Biology subjects

Johnston, S. A.

Publications and source records attributed to Johnston, S. A..

5 recordsLinked to original sources

bioRxiv2018

Redistribution of a glucuronoxylomannan epitope towards the capsule surface coincides with Titanisation in the human fungal pathogen Cryptococcus neoformans

Disseminated infections with the fungal species Cryptococcus neoformans or, less frequently, C. gattii, are a leading cause of mortality in immunocompromised individuals. Central to the virulence of both species is an elaborate polysaccharide capsule that consists predominantly of glucuronoxylomannan (GXM). Due to its abundance, GXM is an ideal target for host antibodies, and several monoclonal antibodies (mAbs) have previously been derived using purified GXM or whole capsular preparations as antigen. In addition to their application in the diagnosis of cryptococcosis, anti-GXM mAbs are invaluable tools for studying capsule structure. In this study, we report the production and characterisation of a novel anti-GXM mAb, Crp127, that unexpectedly reveals a role for GXM remodelling during the process of fungal Titanisation. We show that Crp127 recognises a GXM epitope in an O-acetylation dependent, but xylosylation-independent, manner. The epitope is differentially expressed by the four main serotypes of Cryptococcus neoformans and gattii, is heterogeneously expressed within clonal populations of C. gattii serotype B strains and is typically confined to the central region of the enlarged capsule. Uniquely, however, this epitope redistributes to the capsular surface in Titan cells, a recently recognised subset of giant fungal cells that are produced in the host lung and are critical for successful infection. Crp127 therefore highlights hitherto unexpected features of cryptococcal morphological change and may hold significant therapeutic potential in differentially identifying cryptococcal strains and subtypes.\n\nImportanceCryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, an invasive fungal infection responsible for approximately 200,000 deaths each year and 15% of AIDS-related deaths annually. Whilst the main virulence factor for both species is a highly variable polysaccharide capsule, formation of Titan cells also underlies the pathogenesis of C. neoformans. Previous studies have shown that capsule composition differs between yeast and Titan cells, however no clear distinctions in the expression or localisation of specific capsular epitopes have been made. In this study, we characterise a novel monoclonal antibody (mAb) specific to a capsular epitope that is differentially distributed throughout the capsules produced by yeast and Titan cells. Whilst this epitope is found within the midzone of yeast capsules, the presentation of this epitope on the surface of Titan cell capsules may represent a way in which these cell types are perceived differently by the immune system.

microbiology↗

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↗

bioRxiv2018

Simu-dependent clearance of dying cells regulates macrophage function and inflammation resolution

Macrophages encounter and clear apoptotic cells during normal development and homeostasis, including at numerous sites of pathology. Clearance of apoptotic cells has been intensively studied, but the effects of macrophage-apoptotic cell interactions on macrophage behaviour are poorly understood. Using Drosophila embryos, we have exploited the ease of manipulating cell death and apoptotic cell clearance in this model to identify that the loss of the apoptotic cell clearance receptor Simu leads to perturbation of macrophage migration and inflammatory responses via pathological levels of apoptotic cells. Removal of apoptosis ameliorates these phenotypes, while acute induction of apoptosis phenocopies these defects and reveals that phagocytosis of apoptotic cells is not necessary for their anti-inflammatory action. Furthermore, Simu is necessary for clearance of necrotic debris and rentention of macrophages at wounds. Thus, Simu is a general detector of damaged self and represents a novel molecular player in controlling resolution of inflammation.

cell biology↗

bioRxiv2017

Mycophenolate mofetil increases susceptibility toopportunistic fungal infection independent of lymphocytes

Anti-proliferative agents that target lymphoid cells are common immunosuppressive agents used in the treatment of diverse autoimmune, graft versus host and inflammatory diseases. Mycophenolate mofetil (MMF) is an anti-proliferative agent that targets lymphoid dependence on inosine monophosphate dehydrogenase for the de novo purine synthesis of deoxyguanosine triphosphate (dGTP) for DNA replication. Here we show that MMF has a distinct and specific in vivo effect on macrophages, in the absence of lymphoid cells. This results in increased macrophage cell death that is dependent on the depletion of cellular GTP, independent of DNA synthesis. Furthermore, the macrophage specific effect of MMF treatment causes an increase in susceptibility to the opportunistic fungal infection Cryptococcus neoformans by reducing phagocytosis and increasing the release of intracellular pathogens via macrophage lysis. Our study demonstrates the need for a better mechanistic understanding of immunosuppressive treatments used in clinical practice and of the specific infection risks associated with certain treatment regimens.

immunology↗