Biology subjects

Brown, A. J. P.

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

5 recordsLinked to original sources

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↗