Biology subjects

Fernandes, C. M.

Publications and source records attributed to Fernandes, C. M..

5 recordsLinked to original sources

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↗

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↗

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

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↗

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↗