Publications by year
In Press
Brown A, Pradhan A, Larcombe D, Gow N (In Press). Epitope shaving promotes fungal immune evasion. mBio
2023
Campbell JS, Bebes A, Pradhan A, Yuecel R, Brown A, Wakefield JG (2023). Characterising phagocytes and measuring phagocytosis from live<i>Galleria mellonella</i>larvae.
Abstract:
Characterising phagocytes and measuring phagocytosis from liveGalleria mellonellalarvae
AbstractOver the last 20 years, the larva of the greater waxmoth,Galleria mellonella, has rapidly increased in popularity as anin vivomammalian 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,Galleriaharbour 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 assayGalleriahemocytes, 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 thatGalleriahemocytes 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 theGalleriahemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicatesin vitrodata, showing that cell wall β-1,3-glucan masking byCandida albicanssubverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics inGalleria.
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Larcombe DE, Bohovych IM, Pradhan A, Ma Q, Hickey E, Leaves I, Cameron G, Avelar GM, de Assis LJ, Childers DS, et al (2023). Glucose-enhanced oxidative stress resistance-A protective anticipatory response that enhances the fitness of Candida albicans during systemic infection.
PLoS Pathog,
19(7).
Abstract:
Glucose-enhanced oxidative stress resistance-A protective anticipatory response that enhances the fitness of Candida albicans during systemic infection.
Most microbes have developed responses that protect them against stresses relevant to their niches. Some that inhabit reasonably predictable environments have evolved anticipatory responses that protect against impending stresses that are likely to be encountered in their niches-termed "adaptive prediction". Unlike yeasts such as Saccharomyces cerevisiae, Kluyveromyces lactis and Yarrowia lipolytica and other pathogenic Candida species we examined, the major fungal pathogen of humans, Candida albicans, activates an oxidative stress response following exposure to physiological glucose levels before an oxidative stress is even encountered. Why? Using competition assays with isogenic barcoded strains, we show that "glucose-enhanced oxidative stress resistance" phenotype enhances the fitness of C. albicans during neutrophil attack and during systemic infection in mice. This anticipatory response is dependent on glucose signalling rather than glucose metabolism. Our analysis of C. albicans signalling mutants reveals that the phenotype is not dependent on the sugar receptor repressor pathway, but is modulated by the glucose repression pathway and down-regulated by the cyclic AMP-protein kinase a pathway. Changes in catalase or glutathione levels do not correlate with the phenotype, but resistance to hydrogen peroxide is dependent on glucose-enhanced trehalose accumulation. The data suggest that the evolution of this anticipatory response has involved the recruitment of conserved signalling pathways and downstream cellular responses, and that this phenotype protects C. albicans from innate immune killing, thereby promoting the fitness of C. albicans in host niches.
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Arita GS, Ma Q, Leaves I, Pradhan A, Hickey E, Dambuza I, Bebes A, Vincenzi Conrado PC, Barros Galinari C, Vicente Seixas FA, et al (2023). The impact of ORF19.36.1 in the pathobiology of Candida albicans.
Microbial Pathogenesis,
185Abstract:
The impact of ORF19.36.1 in the pathobiology of Candida albicans
Background: Our previous proteomics data obtained from Candida albicans recovered after serial passage in a murine model of systemic infection revealed that Orf19.36.1 expression correlates with the virulence of the fungus. Therefore, the impact of ORF19.36.1 upon virulence was tested in this study. Materials & Methods: CRISPR-Cas9 technology was used to construct homozygous C. albicans orf19.36.1 null mutants and the phenotypes of these mutants examined in vitro (filamentation, invasion, adhesion, biofilm formation, hydrolase activities) and in vivo assays. Results: the deletion of ORF19.36.1 did not significantly impact the phenotypes examined or the virulence of C. albicans in two infection models. Conclusion: These results suggest that, although Orf19.36.1 expression correlates with virulence, this protein is not essential for C. albicans pathobiology.
Abstract.
2022
Avelar GM, Dambuza IM, Ricci L, Yuecel R, Mackenzie K, Childers DS, Bain JM, Pradhan A, Larcombe DE, Netea MG, et al (2022). Impact of Changes at the Candida Albicans Cell Surface Upon Immunogenicity and Colonisation in the Gastrointestinal Tract.
Avelar GM, Dambuza IM, Ricci L, Yuecel R, Mackenzie K, Childers DS, Bain JM, Pradhan A, Larcombe DE, Netea MG, et al (2022). Impact of changes at the Candida albicans cell surface upon immunogenicity and colonisation in the gastrointestinal tract.
Cell Surf,
8Abstract:
Impact of changes at the Candida albicans cell surface upon immunogenicity and colonisation in the gastrointestinal tract.
The immunogenicity of Candida albicans cells is influenced by changes in the exposure of microbe-associated molecular patterns (MAMPs) on the fungal cell surface. Previously, the degree of exposure on the C. albicans cell surface of the immunoinflammatory MAMP β-(1,3)-glucan was shown to correlate inversely with colonisation levels in the gastrointestinal (GI) tract. This is important because life-threatening systemic candidiasis in critically ill patients often arises from translocation of C. albicans strains present in the patient's GI tract. Therefore, using a murine model, we have examined the impact of gut-related factors upon β-glucan exposure and colonisation levels in the GI tract. The degree of β-glucan exposure was examined by imaging flow cytometry of C. albicans cells taken directly from GI compartments, and compared with colonisation levels. Fungal β-glucan exposure was lower in the cecum than the small intestine, and fungal burdens were correspondingly higher in the cecum. This inverse correlation did not hold for the large intestine. The gut fermentation acid, lactate, triggers β-glucan masking in vitro, leading to attenuated anti-Candida immune responses. Additional fermentation acids are present in the GI tract, including acetate, propionate, and butyrate. We show that these acids also influence β-glucan exposure on C. albicans cells in vitro and, like lactate, they influence β-glucan exposure via Gpr1/Gpa2-mediated signalling. Significantly, C. albicans gpr1Δ gpa2Δ cells displayed elevated β-glucan exposure in the large intestine and a corresponding decrease in fungal burden, consistent with the idea that Gpr1/Gpa2-mediated β-glucan masking influences colonisation of this GI compartment. Finally, extracts from the murine gut and culture supernatants from the mannan grazing gut anaerobe Bacteroides thetaiotaomicron promote β-glucan exposure at the C. albicans cell surface. Therefore, the local microbiota influences β-glucan exposure levels directly (via mannan grazing) and indirectly (via fermentation acids), whilst β-glucan masking appears to promote C. albicans colonisation of the murine large intestine.
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de Assis LJ, Bain JM, Liddle C, Leaves I, Hacker C, Peres da Silva R, Yuecel R, Bebes A, Stead D, Childers DS, et al (2022). Nature of β-1,3-Glucan-Exposing Features on Candida albicans Cell Wall and Their Modulation.
mBio,
13(6).
Abstract:
Nature of β-1,3-Glucan-Exposing Features on Candida albicans Cell Wall and Their Modulation.
Candida albicans exists as a commensal of mucosal surfaces and the gastrointestinal tract without causing pathology. However, this fungus is also a common cause of mucosal and systemic infections when antifungal immune defenses become compromised. The activation of antifungal host defenses depends on the recognition of fungal pathogen-associated molecular patterns (PAMPs), such as β-1,3-glucan. In C. albicans, most β-1,3-glucan is present in the inner cell wall, concealed by the outer mannan layer, but some β-1,3-glucan becomes exposed at the cell surface. In response to host signals, such as lactate, C. albicans induces the Xog1 exoglucanase, which shaves exposed β-1,3-glucan from the cell surface, thereby reducing phagocytic recognition. We show here that β-1,3-glucan is exposed at bud scars and punctate foci on the lateral wall of yeast cells, that this exposed β-1,3-glucan is targeted during phagocytic attack, and that lactate-induced masking reduces β-1,3-glucan exposure at bud scars and at punctate foci. β-1,3-Glucan masking depends upon protein kinase a (PKA) signaling. We reveal that inactivating PKA, or its conserved downstream effectors, Sin3 and Mig1/Mig2, affects the amounts of the Xog1 and Eng1 glucanases in the C. albicans secretome and modulates β-1,3-glucan exposure. Furthermore, perturbing PKA, Sin3, or Mig1/Mig2 attenuates the virulence of lactate-exposed C. albicans cells in Galleria. Taken together, the data are consistent with the idea that β-1,3-glucan masking contributes to Candida pathogenicity. IMPORTANCE Microbes that coexist with humans have evolved ways of avoiding or evading our immunological defenses. These include the masking by these microbes of their "pathogen-associated molecular patterns" (PAMPs), which are recognized as "foreign" and used to activate protective immunity. The commensal fungus Candida albicans masks the proinflammatory PAMP β-1,3-glucan, which is an essential component of its cell wall. Most of this β-1,3-glucan is hidden beneath an outer layer of the cell wall on these microbes, but some can become exposed at the fungal cell surface. Using high-resolution confocal microscopy, we examine the nature of the exposed β-1,3-glucan at C. albicans bud scars and at punctate foci on the lateral cell wall, and we show that these features are targeted by innate immune cells. We also reveal that downstream effectors of protein kinase a (Mig1/Mig2, Sin3) regulate the secretion of major glucanases, modulate the levels of β-1,3-glucan exposure, and influence the virulence of C. albicans in an invertebrate model of systemic infection. Our data support the view that β-1,3-glucan masking contributes to immune evasion and the virulence of a major fungal pathogen of humans.
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Pradhan A, Ma Q, Hickey E, Avelar G, Larcombe D, Bain J, Childers D, Dambuza I, Leaves I, de Assis LJ, et al (2022). S9.4c Diverse environmental inputs mediate changes in β-glucan exposure at the Candida albicans cell surface thereby influencing tissue colonisation during systemic infection. Medical Mycology, 60(Suppl 1).
2021
Pradhan A, Ma Q, de Assis LJ, Leaves I, Larcombe DE, Rodriguez Rondon AV, Nev OA, Brown AJP (2021). Anticipatory Stress Responses and Immune Evasion in Fungal Pathogens.
Trends in Microbiology,
29(5), 416-427.
Abstract:
Anticipatory Stress Responses and Immune Evasion in Fungal Pathogens
In certain niches, microbes encounter environmental challenges that are temporally linked. In such cases, microbial fitness is enhanced by the evolution of anticipatory responses where the initial challenge simultaneously activates pre-emptive protection against the second impending challenge. The accumulation of anticipatory responses in domesticated yeasts, which have been termed 'adaptive prediction', has led to the emergence of 'core stress responses' that provide stress cross-protection. Protective anticipatory responses also seem to be common in fungal pathogens of humans. These responses reflect the selective pressures that these fungi have faced relatively recently in their evolutionary history. Consequently, some pathogens have evolved 'core environmental responses' which exploit host signals to trigger immune evasion strategies that protect them against imminent immune attack.
Abstract.
Bain JM, Alonso MF, Childers DS, Walls CA, Mackenzie K, Pradhan A, Lewis LE, Louw J, Avelar GM, Larcombe DE, et al (2021). Immune cells fold and damage fungal hyphae.
Proc Natl Acad Sci U S A,
118(15).
Abstract:
Immune cells fold and damage fungal hyphae.
Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal pathogens are not a foregone conclusion because some pathogens have evolved mechanisms to evade phagocytic recognition, engulfment, and killing. For example, Candida albicans can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of C. albicans-macrophage interactions, we discovered that macrophages can counteract this by folding fungal hyphae. The folding of fungal hyphae is promoted by Dectin-1, β2-integrin, VASP, actin-myosin polymerization, and cell motility. Folding facilitates the complete engulfment of long hyphae in some cases and it inhibits hyphal growth, presumably tipping the balance toward successful fungal clearance.
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Hatinguais R, Pradhan A, Brown GD, Brown AJP, Warris A, Shekhova E (2021). Mitochondrial Reactive Oxygen Species Regulate Immune Responses of Macrophages to Aspergillus fumigatus.
Front Immunol,
12Abstract:
Mitochondrial Reactive Oxygen Species Regulate Immune Responses of Macrophages to Aspergillus fumigatus.
Reactive Oxygen Species (ROS) are highly reactive molecules that can induce oxidative stress. For instance, the oxidative burst of immune cells is well known for its ability to inhibit the growth of invading pathogens. However, ROS also mediate redox signalling, which is important for the regulation of antimicrobial immunity. Here, we report a crucial role of mitochondrial ROS (mitoROS) in antifungal responses of macrophages. We show that mitoROS production rises in murine macrophages exposed to swollen conidia of the fungal pathogen Aspergillus fumigatus compared to untreated macrophages, or those treated with resting conidia. Furthermore, the exposure of macrophages to swollen conidia increases the activity of complex II of the respiratory chain and raises mitochondrial membrane potential. These alterations in mitochondria of infected macrophages suggest that mitoROS are produced via reverse electron transport (RET). Significantly, preventing mitoROS generation via RET by treatment with rotenone, or a suppressor of site IQ electron leak, S1QEL1.1, lowers the production of pro-inflammatory cytokines TNF-α and IL-1β in macrophages exposed to swollen conidia of A. fumigatus. Rotenone and S1QEL1.1 also reduces the fungicidal activity of macrophages against swollen conidia. Moreover, we have established that elevated recruitment of NADPH oxidase 2 (NOX2, also called gp91phox) to the phagosomal membrane occurs prior to the increase in mitoROS generation. Using macrophages from gp91phox-/- mice, we have further demonstrated that NOX2 is required to regulate cytokine secretion by RET-associated mitoROS in response to infection with swollen conidia. Taken together, these observations demonstrate the importance of RET-mediated mitoROS production in macrophages infected with A. fumigatus.
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2020
Childers DS, Avelar GM, Bain JM, Larcombe DE, Pradhan A, Budge S, Heaney H, Brown AJP (2020). Impact of the Environment upon the Candida albicans Cell Wall and Resultant Effects upon Immune Surveillance.
Curr Top Microbiol Immunol,
425, 297-330.
Abstract:
Impact of the Environment upon the Candida albicans Cell Wall and Resultant Effects upon Immune Surveillance.
The fungal cell wall is an essential organelle that maintains cellular morphology and protects the fungus from environmental insults. For fungal pathogens such as Candida albicans, it provides a degree of protection against attack by host immune defences. However, the cell wall also presents key epitopes that trigger host immunity and attractive targets for antifungal drugs. Rather than being a rigid shield, it has become clear that the fungal cell wall is an elastic organelle that permits rapid changes in cell volume and the transit of large liposomal particles such as extracellular vesicles. The fungal cell wall is also flexible in that it adapts to local environmental inputs, thereby enhancing the fitness of the fungus in these microenvironments. Recent evidence indicates that this cell wall adaptation affects host-fungus interactions by altering the exposure of major cell wall epitopes that are recognised by innate immune cells. Therefore, we discuss the impact of environmental adaptation upon fungal cell wall structure, and how this affects immune recognition, focussing on C. albicans and drawing parallels with other fungal pathogens.
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Brown AJP, Larcombe DE, Pradhan A (2020). Thoughts on the evolution of Core Environmental Responses in yeasts. Fungal Biology, 124(5), 475-481.
2019
Pradhan A, Avelar GM, Bain JM, Childers D, Pelletier C, Larcombe DE, Shekhova E, Netea MG, Brown GD, Erwig L, et al (2019). Non-canonical signalling mediates changes in fungal cell wall PAMPs that drive immune evasion.
Nat Commun,
10(1).
Abstract:
Non-canonical signalling mediates changes in fungal cell wall PAMPs that drive immune evasion.
To colonise their host, pathogens must counter local environmental and immunological challenges. Here, we reveal that the fungal pathogen Candida albicans exploits diverse host-associated signals to promote immune evasion by masking of a major pathogen-associated molecular pattern (PAMP), β-glucan. Certain nutrients, stresses and antifungal drugs trigger β-glucan masking, whereas other inputs, such as nitrogen sources and quorum sensing molecules, exert limited effects on this PAMP. In particular, iron limitation triggers substantial changes in the cell wall that reduce β-glucan exposure. This correlates with reduced phagocytosis by macrophages and attenuated cytokine responses by peripheral blood mononuclear cells. Iron limitation-induced β-glucan masking depends on parallel signalling via the iron transceptor Ftr1 and the iron-responsive transcription factor Sef1, and the protein kinase a pathway. Our data reveal that C. albicans exploits a diverse range of specific host signals to trigger protective anticipatory responses against impending phagocytic attack and promote host colonisation.
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2018
Childers DS, Avelar GM, Bain J, Pradhan A, Larcome D, Netea M, Erwig L, Gow NAR, Brown AJP (2018). Fungal Transformers: Tracking a Moving Target.
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Pradhan A, Avelar GM, Bain JM, Childers DS, Larcombe DE, Netea MG, Shekhova E, Munro CA, Brown GD, Erwig LP, et al (2018). Hypoxia Promotes Immune Evasion by Triggering β-Glucan Masking on the Candida albicans Cell Surface via Mitochondrial and cAMP-Protein Kinase a Signaling.
mBio,
9(6).
Abstract:
Hypoxia Promotes Immune Evasion by Triggering β-Glucan Masking on the Candida albicans Cell Surface via Mitochondrial and cAMP-Protein Kinase a Signaling.
Organisms must adapt to changes in oxygen tension if they are to exploit the energetic benefits of reducing oxygen while minimizing the potentially damaging effects of oxidation. Consequently, organisms in all eukaryotic kingdoms display robust adaptation to hypoxia (low oxygen levels). This is particularly important for fungal pathogens that colonize hypoxic niches in the host. We show that adaptation to hypoxia in the major fungal pathogen of humans Candida albicans includes changes in cell wall structure and reduced exposure, at the cell surface, of β-glucan, a key pathogen-associated molecular pattern (PAMP). This leads to reduced phagocytosis by murine bone marrow-derived macrophages and decreased production of IL-10, RANTES, and TNF-α by peripheral blood mononuclear cells, suggesting that hypoxia-induced β-glucan masking has a significant effect upon C. albicans-host interactions. We show that hypoxia-induced β-glucan masking is dependent upon both mitochondrial and cAMP-protein kinase a (PKA) signaling. The decrease in β-glucan exposure is blocked by mutations that affect mitochondrial functionality (goa1Δ and upc2Δ) or that decrease production of hydrogen peroxide in the inner membrane space (sod1Δ). Furthermore, β-glucan masking is enhanced by mutations that elevate mitochondrial reactive oxygen species (aox1Δ). The β-glucan masking defects displayed by goa1Δ and upc2Δ cells are suppressed by exogenous dibutyryl-cAMP. Also, mutations that inactivate cAMP synthesis (cyr1Δ) or PKA (tpk1Δ tpk2Δ) block the masking phenotype. Our data suggest that C. albicans responds to hypoxic niches by inducing β-glucan masking via a mitochondrial cAMP-PKA signaling pathway, thereby modulating local immune responses and promoting fungal colonization.IMPORTANCE Animal, plant, and fungal cells occupy environments that impose changes in oxygen tension. Consequently, many species have evolved mechanisms that permit robust adaptation to these changes. The fungal pathogen Candida albicans can colonize hypoxic (low oxygen) niches in its human host, such as the lower gastrointestinal tract and inflamed tissues, but to colonize its host, the fungus must also evade local immune defenses. We reveal, for the first time, a defined link between hypoxic adaptation and immune evasion in C. albicans As this pathogen adapts to hypoxia, it undergoes changes in cell wall structure that include masking of β-glucan at its cell surface, and it becomes better able to evade phagocytosis by innate immune cells. We also define the signaling mechanisms that mediate hypoxia-induced β-glucan masking, showing that they are dependent on mitochondrial signaling and the cAMP-protein kinase pathway. Therefore, hypoxia appears to trigger immune evasion in this fungal pathogen.
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2017
Pradhan A, Herrero-de-Dios C, Belmonte R, Budge S, Lopez Garcia A, Kolmogorova A, Lee KK, Martin BD, Ribeiro A, Bebes A, et al (2017). Elevated catalase expression in a fungal pathogen is a double-edged sword of iron.
PLoS Pathog,
13(5).
Abstract:
Elevated catalase expression in a fungal pathogen is a double-edged sword of iron.
Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.
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