Biology subjects

Duvenage, L.

Publications and source records attributed to Duvenage, L..

6 recordsLinked to original sources

bioRxiv2025

Tools for genetic manipulation of the endemic fungal pathogen, Emergomyces africanus, and the application of a fluorescent reporter strain in infection models

AbstractEmergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterisation of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of H. capsulatum were adapted for use in E. africanus. Agrobacterium-mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences, to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall (1,3)-glucan by targeting the transcripts of the -glucan synthase (AGS1). An episomal CRISPR/Cas9 system was evaluated for E. africanus, which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus. ImportanceEmergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognised in recent years and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (1) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (2) methods for knocking down gene expression in E. africanus, and (3) the permanent disruption of genes through CRISPR/Cas9 gene editing.

microbiology↗

bioRxiv2023

The pathogenesis of experimental Emergomycosis in mice.

Emergomyces africanus is a recently identified thermally-dimorphic fungal pathogen that causes disseminated infection in people living with advanced HIV disease. Known as emergomycosis, this disseminated disease is associated with very high case fatality rates. Over the last decade, improved diagnostics and fungal identification in South Africa resulted in a dramatic increase in the number of reported cases. Although the true burden of disease is still unknown, emergomycosis is among the most frequently diagnosed dimorphic fungal infections in Southern Africa; and additional species in the genus have been identified on four continents. Little is known about the pathogenesis and the hosts immune response to this emerging pathogen. Therefore, we established a murine model of pulmonary infection using a clinical isolate, E. africanus (CBS 136260). Both conidia and yeast forms caused pulmonary and disseminated infection in mice with fungus subsequently isolated in culture from lung, spleen, liver, and kidney tissue. Wild-type C57BL/6 mice demonstrated a drop in body weight at two weeks post-infection, corresponding to a peak in fungal burden in the lung, spleen, liver, and kidney. An increase in pro-inflammatory cytokine production was detected in homogenized lung supernatants including IFN-{gamma}, IL-1{beta}, IL-6, IL12-p40 and IL-17 at three- and four-weeks post-infection. Rag-1-deficient mice, lacking mature T and B cells, had an increased fungal burden associated with reduced IFN-{gamma} production. Together our data support a protective T-helper type-1 immune response to E. africanus infection. This may provide a possible explanation for the susceptibility of only a subset of people living with advanced HIV disease despite hypothesized widespread environmental exposure. In summary, we have established a novel murine model of E. africanus disease providing critical insights into the host immune components required for eliminating the infection. Author summaryEmergomyces africanus is a thermally-dimorphic fungus that was recently described as the cause of disseminated infections in persons living with advanced HIV disease in South Africa, where emergomycosis is among the most frequently diagnosed dimorphic fungal diseases. Four additional Emergomyces species have been described causing serious infections around the world, and the infection has a high case fatality rate. Despite the seriousness of this infection, the pathophysiology, immunology, diagnosis, and optimal management of emergomycosis remain largely unknown, hampered by the absence of an animal model. Here, we established a mouse model of pulmonary and disseminated emergomycosis using a clinical isolate of E. africanus with the aim of investigating the host immune response. We found that infection spread from the lungs to other organs within two weeks. We describe the effector immune responses in immune-competent mice aiding in controlled infection. We also highlight the immune-modulating components lacking in immunocompromised mice that promote susceptibilty to infection. Our study provides new insights into the components of the host immune response required for controlling Emergomyces spp. infection.

immunology↗

bioRxiv2021

Cbp1, a rapidly evolving fungal virulence factor, forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel "binocular" fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a rapidly evolving fungal virulence factor that localizes to the cytosol to trigger host cell lysis. Author SummaryThe members of the Ajellomycetaceae fungal family are human pathogens that are responsible for a rising number of mycoses around the world. Calcium binding protein 1 (Cbp1) is a rapidly evolving virulence factor that is present in the genomes of the Ajellomycetaceae species that lead primarily intracellular lifestyles, including Histoplasma, Paracoccidioides, and Emergomyces but not Blastomyces, which remains largely extracellular during infection. Both Paracoccidioides and Histoplasma Cbp1 homologs are able to cause lysis of macrophages whereas Emergomyces homologs cannot. This result is consistent with Emergomyces africanus natural infection of macrophages, during which the yeast cells can replicate but cannot actively lyse the host cell. Despite divergence of the primary sequence of Histoplasma and Paracoccidioides Cbp1 homologs, their protein structures are remarkably similar and reveal a novel fold. During infection, Cbp1 enters the cytosol of the host macrophage, making it the first known virulence factor from an intracellular human fungal pathogen that localizes to the cytosol of the host cell. We also show that Cbp1 forms a complex with another cytosolic virulence factor, Yps-3. Taken together, these studies significantly advance our understanding of Histoplasma virulence.

microbiology↗

bioRxiv2019

An analysis of respiratory function and mitochondrial morphology in Candida albicans

Respiratory function and mitochondrial dynamics have been well characterised in a number of cell types, including the model yeast Saccharomyces cerevisiae, but remain under-researched in fungal pathogens such as Candida albicans. An understanding of mitochondrial activity and morphology is important if we are to understand the role that this organelle plays in adaption and response to stress. Here we examine the respiratory profiles of several prominent pathogenic Candida species and present a useful GFP probe for the study of mitochondrial morphology. We examine mitochondrial morphology under a variety of conditions that Candida species may encounter within the host, such as acidic pH, respiratory and oxidative stress. The GFP probe also allowed for the visualisation of mitochondria during hyphal development, during growth following macrophage engulfment and distribution within biofilms. These data demonstrate that the mitochondrial network of C. albicans is highly responsive to both environmental conditions and developmental cues, suggesting important roles for this organelle in environmental adaption.

cell biology↗

bioRxiv2018

Alternative oxidase induction protects Candida albicans from respiratory stress and promotes hyphal growth

The human fungal pathogen Candida albicans possesses two genes expressing a cyanide-insensitive Alternative Oxidase (Aox) enzymes in addition to classical and parallel electron transfer chains (ETC). In this study, we examine the role of Aox in C. albicans under conditions of respiratory stress, which may be inflicted during its interaction with the human host or co-colonising bacteria. We find that the level of Aox expression is sufficient to modulate resistance to classical ETC inhibition under respiratory stress and are linked to gene expression changes that can promote both survival and pathogenicity. For example we demonstrate that Aox function is important for the regulation of filamentation in C. albicans and observe that cells lacking Aox function lose virulence in a zebrafish infection model. Our investigations also identify that pyocyanin, a phenazine produced by the co-colonising bacterium Pseudomonas aeruginosa, inhibits Aox-based respiration in C. albicans. These results suggest that Aox plays important roles within respiratory stress response pathways which C. albicans may utilise both as a commensal organism and as a pathogen.

microbiology↗

bioRxiv2018

Inhibition of classical and alternative modes of respiration in C. albicans leads to cell wall remodelling and increased macrophage recognition

The human fungal pathogen C. albicans requires respiratory function for normal growth, morphogenesis and virulence. As such the mitochondria represent an enticing target for the development of new antifungal strategies. This possibility is further bolstered by the presence of fungal specific characteristics. However, respiration in C. albicans, as is the case in many fungal organisms, is facilitated by redundant electron transport mechanisms that makes direct inhibition a challenge. In addition, many chemicals known to target the electron transport chain are highly toxic. Here we make use of chemicals with low toxicity in mammals to efficiently inhibit respiration in C. albicans. We find that use of the Nitric Oxide donor, Sodium Nitroprusside (SNP), and the alternative oxidase inhibitor, SHAM, prevent respiration, lead to a loss in viability and to cell wall rearrangements that increase the rate of uptake by macrophages in vitro and in vivo. We propose that SNP+SHAM treatment leads to transcriptional changes that drive cell wall re-arrangement but which also prime cells to activate transition to hyphal growth. In line with this we find that pre-treatment of C. albicans with SNP+SHAM leads to an increase in virulence. Our data reveals strong links between respiration, cell wall remodelling and activation of virulence factors. Our findings also demonstrate that respiration in C. albicans can be efficiently inhibited with chemicals which are not damaging to the mammalian host, but that we need to develop a deeper understanding of the roles of mitochondria in cellular signalling if they are to be developed successfully as a target for new antifungals.\n\nAuthor SummaryCurrent approaches to tackling fungal infections are limited and new targets must be identified to protect against the emergence of resistant strains. We investigate the potential of targeting mitochondria, organelles required for energy production, growth and virulence, in the yeast human fungal pathogen Candida albicans. Our findings suggest that mitochondria can be targeted using drugs that can be tolerated by humans and that this treatment enhances their recognition by immune cells. However release of C. albicans cells from mitochondrial inhibition appears to activate a stress response that increases traits associated with virulence. Our results make it clear that mitochondria are a valid target for the development of anti-fungal strategies but that we must determine the mechanisms by which they regulate stress signalling and virulence ahead of successful therapeutic advance.

microbiology↗