and TFE3, and their target genes were upregulated during A. fumigatus lung infection. Additionally, A. fumigatus infection induced nuclear accumulation of TFEB and TFE3 in macrophages in a process regulated by Dectin-1 and CARD9 signaling. Genetic deletion of Tfeb and Tfe3 impaired macrophage killing of A. fumigatus conidia. However, in a murine immune competent Aspergillus infection model with genetic deficiency of Tfeb and Tfe3 in hematopoietic cells, we surprisingly found that lung myeloid phagocytes had no defects in conidial phagocytosis or killing. Loss of TFEB and TFE3 did not impact murine survival or clearance of A. fumigatus from the lungs. Our findings indicate that myeloid phagocytes activate TFEB and TFE3 in response to A. fumigatus, and while this pathway promotes macrophage fungicidal activity in vitro, genetic loss can be functionally compensated at the portal of infection in the lung, resulting in no measurable defect in fungal control and host survival.

Mitochondrial Reactive Oxygen Species Enhance Alveolar Macrophage Activity against Aspergillus fumigatus but Are Dispensable for Host Protection.

Aspergillus fumigatus is the most common cause of mold pneumonia worldwide, and a significant cause of infectious morbidity and mortality in immunocompromised individuals. The oxidative burst, which generates reactive oxidative species (ROS), plays a pivotal role in host defense against aspergillosis and induces regulated cell death in Aspergillus conidia, the infectious propagules. Beyond the well-established role of NADP (NADPH) oxidase in ROS generation by neutrophils and other innate effector cells, mitochondria represent a major ROS production site in many cell types, though it is unclear whether mitochondrial ROS (mtROS) contribute to antifungal activity in the lung. Following A. fumigatus infection, we observed that innate effector cells, including alveolar macrophages (AMs), monocyte-derived dendritic cells (Mo-DCS), and neutrophils, generated mtROS, primarily in fungus-infected cells. To examine the functional role of mtROS, specifically the H2O2 component, in pulmonary host defense against A. fumigatus, we infected transgenic mice that expressed a mitochondrion-targeted catalase. Using a reporter of fungal viability during interactions with leukocytes, mitochondrial H2O2 (mtH2O2) was essential for optimal AM, but not for neutrophil phagocytic and conidiacidal activity in the lung. Catalase-mediated mtH2O2 neutralization did not lead to invasive aspergillosis in otherwise immunocompetent mice and did not shorten survival in mice that lack NADPH oxidase function. Collectively, these studies indicate that mtROS-associated defects in AM antifungal activity can be functionally compensated by the action of NADPH oxidase and by nonoxidative effector mechanisms during murine A. fumigatus lung infection. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes invasive disease in humans with defects in immune function. Airborne conidia, the infectious propagules, are ubiquitous and inhaled on a daily basis. In the respiratory tree, conidia are killed by the coordinated actions of phagocytes, including alveolar macrophages, neutrophils, and monocyte-derived dendritic cells. The oxidative burst represents a central killing mechanism and relies on the assembly of the NADPH oxidase complex on the phagosomal membrane. However, NADPH oxidase-deficient leukocytes have significant residual fungicidal activity in vivo, indicating the presence of alternative effector mechanisms. Here, we report that murine innate immune cells produce mitochondrial reactive oxygen species (mtROS) in response to fungal interactions. Neutralizing the mtROS constituent hydrogen peroxide (H2O2) via a catalase expressed in mitochondria of innate immune cells substantially diminished fungicidal properties of alveolar macrophages, but not of other innate immune cells. These data indicate that mtH2O2 represent a novel AM killing mechanism against Aspergillus conidia. mtH2O2 neutralization is compensated by other killing mechanisms in the lung, demonstrating functional redundancy at the level of host defense in the respiratory tree. These findings have important implications for the development of host-directed therapies against invasive aspergillosis in susceptible patient populations.

Fungal Bioreporters to Monitor Outcomes of Blastomyces: Host-Cell Interactions.

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the North American endemic dimorphic fungus, Blastomyces dermatitidis.

Fungal Bioreporters to Monitor Outcomes of Aspergillus: Host-Cell Interactions.

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the filamentous mold Aspergillus fumigatus.

Response to Comment on "Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death".

Aouacheria et al question the interpretation of contemporary assays to monitor programmed cell death with apoptosis-like features (A-PCD) in Aspergillus fumigatus Although our study focuses on fungal A-PCD for host immune surveillance and infectious outcomes, the experimental approach incorporates multiple independent A-PCD markers and genetic manipulations based on fungal rather than mammalian orthologs to circumvent the limitations associated with any single approach.

Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death.

Humans inhale mold conidia daily and typically experience lifelong asymptomatic clearance. Conidial germination into tissue-invasive hyphae can occur in individuals with defects in myeloid function, although the mechanism of myeloid cell-mediated immune surveillance remains unclear. By monitoring fungal physiology in vivo, we demonstrate that lung neutrophils trigger programmed cell death with apoptosis-like features in Aspergillus fumigatus conidia, the most prevalent human mold pathogen. An antiapoptotic protein, AfBIR1, opposes this process by inhibiting fungal caspase activation and DNA fragmentation in the murine lung. Genetic and pharmacologic studies indicate that AfBIR1 expression and activity underlie conidial susceptibility to NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-dependent killing and, in turn, host susceptibility to invasive aspergillosis. Immune surveillance exploits a fungal apoptosis-like programmed cell death pathway to maintain sterilizing immunity in the lung.

Translocation from nuclei to cytoplasm is necessary for anti A-PCD activity and turnover of the Type II IAP BcBir1.

Type II inhibitors of apoptosis (IAPs) belong to a subgroup of IAP-related proteins. While IAPs are restricted to animals, Type II IAPs are found in other phyla, including fungi. BcBir1, a Type II IAP from Botrytis cinerea has anti apoptotic-like programmed cell death (A-PCD) activity, which is important for pathogenicity of this fungus. Here we report on the role of sub-cellular localization of BcBir1 in protein turnover and anti A-PCD activity. Expression of BcBir1 in Saccharomyces cerevisiae had no effect on sensitivity of the yeast cells to A-PCD-inducing conditions, whereas expression of a truncated N' part reduced sensitivity of the cells to these conditions. The full-length BcBir1 protein was detected only in the yeast nucleus, whereas the N' part was observed both in the nucleus and cytoplasm. In B. cinerea, BcBir1 was mainly nuclear under optimal conditions, whereas under A-PCD-inducing conditions it shuttled to the cytoplasm and then it was completely degraded. Collectively, our results show that anti A-PCD activity of BcBir1 occurs in the cytoplasm, the C' end mediates regulation of steady state level of BcBir1 in the nucleus, and the N' end mediates anti A-PCD activity as well as fast degradation of BcBir1 in the cytoplasm.

Measurement of apoptosis by SCAN©, a system for counting and analysis of fluorescently labelled nuclei.

Apoptosis-like programmed cell death (A-PCD) is a universal process common to all types of eukaryotic organisms. Because A-PCD-associated processes are conserved, it is possible to define A-PCD by a standard set of markers. Many of the popular methods to measure A-PCD make use of fluorescent ligands that change in intensity or cellular localization during A-PCD. In single cell organisms, it is possible to quantify levels of A-PCD by scoring the number of apoptotic cells using flow cytometry instruments. In a multicellular organism, quantification of A-PCD is more problematic due to the complex nature of the tissue. The situation is further complicated in filamentous fungi, in which nuclei are divided between compartments, each containing a number of nuclei, which can also migrate between the compartments. We developed SCAN©, a System for Counting and Analysis of Nuclei, and used it to measure A-PCD according to two markers - chromatin condensation and DNA strand breaks. The package includes three modules designed for counting the number of nuclei in multi-nucleated domains, scoring the relative number of nuclei with condensed chromatin, and calculating the relative number of nuclei with DNA strand breaks. The method provides equal or better results compared with manual counting, the analysis is fast and can be applied on large data sets. While we demonstrated the utility of the software for measurement of A-PCD in fungi, the method is readily adopted for measurement of A-PCD in other types of multicellular specimens.

Apoptotic-like programed cell death in fungi: the benefits in filamentous species.

Studies conducted in the early 1990s showed for the first time that Saccharomyces cerevisiae can undergo cell death with hallmarks of animal apoptosis. These findings came as a surprise, since suicide machinery was unexpected in unicellular organisms. Today, apoptosis in yeast is well-documented. Apoptotic death of yeast cells has been described under various conditions and S. cerevisiae homologs of human apoptotic genes have been identified and characterized. These studies also revealed fundamental differences between yeast and animal apoptosis; in S. cerevisiae apoptosis is mainly associated with aging and stress adaptation, unlike animal apoptosis, which is essential for proper development. Further, many apoptosis regulatory genes are either missing, or highly divergent in S. cerevisiae. Therefore, in this review we will use the term apoptosis-like programed cell death (PCD) instead of apoptosis. Despite these significant differences, S. cerevisiae has been instrumental in promoting the study of heterologous apoptotic proteins, particularly from human. Work in fungi other than S. cerevisiae revealed differences in the manifestation of PCD in single cell (yeasts) and multicellular (filamentous) species. Such differences may reflect the higher complexity level of filamentous species, and hence the involvement of PCD in a wider range of processes and life styles. It is also expected that differences might be found in the apoptosis apparatus of yeast and filamentous species. In this review we focus on aspects of PCD that are unique or can be better studied in filamentous species. We will highlight the similarities and differences of the PCD machinery between yeast and filamentous species and show the value of using S. cerevisiae along with filamentous species to study apoptosis.

Apoptosis-like programmed cell death in the grey mould fungus Botrytis cinerea: genes and their role in pathogenicity.

A considerable number of fungal homologues of human apoptotic genes have been identified in recent years. Nevertheless, we are far from being able to connect the different pieces and construct a primary structure of the fungal apoptotic regulatory network. To get a better picture of the available fungal components, we generated an automatic search protocol that is based on protein sequences together with a domain-centred approach. We used this protocol to search all the available fungal databases for domains and homologues of human apoptotic proteins. Among all known apoptotic domains, only the BIR [baculovirus IAP (inhibitor of apoptosis protein) repeat] domain was found in fungi. A single protein with one or two BIR domains is present in most (but not all) fungal species. We isolated the BIR-containing protein from the grey mould fungus Botrytis cinerea and determined its role in apoptosis and pathogenicity. We also isolated and analysed BcNMA, a homologue of the yeast NMA11 gene. Partial knockout or overexpression strains of BcBIR1 confirmed that BcBir1 is anti-apoptotic and this activity was assigned to the N'-terminal part of the protein. Plant infection assays showed that the fungus undergoes massive PCD (programmed cell death) during early stages of infection. Further studies showed that fungal virulence was fully correlated with the ability of the fungus to cope with plant-induced PCD. Together, our result show that BcBir1 is a major regulator of PCD in B. cinerea and that proper regulation of the host-induced PCD is essential for pathogenesis in this and other similar fungal pathogens.

Anti-apoptotic machinery protects the necrotrophic fungus Botrytis cinerea from host-induced apoptotic-like cell death during plant infection.

Necrotrophic fungi are unable to occupy living plant cells. How such pathogens survive first contact with living host tissue and initiate infection is therefore unclear. Here, we show that the necrotrophic grey mold fungus Botrytis cinerea undergoes massive apoptotic-like programmed cell death (PCD) following germination on the host plant. Manipulation of an anti-apoptotic gene BcBIR1 modified fungal response to PCD-inducing conditions. As a consequence, strains with reduced sensitivity to PCD were hyper virulent, while strains in which PCD was over-stimulated showed reduced pathogenicity. Similarly, reduced levels of PCD in the fungus were recorded following infection of Arabidopsis mutants that show enhanced susceptibility to B. cinerea. When considered together, these results suggest that Botrytis PCD machinery is targeted by plant defense molecules, and that the fungal anti-apoptotic machinery is essential for overcoming this host-induced PCD and hence, for establishment of infection. As such, fungal PCD machinery represents a novel target for fungicides and antifungal drugs.

Botrytis cinerea BcNma is involved in apoptotic cell death but not in stress adaptation.

Apoptotic-like programmed cell death (PCD) occurs naturally in fungi during development and might also be induced by external conditions. Candidate apoptotic genes have been characterized in several model fungal species but not in plant pathogenic fungi. Here we report on the isolation and characterization of BcNMA, an orthologue of the human pro-apoptotic gene HtrA2 from the plant pathogen Botrytis cinerea. The predicted BcNma protein shows high homology to the previously characterized Nma111p from Saccharomyces cerevisiae and despite some structural differences it complemented the function of Nma111p in Δnma111 mutant strains. BcNMA-over-expression and mutant strains had enhanced or reduced appearance of apoptotic markers, respectively. However there was no difference in growth response of the wild type and BcNMA-transgenic strains to application of various stresses, and the effect on pathogenicity was marginal in both the over-expression and mutant strains. When considered together these results suggest that although BcNma has a pro-apoptotic activity, it is not a major regulator of apoptosis. The protein probably has additional roles that are unrelated to apoptosis, which lead to the pleotrophic phenotype of the transgenic strains and lack of a clear effect on stress adaptation and pathogenicity.

Fungal apoptosis: function, genes and gene function.

Cells of all living organisms are programmed to self-destruct under certain conditions. The most well known form of programmed cell death is apoptosis, which is essential for proper development in higher eukaryotes. In fungi, apoptotic-like cell death occurs naturally during aging and reproduction, and can be induced by environmental stresses and exposure to toxic metabolites. The core apoptotic machinery in fungi is similar to that in mammals, but the apoptotic network is less complex and of more ancient origin. Only some of the mammalian apoptosis-regulating proteins have fungal homologs, and the number of protein families is drastically reduced. Expression in fungi of animal proteins that do not have fungal homologs often affects apoptosis, suggesting functional conservation of these components despite the absence of protein-sequence similarity. Functional analysis of Saccharomyces cerevisiae apoptotic genes, and more recently of those in some filamentous species, has revealed partial conservation, along with substantial differences in function and mode of action between fungal and human proteins. It has been suggested that apoptotic proteins might be suitable targets for novel antifungal treatments. However, implementation of this approach requires a better understanding of fungal apoptotic networks and identification of the key proteins regulating apoptotic-like cell death in fungi.