Biology subjects

Del Poeta, M.

Publications and source records attributed to Del Poeta, M..

11 recordsLinked to original sources

bioRxiv2026

Micafungin exposure drives multidrug resistance in Clavispora lusitaniae

Fungal infections are an escalating global health concern, with rare Candida species posing an urgent threat due to emerging multidrug resistance. Clavispora (Candida) lusitaniae is an uncommon pathogen in which multidrug resistance has been documented during antifungal therapy, yet the selective forces driving this phenotype remain unclear. Here, we show that exposure to the echinocandin micafungin (MCF) alone can select for multidrug resistance in C. lusitaniae. Through controlled evolution experiments we identified individual point mutations in genes encoding ergosterol biosynthesis enzymes (ERGs), sterol trafficking proteins (OSH2), and the echinocandin drug target (FKS1) that confer a significant fitness benefit to one or more classes of antifungals. We find that ERG3 loss-of-function is the primary and independent driver of pan-antifungal resistance to echinocandins, azoles and polyenes. The ERG3 mutants have <1% ergosterol, increased levels of non-toxic sterol intermediates, and increased chitin content, consistent with both cell membrane and cell wall remodeling that enables the fungal pathogen to evade all three drug classes. The convergence of sterol reprogramming and compensatory cell wall remodeling that occurs during adaptation to echinocandin monotherapy can evolve through a single point mutation and parallels our recent case study of acquired multidrug resistance. IMPORTANCEMultidrug resistance in Candida species severely limits treatment options and increases mortality, particularly in immunocompromised patients. Despite increasing reports of multidrug resistance, the molecular mechanisms driving multidrug resistance remain poorly understood. We find that in vitro MCF exposure alone can drive multidrug resistance in C. lusitaniae via acquisition of de novo point mutations in ERG3, an observation that parallels our recent patient case study. By identifying causative mutations and associated physiological changes, we provide mechanistic insight into the emergence of multidrug resistance and highlight the need for surveillance strategies that account for resistance evolution under echinocandin monotherapy.

microbiology↗

bioRxiv2026

α-1,3-Glucan-Driven Remodeling of the Conidial Cell Wall in an Aspergillus fumigatus Vaccine Strain Alters Innate Immune Recognition

Aspergillus fumigatus is a major cause of invasive aspergillosis in immunocompromised patients, where current antifungal therapies are limited by toxicity, drug resistance, and lack of durable protection, and no vaccines are available. A mutant lacking the sterylglucosidase-encoding gene (sglA) has emerged as a candidate that induces protective immune responses, but the structural basis for this phenotype remains unclear. Here, we use cellular solid-state NMR spectroscopy to compare the organization of the conidial cell wall in {Delta}sglA and its wild-type counterpart. The {Delta}sglA conidial cell wall displays extensive remodeling, including increased -1,3-glucan content and structural polymorphism, strengthened interactions with {beta}-glucans, reduced hydration, and restricted molecular motion, together consolidating a more rigid scaffold with limited {beta}-glucan accessibility. These structural changes are associated with altered neutrophil responses and a shift in innate immune signaling. This work links cell-wall reorganization to altered immune recognition in this vaccine candidate, with implications for future immunotherapeutic strategies. TEASERMolecular-level Insights from a fungal vaccine candidate show how cell-wall remodeling could affect immune response.

biochemistry↗

bioRxiv2025

Sphingolipid Homeostasis, Mitochondrial Activity, and PKA Signaling Drive an Azole-Tolerant State

Antifungal therapies frequently fail, resulting in persistent infections caused by the important opportunistic pathogen, Candida albicans, which is highly tolerant to azole drugs. Tolerance arises from a subpopulation of cells that survive prolonged drug exposure, yet the underlying mechanisms remain poorly defined. Here, transcription factor mutant library screens identified Mnl1 as a key repressor of tolerance. Loss of MNL1 elevated cAMP levels and activated the PKA pathway, linking Mnl1 to growth control and stress responses. Additionally, mnl1 cells exhibited altered sphingolipid composition, implicating Mnl1 in regulating membrane permeability, a key determinant of drug efflux and membrane integrity. Increased tolerance in mnl1 cells was also linked to increased mitochondrial function. Thus, Mnl1 regulates tolerance through coordinated control of sphingolipid metabolism, mitochondrial activity, and PKA signaling, highlighting that tolerant cells adopt a distinct physiological state primed to withstand antifungal stress.

microbiology↗

bioRxiv2024

Step-wise evolution of azole resistance through copy number variation followed by KSR1 loss of heterozygosity in Candida albicans.

Antimicrobial drug resistance poses a global health threat, requiring a deeper understanding of the evolutionary processes that lead to its emergence in pathogens. Complex evolutionary dynamics involve multiple mutations that can result in cooperative or competitive (clonal interference) effects. Candida albicans, a major fungal pathogen, displays high rates of copy number variation (CNV) and loss of heterozygosity (LOH). CNV and LOH events involve large numbers of genes and could synergize during evolutionary adaptation. Understanding the contributions of CNV and LOH to antifungal drug adaptation is challenging, especially in the context of whole-population genome sequencing. Here, we document the sequential evolution of fluconazole tolerance and then resistance in a C. albicans isolate involving an initial CNV on chromosome 4, followed by an LOH on chromosome R that involves KSR1. Similar LOH events involving KSR1, which encodes a reductase involved in sphingolipid biosynthesis, were also detected in independently evolved fluconazole resistant isolates. We dissect the specific KSR1 codons that affect fluconazole resistance and tolerance. The combination of the chromosome 4 CNV and KSR1 LOH results in a >500-fold increase in azole resistance, illustrating a compelling example of rapid, yet step-wise, interplay between CNV and LOH in drug resistance evolution.

microbiology↗

bioRxiv2022

Three models of vaccination strategies against cryptococcosis in immunocompromised hosts using heat-killed Cryptococcus neoformans Δsgl1

Vaccines are one of the greatest medical accomplishments to date, yet no fungal vaccines are currently available in humans mainly because opportunistic mycoses generally occur during immunodeficiencies necessary for vaccine protection. In previous studies, a live, attenuated Cryptococcus neoformans {Delta}sgl1 mutant accumulating sterylglucosides was found to be avirulent and protected mice from a subsequent lethal infection even in absence of CD4+ T cells, a condition most associated with cryptococcosis (e.g., HIV). Here, we tested three strategies of vaccination against cryptococcosis. First, in our preventative model, protection was achieved even after a 3-fold increase of the vaccination window. Second, because live C. neoformans {Delta}sgl1-vaccinated mice challenged more than once with WT strain had a significant decrease in lung fungal burden, we tested C. neoformans {Delta}sgl1 as an immunotherapeutic. We found that therapeutic administrations of HK C. neoformans {Delta}sgl1 subsequent to WT challenge significantly improve the lung fungal burden. Similarly, therapeutic administration of HK C. neoformans {Delta}sgl1 post WT challenge resulted in 100% or 70% survival depending on the time of vaccine administration, suggesting that HK {Delta}sgl1 is a robust immunotherapeutic option. Third, we investigated a novel model of vaccination in preventing reactivation from lung granuloma using C. neoformans {Delta}gcs1. Remarkably, we show that administration of HK {Delta}sgl1 prevents mice from reactivating {Delta}gcs1 upon inducing immunosuppression with corticosteroids or by depleting CD4+ T cells. Our results suggest that HK {Delta}sgl1 represents a clinically relevant, efficacious vaccine that confers robust host protection in three models of vaccination against cryptococcosis even during CD4-deficiency. ImportanceCryptococcosis results in [~]180,000 global deaths per year in immunocompromised individuals. Current antifungal treatment options are potentially toxic, lacking in areas of need, and exhibit limited efficacy. In addition to these lackluster therapeutic options, no fungal vaccines are currently available for clinical use. Due to the increasing rate of immunocompromised individuals, there is a dire need for the development of improved antifungal therapeutics. Presently, we have demonstrated the high efficacy of a clinically relevant heat-killed mutant strain of Cryptococcus neoformans in inducing advantageous host protection in three models of vaccination against cryptococcosis during immunodeficiencies most associated with this disease.

microbiology↗

bioRxiv2021

Microdomain protein Nce102 is a local sensor of plasma membrane sphingolipid balance

Sphingolipids are essential building blocks of eukaryotic membranes and important signalling molecules, tightly regulated in response to environmental and physiological inputs. Mechanism of sphingolipid level perception at the plasma membrane remains unclear. In Saccharomyces cerevisiae, Nce102 protein has been proposed to function as sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that production of Nce102 increases following sphingolipid biosynthesis inhibition and Nce102 is internalized when excess sphingolipid precursors are supplied. This suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. Physiological role of Nce102 in regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of complex sphingolipids and long-chain bases in nce102{Delta} deletion mutant. Nce102 behaves analogously in human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species.

cell biology↗

bioRxiv2021

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores, whereby macrophages either aid in clearance or serve as a niche for its dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high order plasma membrane domains enriched in cholesterol and sphingomyelin) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages ability to phagocytose C. neoformans. We found that cholesterol or sphingomyelin depletion resulted in significantly deficient IgG-mediated phagocytosis of the fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency while a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable sphingomyelin (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis whereas raft-inhibiting conformation (cis-AzoSM) significantly blunted phagocytosis in a reversible manner. We observed that the effect on phagocytosis may be mediated by facilitating Fc{gamma} receptor (Fc{gamma}R) function, whereby IgG immune complexes cross-link to Fc{gamma}RIII, resulting in tyrosine phosphorylation of FcR {gamma}-subunit (FcR{gamma}), an important accessory protein in the Fc{gamma}R signaling cascade. Correspondingly, cholesterol or sphingomyelin depletion resulted in decreased FcR{gamma} phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcR{gamma} phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating Fc{gamma}RIII-mediated phagocytosis of C. neoformans.

microbiology↗

bioRxiv2021

Screening of chemical libraries for new antifungal drugs against Aspergillus fumigatus reveals the potential mechanism of action of miltefosine

Aspergillus fumigatus is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation called invasive pulmonary aspergillosis (IPA) in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant A. fumigatus isolates has significantly increased over the last decades. Therefore, new strategies are necessary to combat aspergillosis and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze A. fumigatus in vitro susceptibility to 1,127 compounds. A. fumigatus was more susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against A. fumigatus. By screening an A. fumigatus transcription factor null library, we identified a single mutant, which has the rmiA (resistant to miltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and the {Delta}rmiA strains and the Chromatin Immunoprecipitation coupled to next generation sequencing (ChIP-Seq) of a RmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipids pathway that are directly or indirectly regulated by RmiA. Sphingolipids analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of RmiA represents the first genetic element described and characterized which plays a direct role in miltefosine response in fungi. Author summaryThe filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over the last decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungals agents. By using a repurposing strategy, we identified 10 different compounds that impact A. fumigatus survival. One of these compounds, miltefosine, demonstrated fungicidal activity against A. fumigatus. The mechanism of action of miltefosine is unknown and aiming to get more insights about it, we identified a transcription factor RmiA (Resistant to miltefosine) important for miltefosine resistance. Our results suggest that miltefosine plays antifungal activity against A. fumigatus interfering in the sphingolipids biosynthesis.

microbiology↗

bioRxiv2021

Extracellular vesicles regulate yeast growth, biofilm formation, and yeast-to-hypha differentiation in Candida albicans

The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on virulence. In this study, we investigated the influence of fungal extracellular vesicles (EVs) during yeast growth, biofilm formation, and morphogenesis in C. albicans. Addition of C. albicans EVs (Ca EVs) to the culture medium positively affected yeast growth. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that Ca EVs inhibited biofilm formation by C. albicans in vitro. By time-lapse microscopy and SEM, we showed that Ca EV-treatment stops filamentation promoting pseudohyphae formation with multiple sites for yeast budding. The ability of Ca EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. Ca EVs from distinct strains robustly inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 hours. A minor inhibitory effect was promoted by EVs from S. cerevisiae and H. capsulatum only after 24 hours of incubation. The inhibitory effect of Ca EVs was promoted by a combination of lipid compounds identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, Ca EVs were also able to reverse filamentation, transforming hyphal growth to yeast forms. Transcriptomic analysis demonstrated that treatment with Ca EVs modified the expression of more than 300 genes. The most effectively upregulated pathways were related to DNA metabolism. The downregulated genes were mostly associated with extracellular and adhesion proteins. Finally, yeast cells treated with Ca EVs for 24 hours lost their agar invasive ability and were avirulent when inoculated in Galleria mellonella larvae. In summary, our results indicate that fungal EVs can profoundly modify C. albicans growth and regulate yeast-to-hypha differentiation inhibiting biofilm formation and virulence.

microbiology↗

bioRxiv2020

Internalization of the host alkaline pH signal in a fungal pathogen

The ability for cells to internalize extracellular cues allows them to adapt to novel and stressful environments. This adaptability is especially important for microbial pathogens that must sense and respond to drastic changes when encountering the human host. Cryptococcus neoformans is an environmental fungus and opportunistic pathogen that naturally lives in slightly acidic reservoirs, but must adapt to the relative increase in alkalinity in the human host in order to effectively cause disease. The fungal-specific Rim alkaline response signaling pathway effectively converts this extracellular signal into an adaptive cellular response allowing the pathogen to survive in its new environment. The newly identified Rra1 protein, the most upstream component of the C. neoformans Rim pathway, is an essential component of this alkaline response. Previous work connected Rra1-mediated signaling to the dynamics of the plasma membrane. Here we identify the specific mechanisms of Rim pathway signaling through detailed studies of the activation of the Rra1 protein. Specifically, we observe that the Rra1 protein is internalized and recycled in a pH-dependent manner, and that this dynamic pattern of localization further depends on specific residues in its C-terminal tail, clathrin-mediated endocytosis, and the integrity of the plasma membrane. The data presented here continue to unravel the complex and intricate processes of pH-sensing in a relevant human fungal pathogen. These studies will further elucidate general mechanisms by which cells respond to and internalize extracellular stress signals. Author SummaryThe work described here explores the genetics and mechanics of a cellular signaling pathway in a relevant human fungal pathogen, Cryptococcus neoformans. The findings presented in this manuscript untangle the complex interactions involved in the activation of a fungal-specific alkaline response pathway, the Rim pathway. Specifically, we find that C. neoformans is able to sense an increase in pH within the human host, internalize a membrane-bound pH-sensor, and activate a downstream signaling pathway enabling this pathogen to adapt to a novel host environment and effectively cause disease. Revealing the mechanisms of Rim pathway activation within the larger context of the fungal cell allows us to understand how and when this microorganism interprets relevant host signals. Furthermore, understanding how this pathogenic organism converts extracellular stress signals into an adaptive cellular response will elucidate more general mechanisms of microbial environmental sensing and stress response.

cell biology↗

bioRxiv2020

Fenbendazole controls in vitro growth, virulence potential and animal infection in the Cryptococcusmodel

The human diseases caused by the fungal pathogens Cryptococcus neoformans and C. gattii are associated with high indices of mortality, and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anti-cryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anti-cryptococcal benzimidazole. Fenbendazole was inhibitory against 30 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anti-cryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anti-cryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mice model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

microbiology↗