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.

Multiple phosphorylation sites regulate the activity of the repressor Mig1 in Candida albicans.

IMPORTANCE: The SNF1 protein kinase signaling pathway, which is highly conserved in eukaryotic cells, is important for metabolic adaptations in the pathogenic yeast Candida albicans. However, so far, it has remained elusive how SNF1 controls the activity of one of its main effectors, the repressor protein Mig1 that inhibits the expression of genes required for the utilization of alternative carbon sources when glucose is available. In this study, we have identified multiple phosphorylation sites in Mig1 that contribute to its inactivation. Mutation of these sites strongly increased Mig1 repressor activity in the absence of SNF1, but SNF1 could still sufficiently inhibit the hyperactive Mig1 to enable growth on alternative carbon sources. These findings reveal features of Mig1 that are important for controlling its repressor activity. Furthermore, they demonstrate that both SNF1 and additional protein kinases regulate Mig1 in this pathogenic yeast.

The influenza A virus promotes fungal growth of Aspergillus fumigatus via direct interaction in vitro.

Seasonal influenza A virus (IAV) infections still pose a major burden for public health worldwide. Severe disease progression or even death is often related to superinfections of the virus and a secondary bacterial pathogen. However, fungi, especially Aspergillus fumigatus, are also frequently diagnosed during IAV infection. Although, clinical studies have reported the severity of influenza-associated pulmonary aspergillosis, the molecular mechanisms underlying this type of disease are poorly understood. Here, a new in vitro model is introduced that allows the investigation of complex pathogen-host and pathogen-pathogen interactions during coinfection of lung epithelial cells with IAV and A. fumigatus. Our data reveal a reduced IAV load and IAV-induced cytokine and chemokine expression in the presence of A. fumigatus. At the same time, IAV infection promotes the growth of A. fumigatus. Even in the absence of the human host cell, purified IAV particles are able to induce hyphal growth, due to a direct interaction of the virus particles with the fungal surface. Thus, our study gives first insights into the complex interplay between IAV, A. fumigatus and the host cell as well as the two pathogens alone.

Distinct transcriptional responses to fludioxonil in Aspergillus fumigatus and its ΔtcsC and Δskn7 mutants reveal a crucial role for Skn7 in the cell wall reorganizations triggered by this antifungal.

BACKGROUND: Aspergillus fumigatus is a major fungal pathogen that causes severe problems due to its increasing resistance to many therapeutic agents. Fludioxonil is a compound that triggers a lethal activation of the fungal-specific High Osmolarity Glycerol pathway. Its pronounced antifungal activity against A. fumigatus and other pathogenic molds renders this agent an attractive lead substance for the development of new therapeutics. The group III hydride histidine kinase TcsC and its downstream target Skn7 are key elements of the multistep phosphorelay that represents the initial section of the High Osmolarity Glycerol pathway. Loss of tcsC results in resistance to fludioxonil, whereas a Δskn7 mutant is partially, but not completely resistant. RESULTS: In this study, we compared the fludioxonil-induced transcriptional responses in the ΔtcsC and Δskn7 mutant and their parental A. fumigatus strain. The number of differentially expressed genes correlates well with the susceptibility level of the individual strains. The wild type and, to a lesser extend also the Δskn7 mutant, showed a multi-faceted stress response involving genes linked to ribosomal and peroxisomal function, iron homeostasis and oxidative stress. A marked difference between the sensitive wild type and the largely resistant Δskn7 mutant was evident for many cell wall-related genes and in particular those involved in the biosynthesis of chitin. Biochemical data corroborate this differential gene expression that does not occur in response to hyperosmotic stress. CONCLUSIONS: Our data reveal that fludioxonil induces a strong and TcsC-dependent stress that affects many aspects of the cellular machinery. The data also demonstrate a link between Skn7 and the cell wall reorganizations that foster the characteristic ballooning and the subsequent lysis of fludioxonil-treated cells.

Selection of cross-reactive T cells by commensal and food-derived yeasts drives cytotoxic TH1 cell responses in Crohn's disease.

Aberrant CD4+ T cell reactivity against intestinal microorganisms is considered to drive mucosal inflammation in inflammatory bowel diseases. The disease-relevant microbial species and the corresponding microorganism-specific, pathogenic T cell phenotypes remain largely unknown. In the present study, we identified common gut commensal and food-derived yeasts, as direct activators of altered CD4+ T cell reactions in patients with Crohn's disease (CD). Yeast-responsive CD4+ T cells in CD display a cytotoxic T helper cell (TH1 cell) phenotype and show selective expansion of T cell clones that are highly cross-reactive to several commensal, as well as food-derived, fungal species. This indicates cross-reactive T cell selection by repeated encounter with conserved fungal antigens in the context of chronic intestinal disease. Our results highlighted a role of yeasts as drivers of aberrant CD4+ T cell reactivity in patients with CD and suggest that both gut-resident fungal commensals and daily dietary intake of yeasts might contribute to chronic activation of inflammatory CD4+ T cell responses in patients with CD.

A phylogenetic approach to explore the Aspergillus fumigatus conidial surface-associated proteome and its role in pathogenesis.

Aspergillus fumigatus , an important pulmonary fungal pathogen causing several diseases collectively called aspergillosis, relies on asexual spores or conidia for initiating host infection. Here, we used a phylogenomic approach to compare proteins in the conidial surface of A. fumigatus , two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis , and the cryptic pathogen Aspergillus lentulus . After identifying 62 proteins uniquely expressed on the A. fumigatus conidial surface, we deleted 42 genes encoding conidial proteins. We found deletion of 33 of these genes altered susceptibility to macrophage killing, penetration and damage to epithelial cells, and cytokine production. Notably, a gene that encodes glycosylasparaginase, which modulates levels of the host pro-inflammatory cytokine IL-1ß, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins and effectors are important for evasion and modulation of the immune response at the onset of fungal infection.

Bacterial secretion systems contribute to rapid tissue decay in button mushroom soft rot disease.

The soft rot pathogen Janthinobacterium agaricidamnosum causes devastating damage to button mushrooms (Agaricus bisporus), one of the most cultivated and commercially relevant mushrooms. We previously discovered that this pathogen releases the membrane-disrupting lipopeptide jagaricin. This bacterial toxin, however, could not solely explain the rapid decay of mushroom fruiting bodies, indicating that J. agaricidamnosum implements a more sophisticated infection strategy. In this study, we show that secretion systems play a crucial role in soft rot disease. By mining the genome of J. agaricidamnosum, we identified gene clusters encoding a type I (T1SS), a type II (T2SS), a type III (T3SS), and two type VI secretion systems (T6SSs). We targeted the T2SS and T3SS for gene inactivation studies, and subsequent bioassays implicated both in soft rot disease. Furthermore, through a combination of comparative secretome analysis and activity-guided fractionation, we identified a number of secreted lytic enzymes responsible for mushroom damage. Our findings regarding the contribution of secretion systems to the disease process expand the current knowledge of bacterial soft rot pathogens and represent a significant stride toward identifying targets for their disarmament with secretion system inhibitors. IMPORTANCE The button mushroom (Agaricus bisporus) is the most popular edible mushroom in the Western world. However, mushroom crops can fall victim to serious bacterial diseases that are a major threat to the mushroom industry, among them being soft rot disease caused by Janthinobacterium agaricidamnosum. Here, we show that the rapid dissolution of mushroom fruiting bodies after bacterial invasion is due to degradative enzymes and putative effector proteins secreted via the type II secretion system (T2SS) and the type III secretion system (T3SS), respectively. The ability to degrade mushroom tissue is significantly attenuated in secretion-deficient mutants, which establishes that secretion systems are key factors in mushroom soft rot disease. This insight is of both ecological and agricultural relevance by shedding light on the disease processes behind a pathogenic bacterial-fungal interaction which, in turn, serves as a starting point for the development of secretion system inhibitors to control disease progression.

Genome-scale metabolic modeling of Aspergillus fumigatus strains reveals growth dependencies on the lung microbiome.

Aspergillus fumigatus, an opportunistic human pathogen, frequently infects the lungs of people with cystic fibrosis and is one of the most common causes of infectious-disease death in immunocompromised patients. Here, we construct 252 strain-specific, genome-scale metabolic models of this important fungal pathogen to study and better understand the metabolic component of its pathogenic versatility. The models show that 23.1% of A. fumigatus metabolic reactions are not conserved across strains and are mainly associated with amino acid, nucleotide, and nitrogen metabolism. Profiles of non-conserved reactions and growth-supporting reaction fluxes are sufficient to differentiate strains, for example by environmental or clinical origin. In addition, shotgun metagenomics analysis of sputum from 40 cystic fibrosis patients (15 females, 25 males) before and after diagnosis with an A. fumigatus colonization suggests that the fungus shapes the lung microbiome towards a more beneficial fungal growth environment associated with aromatic amino acid availability and the shikimate pathway. Our findings are starting points for the development of drugs or microbiome intervention strategies targeting fungal metabolic needs for survival and colonization in the non-native environment of the human lung.

Streptomyces polyketides mediate bacteria-fungi interactions across soil environments.

Although the interaction between prokaryotic and eukaryotic microorganisms is crucial for the functioning of ecosystems, information about the processes driving microbial interactions within communities remains scarce. Here we show that arginine-derived polyketides (arginoketides) produced by Streptomyces species mediate cross-kingdom microbial interactions with fungi of the genera Aspergillus and Penicillium, and trigger the production of natural products. Arginoketides can be cyclic or linear, and a prominent example is azalomycin F produced by Streptomyces iranensis, which induces the cryptic orsellinic acid gene cluster in Aspergillus nidulans. Bacteria that synthesize arginoketides and fungi that decode and respond to this signal were co-isolated from the same soil sample. Genome analyses and a literature search indicate that arginoketide producers are found worldwide. Because, in addition to their direct impact, arginoketides induce a secondary wave of fungal natural products, they probably contribute to the wider structure and functioning of entire soil microbial communities.

Influenza Virus-Induced Paracrine Cellular Senescence of the Lung Contributes to Enhanced Viral Load.

Aging is a major risk factor associated with increased morbidity and mortality rates observed during respiratory infections. In this study, we investigated the role of influenza virus infections in the establishment of premature cellular senescence and paracrine macrophage-activated inflammation. We observed in our murine model a premature aging by the appearance of senescent cells in the lungs after 21 d of influenza A virus infection. By using murine ex vivo lung models, the influence of TNF-α on the establishment of cellular senescence was detectable. Our findings were proven by using conditioned media of infected human monocyte-derived macrophages on primary lung fibroblasts. Here, a distinct expression of senescence-associated parameters could be confirmed. Furthermore, senescent cells in the lungs strongly influenced subsequent viral infections. Our data demonstrated a higher viral load in senescent primary lung fibroblasts, indicating an intracellular effect on viral replication. Transcriptomic data revealed an increased regulation of JAK/STAT signaling in senescent IAV-infected cells accompanied with increased TRAIL expression. Additionally, senescent cells indicating low pH values, accelerating viral replication. Our study provides new insights into pathomechanisms of virus-induced cellular senescence. Hence, IAV infection induces premature senescence and subsequent infections in senescent cells lead to an increased viral replication.

Disruption of the Aspergillus fumigatus RNA interference machinery alters the conidial transcriptome.

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

A New Phenotype in Candida-Epithelial Cell Interaction Distinguishes Colonization- versus Vulvovaginal Candidiasis-Associated Strains.

Vulvovaginal candidiasis (VVC) affects nearly 3/4 of women during their lifetime, and its symptoms seriously reduce quality of life. Although Candida albicans is a common commensal, it is unknown if VVC results from a switch from a commensal to pathogenic state, if only some strains can cause VVC, and/or if there is displacement of commensal strains with more pathogenic strains. We studied a set of VVC and colonizing C. albicans strains to identify consistent in vitro phenotypes associated with one group or the other. We find that the strains do not differ in overall genetic profile or behavior in culture media (i.e., multilocus sequence type [MLST] profile, rate of growth, and filamentation), but they show strikingly different behaviors during their interactions with vaginal epithelial cells. Epithelial infections with VVC-derived strains yielded stronger fungal proliferation and shedding of fungi and epithelial cells. Transcriptome sequencing (RNA-seq) analysis of representative epithelial cell infections with selected pathogenic or commensal isolates identified several differentially activated epithelial signaling pathways, including the integrin, ferroptosis, and type I interferon pathways; the latter has been implicated in damage protection. Strikingly, inhibition of type I interferon signaling selectively increases fungal shedding of strains in the colonizing cohort, suggesting that increased shedding correlates with lower interferon pathway activation. These data suggest that VVC strains may intrinsically have enhanced pathogenic potential via differential elicitation of epithelial responses, including the type I interferon pathway. Therefore, it may eventually be possible to evaluate pathogenic potential in vitro to refine VVC diagnosis. IMPORTANCE Despite a high incidence of VVC, we still have a poor understanding of this female-specific disease whose negative impact on women's quality of life has become a public health issue. It is not yet possible to determine by genotype or laboratory phenotype if a given Candida albicans strain is more or less likely to cause VVC. Here, we show that Candida strains causing VVC induce more fungal shedding from epithelial cells than strains from healthy women. This effect is also accompanied by increased epithelial cell detachment and differential activation of the type I interferon pathway. These distinguishing phenotypes suggest it may be possible to evaluate the VVC pathogenic potential of fungal isolates. This would permit more targeted antifungal treatments to spare commensals and could allow for displacement of pathogenic strains with nonpathogenic colonizers. We expect these new assays to provide a more targeted tool for identifying fungal virulence factors and epithelial responses that control fungal vaginitis.

Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway.

The decision whether endosomes enter the degradative or recycling pathway in mammalian cells is of fundamental importance for pathogen killing, and its malfunctioning has pathological consequences. We discovered that human p11 is a critical factor for this decision. The HscA protein present on the conidial surface of the human-pathogenic fungus Aspergillus fumigatus anchors p11 on conidia-containing phagosomes (PSs), excludes the PS maturation mediator Rab7, and triggers binding of exocytosis mediators Rab11 and Sec15. This reprogramming redirects PSs to the non-degradative pathway, allowing A. fumigatus to escape cells by outgrowth and expulsion as well as transfer of conidia between cells. The clinical relevance is supported by the identification of a single nucleotide polymorphism in the non-coding region of the S100A10 (p11) gene that affects mRNA and protein expression in response to A. fumigatus and is associated with protection against invasive pulmonary aspergillosis. These findings reveal the role of p11 in mediating fungal PS evasion.

The Lipid Raft-Associated Protein Stomatin Is Required for Accumulation of Dectin-1 in the Phagosomal Membrane and for Full Activity of Macrophages against Aspergillus fumigatus.

Alveolar macrophages belong to the first line of defense against inhaled conidia of the human-pathogenic fungus Aspergillus fumigatus. In lung alveoli, they contribute to phagocytosis and elimination of conidia. As a counterdefense, conidia have a gray-green pigment that enables them to survive in phagosomes of macrophages for some time. Previously, we showed that this conidial pigment interferes with the formation of flotillin-dependent lipid raft microdomains in the phagosomal membrane, thereby preventing the formation of functional phagolysosomes. Besides flotillins, stomatin is a major component of lipid rafts and can be targeted to the membrane. However, only limited information on stomatin is available, in particular on its role in defense against pathogens. To determine the function of this integral membrane protein, a stomatin-deficient macrophage line was generated by CRISPR/Cas9 gene editing. Immunofluorescence microscopy and flow cytometry revealed that stomatin contributes to the phagocytosis of conidia and is important for recruitment of the ß-glucan receptor dectin-1 to both the cytoplasmic membrane and phagosomal membrane. In stomatin knockout cells, fusion of phagosomes and lysosomes, recruitment of the vATPase to phagosomes, and tumor necrosis factor alpha (TNF-α) levels were reduced when cells were infected with pigmentless conidia. Thus, our data suggest that stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. IMPORTANCE Stomatin is an integral membrane protein that contributes to the uptake of microbes, e.g., spores of the human-pathogenic fungus Aspergillus fumigatus. By generation of a stomatin-deficient macrophage line by advanced genetic engineering, we found that stomatin is involved in the recruitment of the ß-glucan receptor dectin-1 to the phagosomal membrane of macrophages. Furthermore, stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. The data provide new insights on the important role of stomatin in the immune response against human-pathogenic fungi.

Antigen specificity and cross-reactivity drive functionally diverse anti-Aspergillus fumigatus T cell responses in cystic fibrosis.

BACKGROUNDThe fungus Aspergillus fumigatus causes a variety of clinical phenotypes in patients with cystic fibrosis (pwCF). Th cells orchestrate immune responses against fungi, but the types of A. fumigatus-specific Th cells in pwCF and their contribution to protective immunity or inflammation remain poorly characterized.METHODSWe used antigen-reactive T cell enrichment (ARTE) to investigate fungus-reactive Th cells in peripheral blood of pwCF and healthy controls.RESULTSWe show that clonally expanded, high-avidity A. fumigatus-specific effector Th cells, which were absent in healthy donors, developed in pwCF. Individual patients were characterized by distinct Th1-, Th2-, or Th17-dominated responses that remained stable over several years. These different Th subsets target different A. fumigatus proteins, indicating that differential antigen uptake and presentation directs Th cell subset development. Patients with allergic bronchopulmonary aspergillosis (ABPA) are characterized by high frequencies of Th2 cells that cross-recognize various filamentous fungi.CONCLUSIONOur data highlight the development of heterogenous Th responses targeting different protein fractions of a single fungal pathogen and identify the development of multispecies cross-reactive Th2 cells as a potential risk factor for ABPA.FUNDINGGerman Research Foundation (DFG), under Germany's Excellence Strategy (EXC 2167-390884018 "Precision Medicine in Chronic Inflammation" and EXC 2051-390713860 "Balance of the Microverse"); Oskar Helene Heim Stiftung; Christiane Herzog Stiftung; Mukoviszidose Institut gGmb; German Cystic Fibrosis Association Mukoviszidose e.V; German Federal Ministry of Education and Science (BMBF) InfectControl 2020 Projects AnDiPath (BMBF 03ZZ0838A+B).

Polymer-based particles against pathogenic fungi: A non-uptake delivery of compounds.

The therapy of life-threatening fungal infections is limited and needs urgent improvement. This is in part due to toxic side effects of clinically used antifungal compounds or their limited delivery to fungal structures. Until today, it is a matter of debate how drugs or drug-delivery systems can efficiently reach the intracellular lumen of fungal cells and how this can be improved. Here, we addressed both questions by applying two different polymeric particles for delivery of compounds. Their formulation was based on two biocompatible polymers, i.e., poly(lactic-co-glycolic acid)50:50 and poly(methyl methacrylate-stat-methacrylic acid)90:10 yielding particles with hydrodynamic diameters ranging from 100 to 300 nm. The polymers were covalently labeled with the fluorescent dye DY-550 to monitor the interaction between particles and fungi by confocal laser scanning microscopy. Furthermore, the fluorescent dye coumarin-6 and the antifungal drug itraconazole were successfully encapsulated in particles to study the fate of both the cargo and the particle when interacting with the clinically most important human-pathogenic fungi Aspergillus fumigatus, A. terreus, Candida albicans, and Cryptococcus neoformans. While the polymers were exclusively located on the fungal surface, the encapsulated cargo was efficiently transported into fungal hyphae, indicated by increased intracellular fluorescence signals due to coumarin-6. In accordance with this finding, compared to the pristine drug a reduced minimal inhibitory concentration for itraconazole was determined, when it was encapsulated. Together, the herein used polymeric particles were not internalized by pathogenic fungi but were able to efficiently deliver hydrophobic cargos into fungal cells.

Functional modulation of chemical mediators in microbial communities.

Interactions between microorganisms are often mediated by specialized metabolites. Although the structures and biosynthesis of these compounds may have been elucidated, microbes exist within complex microbiomes and chemical signals can thus also be subject to community-dependent modifications. Increasingly powerful chemical and biological tools allow to shed light on this poorly understood aspect of chemical ecology. We provide an overview of loss-of-function and gain-of-function chemical mediator (CM) modifications within microbial multipartner relationships. Although loss-of-function modifications are abundant in the literature, few gain-of-function modifications have been described despite their important role in microbial interactions. Research in this field holds great potential for our understanding of microbial interactions and may also provide novel tools for targeted interference with microbial signaling.

Targeting of phagolysosomes containing conidia of the fungus Aspergillus fumigatus with polymeric particles.

Conidia of the airborne human-pathogenic fungus Aspergillus fumigatus are inhaled by humans. In the lung, they are phagocytosed by alveolar macrophages and intracellularly processed. In macrophages, however, conidia can interfere with the maturation of phagolysosomes to avoid their elimination. To investigate whether polymeric particles (PPs) can reach this intracellular pathogen in macrophages, we formulated dye-labeled PPs with a size allowing for their phagocytosis. PPs were efficiently taken up by RAW 264.7 macrophages and were found in phagolysosomes. When macrophages were infected with conidia prior to the addition of PPs, we found that they co-localized in the same phagolysosomes. Mechanistically, the fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was observed. Increasing concentrations of PPs increased fusion events, resulting in 14% of phagolysosomes containing both conidia and PPs. We demonstrate that PPs can reach conidia-containing phagolysosomes, making these particles a promising carrier system for antimicrobial drugs to target intracellular pathogens. KEY POINTS: • Polymer particles of a size larger than 500 nm are internalized by macrophages and localized in phagolysosomes. • These particles can be delivered to Aspergillus fumigatus conidia-containing phagolysosomes of macrophages. • Enhanced phagolysosome fusion by the use of vacuolin1 can increase particle delivery.

CORK1, A LRR-Malectin Receptor Kinase, Is Required for Cellooligomer-Induced Responses in Arabidopsis thaliana.

Cell wall integrity (CWI) maintenance is central for plant cells. Mechanical and chemical distortions, pH changes, and breakdown products of cell wall polysaccharides activate plasma membrane-localized receptors and induce appropriate downstream responses. Microbial interactions alter or destroy the structure of the plant cell wall, connecting CWI maintenance to immune responses. Cellulose is the major polysaccharide in the primary and secondary cell wall. Its breakdown generates short-chain cellooligomers that induce Ca2+-dependent CWI responses. We show that these responses require the malectin domain-containing CELLOOLIGOMER-RECEPTOR KINASE 1 (CORK1) in Arabidopsis and are preferentially activated by cellotriose (CT). CORK1 is required for cellooligomer-induced cytoplasmic Ca2+ elevation, reactive oxygen species (ROS) production, mitogen-associated protein kinase (MAPK) activation, cellulose synthase phosphorylation, and the regulation of CWI-related genes, including those involved in biosynthesis of cell wall material, secondary metabolites and tryptophan. Phosphoproteome analyses identified early targets involved in signaling, cellulose synthesis, the endoplasmic reticulum/Golgi secretory pathway, cell wall repair and immune responses. Two conserved phenylalanine residues in the malectin domain are crucial for CORK1 function. We propose that CORK1 is required for CWI and immune responses activated by cellulose breakdown products.

Impaired amino acid uptake leads to global metabolic imbalance of Candida albicans biofilms.

Candida albicans biofilm maturation is accompanied by enhanced expression of amino acid acquisition genes. Three state-of-the-art omics techniques were applied to detail the importance of active amino acid uptake during biofilm development. Comparative analyses of normoxic wild-type biofilms were performed under three metabolically challenging conditions: aging, hypoxia, and disabled amino acid uptake using a strain lacking the regulator of amino acid permeases Stp2. Aging-induced amino acid acquisition and stress responses to withstand the increasingly restricted environment. Hypoxia paralyzed overall energy metabolism with delayed amino acid consumption, but following prolonged adaptation, the metabolic fingerprints aligned with aged normoxic biofilms. The extracellular metabolome of stp2Δ biofilms revealed deficient uptake for 11 amino acids, resulting in extensive transcriptional and metabolic changes including induction of amino acid biosynthesis and carbohydrate and micronutrient uptake. Altogether, this study underscores the critical importance of a balanced amino acid homeostasis for C. albicans biofilm development.