Biology subjects

Brown, A. J. P.

Publications and source records attributed to Brown, A. J. P..

8 recordsLinked to original sources

bioRxiv2026

FKS1/2-variant independent mechanisms underlying the emergence of resistance in echinocandin-refractory Candida auris infections

The emerging fungus Candida auris is a drug resistant global public health threat and WHO critical priority pathogen. Recommended first-line invasive candidiasis treatment is echinocandin monotherapy, but C. auris can develop on-treatment resistance via FKS1/2 gene mutations and additional, previously unexplained mechanisms. To better understand echinocandin failure in C. auris, we sequenced the genomes of echinocandin refractory FKS1/2 wild-type C. auris serial isolates from two critically unwell patients in London, UK. Population analysis profiling revealed echinocandin heteroresistance, and in vitro culture of clinical isolates at supra-MIC concentrations of anidulafungin (8 g/ml) exhibited morphotypic heterogeneity. Small colony variants (SCVs) and large colony variants (LCVs) showed elevated MICs with polyploidy (to 4n and above) alongside adaptive changes in cell wall {beta}-1,3-glucan content. LCVs contained significantly more mutations in calcineurin-related stress tolerance pathway gene CRZ1 compared to clinical parents and SCVs, associated with further increases in MIC. These findings indicate progressive step-wise accrual of adaptation to echinocandins, including genomic instability, alterations in stress tolerance pathways, and cell wall remodeling, paving the way for resistance emergence. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/700071v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@14460b7org.highwire.dtl.DTLVardef@29a598org.highwire.dtl.DTLVardef@36d187org.highwire.dtl.DTLVardef@f62c8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Lactic acid influences iron assimilation by a fungal pathogen via the iron reductive uptake pathway

Candida albicans is a fungal commensal of humans that often causes mucosal infections in otherwise healthy individuals, and also serious infections in immunocompromised patients. The capacity of this fungus to colonise and cause disease relies on its ability to grow within the host, adapting to various nutrient restrictions and physicochemical conditions. The presence of alternative carbon sources, such as the lactate produced by the local microbiota, influences C. albicans antifungal drug resistance and immune evasion. In this study, we used genome-wide transcriptomic analysis to investigate the effect of lactate exposure upon metabolic rewiring. We provide evidence that C. albicans cells respond to growth in the presence of lactate at pH 5 by regulating genes encoding micronutrient transporters, notably iron transporters. More specifically, lactate triggers the downregulation of genes on the reductive iron uptake pathway, inferring a diminished requirement for high-affinity iron uptake. This is supported by the observation that lactate promotes the intracellular accumulation of iron by C. albicans cells. Lactate even enhances the growth of iron-transport defective C. albicans cells under iron-limited conditions. Lactate is known to activate protein kinase A (PKA) signalling. However, lactate-induced iron assimilation is PKA-independent. This work provides new insights into the role of lactate in iron homeostasis - two important factors that promote C. albicans virulence in the mammalian host, where nutritional immunity is a key antimicrobial strategy. ImportanceCandida albicans is a major opportunistic fungal pathogen capable of causing life- threatening infections, particularly in immunocompromised individuals. Its ability to adapt to diverse host environments underlies its success as a commensal and pathogen. This study provides new insights into the metabolic flexibility of C. albicans, with a specific focus on how lactate, a common carbon source in host niches, influences iron acquisition and homeostasis. Our findings reveal that, during growth at pH 5, lactate modulates the expression of micronutrient transporters and enhances iron assimilation in C. albicans. These results suggest a role of lactate in promoting iron uptake, potentially facilitating fungal colonization and persistence within the host. By elucidating the molecular and phenotypic consequences of lactate exposure upon iron metabolism, this study contributes to a deeper understanding of host-pathogen interactions.

cell biology↗

bioRxiv2025

Expansion, functional diversification and gene fusion events in the Ato protein family

Candida albicans, a commensal opportunistic pathogen, exhibits remarkable metabolic flexibility and adaptability to environmental changes. In glucose-limited niches, it utilizes alternative carbon sources such as carboxylic acids, which may influence its pathogenicity. In Saccharomyces cerevisiae, the uptake of monocarboxylates occurs through regulated plasma membrane (PM) transport proteins, such as Ato1 (Ady2), which belongs to the Acetate Uptake Transporter (AceTr) family. In C. albicans, these proteins are notably expanded, consisting of ten Ato-like proteins (ATO1-ATO10), whose functions remain unknown. Here, we investigated the role of Ato proteins in carboxylic acid utilization by C. albicans using in-silico and functional analysis. Our data revealed that several C. albicans Atos retain conserved AceTr motifs but possess distinct structural features, including differences in pore radius and binding sites for acetate and lactate. Expression analysis revealed that Ato1, Ato2, Ato3, and Ato6 exhibit distinct cellular localization and expression levels on the plasma membrane, depending on the presence or absence of monocarboxylates. Remarkably, deletion of ATO1 impaired Ato2 and Ato3 expression and caused ER retention of a distinct form of Ato2, suggesting a central regulatory role for Ato1 in the Ato transport system. Finally, we identified a novel Ato-related protein family in vertebrates. This family has three consecutive 6-helix transport domains and a unique C-terminal fusion with Sua5/YciO/YrdC, an enzyme involved in tRNA modification. Overall, our data suggests that the Ato protein family might play a critical role in the utilization of acetic or lactic acids in C. albicans. It also proposes potential functional redundancy among its members, which may contribute to rapid environmental adaptation and pathogenicity.

microbiology↗

bioRxiv2023

Characterising phagocytes and measuring phagocytosis from live Galleria mellonella larvae

Over the last 20 years, the larva of the greater waxmoth, Galleria mellonella, has rapidly increased in popularity as an in vivo mammalian replacement model organism for the study of human pathogens. Despite this, experimental readouts of response to infection are generally limited to observing the melanisation cascade - where the organism turns black as part of the systemic immune response - and quantifying larval death over time. As an invertebrate, Galleria harbour an innate immune system comprised of both humoral components and a repertoire of innate immune cells - termed hemocytes. Though information on subtypes of hemocytes exist, there are conflicting reports on their exact number and function. Flow cytometry has previously been used to assay Galleria hemocytes, but protocols include both centrifugation and fixation - physical methods which have the potential to affect hemocyte morphology prior to analysis. Here, we present a method for live hemocyte analysis by flow cytometry, revealing that Galleria hemocytes constitute only a single resolvable population, based on relative size or internal complexity. Using fluorescent zymosan particles, we extend our method to show that up to 80% of the Galleria hemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates in vitro data, showing that cell wall {beta}-1,3-glucan masking by Candida albicans subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in Galleria.

cell biology↗

bioRxiv2023

METABOLIC MODELLING AS A POWERFUL TOOL TO IDENTIFY CRITICAL COMPONENTS OF THE PNEUMOCYSTIS GROWTH MEDIUM

Establishing suitable in vitro culture conditions for microorganisms is crucial for dissecting their biology and empowering potential applications. However, a significant number of bacterial and fungal species, including Pneumocystis jirovecii, remain unculturable, hampering research efforts. P. jirovecii is a deadly pathogen of humans that causes life-threatening pneumonia in immunocompromised individuals and transplant patients. Despite the major impact of Pneumocystis on human health, we remain ignorant about the pathobiology of this fungus. This is largely due to the fact that its experimental dissection has been constrained by the inability to culture the organism in vitro. We present a comprehensive in silico genome-scale metabolic model of Pneumocystis growth and metabolism, with a view to identifying metabolic requirements and imbalances that hinder growth in vitro. We utilise recently published genome data and available information in the literature as well as bioinformatics and software tools to develop and validate the model. In addition, we employ Flux Balance Analysis and Reinforcement Learning approaches to make predictions regarding metabolic fluxes and to identify critical components of the Pneumocystis growth medium. Our findings offer insights into the biology of Pneumocystis and provide a novel strategy to overcome the longstanding challenge of culturing this pathogen in vitro.

systems biology↗

bioRxiv2023

Candida auris undergoes adhesin-dependent and -independent cellular aggregation

Candida auris is a fungal pathogen of humans responsible for nosocomial infections with high mortality rates. High levels of resistance to antifungal drugs and environmental persistence mean these infections are difficult to treat and eradicate from a healthcare setting. Understanding the life cycle and the genetics of this fungus underpinning clinically relevant traits, such as antifungal resistance and virulence, is of the utmost importance to develop novel treatments and therapies. Epidemiological and genomic studies have identified five geographical clades (I-V), which display phenotypic and genomic differences. Aggregation of cells, a phenotype primarily of clade III strains, has been linked to reduced virulence in mouse and Galleria mellonella infection models. The aggregation phenotype has thus been associated with conferring an advantage for (skin) colonisation rather than for systemic infection. However, strains with different clade affiliations were compared to infer the effects of different morphologies on virulence. This makes it difficult to distinguish morphology-dependent causes from clade-specific or even strain-specific genetic factors. Here, we identify two different types of aggregation: one induced by antifungal treatment which is a result of a cell separation defect; and a second which is controlled by growth conditions and only occurs in strains with the ability to aggregate. The latter aggregation type depends on an ALS-family adhesin which is differentially expressed during aggregation in an aggregative C. auris strain. Finally, we demonstrate that macrophages cannot clear aggregates, suggesting that aggregation might after all provide a benefit during systemic infection and could facilitate long-term persistence in the host. Author SummaryCandida auris is a single-celled fungus, a yeast, that can cause severe infections in hospital patients. This fungus is difficult to treat because it is resistant to many antifungal drugs. Therefore, to understand the processes that enhance the virulence of this yeast with a view to developing new treatments. Previous studies have found that C. auris can form aggregates, or clumps of cells, which may play a role in how the fungus infects people. In this study, we identified two different types of aggregation in C. auris, one triggered by antifungal treatment, and another controlled by growth conditions. This discovery allowed us to study aggregate formation in the same genetic background. In doing so, we found that a certain protein, an ALS-family adhesin, is involved in the aggregation process. Surprisingly, we also discovered that aggregates may promote infection by making it harder for the immune system to clear the yeast. This new understanding could help researchers develop better ways to fight C. auris infections.

microbiology↗

bioRxiv2022

Heightened efficacy of anidulafungin when used in combination with manogepix or 5-flucytosine against Candida auris in vitro

Candida auris is an emerging, multi-drug resistant fungal pathogen that causes refractory colonisation and life-threatening invasive nosocomial infections. The high proportion of C. auris isolates that display antifungal resistance severely limits treatment options. Combination therapies provide a possible strategy to enhance antifungal efficacy and prevent the emergence of further resistance. Therefore, we examined drug combinations using antifungals that are already in clinical use or undergoing clinical trials. Using checkerboard assays we screened combinations of 5-flucytosine and manogepix (the active form of the novel antifungal drug fosmanogepix) with anidulafungin, amphotericin B or voriconazole against drug resistant and susceptible C. auris isolates from clades I and III. Fractional inhibitory concentration indices (FICI values) of 0.28-0.75 and 0.36-1.02 were observed for combinations of anidulafungin with manogepix or 5-flucytosine, respectively, indicating synergistic activity. The high potency of these anidulafungin combinations was confirmed using live-cell microfluidics-assisted imaging of fungal growth. In summary, combinations of anidulafungin with manogepix or 5-flucytosine show great potential against both resistant and susceptible C. auris isolates.

microbiology↗

bioRxiv2022

CRISPR-based tools for genetic manipulation in pathogenic Sporothrix species

Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis. Although certain virulence factors have been proposed as potential sporotrichosis determinants, the scarcity of molecular tools for reverse genetics studies on Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous expression in S. brasiliensis, S. schenckii and S. chilensis. Combined with CRISPR/Cas9 gene editing, this transformation protocol allowed the deletion of the putative DHN-melanin synthase gene pks1, which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU80 homologue that is critical for non-homologous end-joining DNA repair. The use of S. brasiliensis {Delta}ku80 strains enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species.

molecular biology↗