Pseudomonas aeruginosa inhibits Rhizopus microsporus germination through sequestration of free environmental iron.

Rhizopus spp are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infection. Key to pathogenesis is the ability of fungal spores to swell, germinate, and penetrate surrounding tissues. Antibiotic treatment in at-risk patients increases the probability of the patient developing mucormycosis, suggesting that bacteria have the potential to control the growth of the fungus. However, research into polymicrobial relationships involving Rhizopus spp has not been extensively explored. Here we show that co-culturing Rhizopus microsporus and Pseudomonas aeruginosa results in the inhibition of spore germination. This inhibition was mediated via the secretion of bacterial siderophores, which induced iron stress on the fungus. Addition of P. aeruginosa siderophores to R. microsporus spores in the zebrafish larval model of infection resulted in inhibition of fungal germination and reduced host mortality. Therefore, during infection antibacterial treatment may relieve bacterial imposed nutrient restriction resulting in secondary fungal infections.

Robust Phagocyte Recruitment Controls the Opportunistic Fungal Pathogen Mucor circinelloides in Innate Granulomas In Vivo.

Mucormycosis is an emerging fungal infection with extremely high mortality rates in patients with defects in their innate immune response, specifically in functions mediated through phagocytes. However, we currently have a limited understanding of the molecular and cellular interactions between these innate immune effectors and mucormycete spores during the early immune response. Here, the early events of innate immune recruitment in response to infection by Mucor circinelloides spores are modeled by a combined in silico modeling approach and real-time in vivo microscopy. Phagocytes are rapidly recruited to the site of infection in a zebrafish larval model of mucormycosis. This robust early recruitment protects from disease onset in vivoIn silico analysis identified that protection is dependent on the number of phagocytes at the infection site, but not the speed of recruitment. The mathematical model highlights the role of proinflammatory signals for phagocyte recruitment and the importance of inhibition of spore germination for protection from active fungal disease. These in silico data are supported by an in vivo lack of fungal spore killing and lack of reactive oxygen burst, which together result in latent fungal infection. During this latent stage of infection, spores are controlled in innate granulomas in vivo Disease can be reactivated by immunosuppression. Together, these data represent the first in vivo real-time analysis of innate granuloma formation during the early stages of a fungal infection. The results highlight a potential latent stage during mucormycosis that should urgently be considered for clinical management of patients.IMPORTANCE Mucormycosis is a dramatic fungal infection frequently leading to the death of patients. We know little about the immune response to the fungus causing this infection, although evidence points toward defects in early immune events after infection. Here, we dissect this early immune response to infectious fungal spores. We show that specialized white blood cells (phagocytes) rapidly respond to these spores and accumulate around the fungus. However, we demonstrate that the mechanisms that enable phagocytes to kill the fungus fail, allowing for survival of spores. Instead a cluster of phagocytes resembling an early granuloma is formed around spores to control the latent infection. This study is the first detailed analysis of early granuloma formation during a fungal infection highlighting a latent stage that needs to be considered for clinical management of patients.

The mucormycete-host interface.

Mucormycosis is a fungal infection with fulminant angioinvasion leading to high morbidity and mortality in susceptible individuals. The major predisposing conditions are uncontrolled diabetes, neutropenia, malignancies, receipt of a transplant and traumatic injury [1]. Over the past decade, mucormycosis has become an emerging fungal infection due to the increase in patient groups presenting with these pre-disposing conditions and our medical advances in diagnosing the infection [2-4]. Yet, we currently lack clinical interventions to treat mucormycosis effectively. This in turn is due to a lack of understanding of mucormycosis pathogenesis. Here, we discuss our current understanding of selected aspects of interactions at the mucormycete-host interface. We will highlight open questions that might guide future research directions for investigations into the pathogenesis of mucormycosis and potential innovative therapeutic approaches.

Using Flow Cytometry to Analyze Cryptococcus Infection of Macrophages.

Flow cytometry is a powerful analytical technique, which is increasingly being used to study the interaction between host cells and intracellular pathogens. Flow cytometry is capable of measuring a greater number of infected cells within a sample compared to alternative techniques such as fluorescence microscopy. This means that robust quantification of rare events during infection is possible. Our lab and others have developed flow cytometry methods to study interactions between host cells and intracellular pathogens, such as Cryptococcus neoformans, to quantify phagocytosis, intracellular replication, and non-lytic expulsion or "vomocytosis" from the phagosome. Herein we describe these methods and how they can be applied to the study of C. neoformans as well as other similar intracellular pathogens.

Novel cell-based in vitro screen to identify small-molecule inhibitors against intracellular replication of Cryptococcus neoformans in macrophages.

The fungal pathogen Cryptococcus neoformans poses a major threat to immunocompromised patients and is a leading killer of human immunodeficiency virus (HIV)-infected patients worldwide. Cryptococci are known to manipulate host macrophages and can either remain latent or proliferate intracellularly within the host phagocyte, a favourable niche that also renders them relatively insensitive to antifungal agents. Here we report an attempt to address this limitation by using a fluorescence-based drug screening method to identify potential inhibitors of intracellular proliferation of C. neoformans. The Prestwick Chemical Library(®) of FDA-approved small molecules was screened for compounds that limit the intracellular replication of a fluorescently-tagged C. neoformans reference strain (H99-GFP) in macrophages. Preliminary screening revealed 19 of 1200 compounds that could significantly reduce intracellular growth of the pathogen. Secondary screening and host cell cytotoxicity assays highlighted fendiline hydrochloride as a potential drug candidate for the development of future anticryptococcal therapies. Live cell imaging demonstrated that this Ca(2+) channel blocker strongly enhanced phagosome maturation in macrophages leading to improved fungal killing and reduced intracellular replication. Whilst the relatively high dose of fendiline hydrochloride required renders it unfit for clinical deployment against cryptococcosis, this study highlights a novel approach for identifying new lead compounds and unravels a pharmacologically promising scaffold towards the development of novel antifungal therapies for this neglected disease.

Cryptococcus neoformans Thermotolerance to Avian Body Temperature Is Sufficient For Extracellular Growth But Not Intracellular Survival In Macrophages.

Cryptococcus neoformans is a fatal fungal pathogen of humans that efficiently parasitises macrophages. Birds can be colonised by cryptococci and can transmit cryptococcosis to humans via inhalation of inoculated bird excreta. However, colonisation of birds appears to occur in the absence of symptomatic infection. Here, using a pure population of primary bird macrophages, we demonstrate a mechanism for this relationship. We find that bird macrophages are able to suppress the growth of cryptococci seen in mammalian cells despite C. neoformans being able to grow at bird body temperature, and are able to escape from bird macrophages by vomocytosis. A small subset of cryptococci are able to adapt to the inhibitory intracellular environment of bird macrophages, exhibiting a large cell phenotype that rescues growth suppression. Thus, restriction of intracellular growth combined with survival at bird body temperature explains the ability of birds to efficiently spread C. neoformans in the environment whilst avoiding systemic disease.

A zebrafish larval model reveals early tissue-specific innate immune responses to Mucor circinelloides.

Mucormycosis is an emerging fungal infection that is clinically difficult to manage, with increasing incidence and extremely high mortality rates. Individuals with diabetes, suppressed immunity or traumatic injury are at increased risk of developing disease. These individuals often present with defects in phagocytic effector cell function. Research using mammalian models and phagocytic effector cell lines has attempted to decipher the importance of the innate immune system in host defence against mucormycosis. However, these model systems have not been satisfactory for direct analysis of the interaction between innate immune effector cells and infectious sporangiospores in vivo. Here, we report the first real-time in vivo analysis of the early innate immune response to mucormycete infection using a whole-animal zebrafish larval model system. We identified differential host susceptibility, dependent on the site of infection (hindbrain ventricle and swim bladder), as well as differential functions of the two major phagocyte effector cell types in response to viable and non-viable spores. Larval susceptibility to mucormycete spore infection was increased upon immunosuppressant treatment. We showed for the first time that macrophages and neutrophils were readily recruited in vivo to the site of infection in an intact host and that spore phagocytosis can be observed in real-time in vivo. While exploring innate immune effector recruitment dynamics, we discovered the formation of phagocyte clusters in response to fungal spores that potentially play a role in fungal spore dissemination. Spores failed to activate pro-inflammatory gene expression by 6 h post-infection in both infection models. After 24 h, induction of a pro-inflammatory response was observed only in hindbrain ventricle infections. Only a weak pro-inflammatory response was initiated after spore injection into the swim bladder during the same time frame. In the future, the zebrafish larva as a live whole-animal model system will contribute greatly to the study of molecular mechanisms involved in the interaction of the host innate immune system with fungal spores during mucormycosis.

Human Fungal Pathogens of Mucorales and Entomophthorales.

In recent years, we have seen an increase in the number of immunocompromised cohorts as a result of infections and/or medical conditions, which has resulted in an increased incidence of fungal infections. Although rare, the incidence of infections caused by fungi belonging to basal fungal lineages is also continuously increasing. Basal fungal lineages diverged at an early point during the evolution of the fungal lineage, in which, in a simplified four-phylum fungal kingdom, Zygomycota and Chytridiomycota belong to the basal fungi, distinguishing them from Ascomycota and Basidiomycota. Currently there are no known human infections caused by fungi in Chytridiomycota; only Zygomycotan fungi are known to infect humans. Hence, infections caused by zygomycetes have been called zygomycosis, and the term "zygomycosis" is often used as a synonym for "mucormycosis." In the four-phylum fungal kingdom system, Zygomycota is classified mainly based on morphology, including the ability to form coenocytic (aseptated) hyphae and zygospores (sexual spores). In the Zygomycota, there are 10 known orders, two of which, the Mucorales and Entomophthorales, contain species that can infect humans, and the infection has historically been known as zygomycosis. However, recent multilocus sequence typing analyses (the fungal tree of life [AFTOL] project) revealed that the Zygomycota forms not a monophyletic clade but instead a polyphyletic clade, whereas Ascomycota and Basidiomycota are monophyletic. Thus, the term "zygomycosis" needed to be further specified, resulting in the terms "mucormycosis" and "entomophthoramycosis." This review covers these two different types of fungal infections.

'Division of labour' in response to host oxidative burst drives a fatal Cryptococcus gattii outbreak.

Cryptococcus gattii is an emerging intracellular pathogen and the cause of the largest primary outbreak of a life-threatening fungal disease in a healthy population. Outbreak strains share a unique mitochondrial gene expression profile and an increased ability to tubularize their mitochondria within host macrophages. However, the underlying mechanism that causes this lineage of C. gattii to be virulent in immunocompetent individuals remains unexplained. Here we show that a subpopulation of intracellular C. gattii adopts a tubular mitochondrial morphology in response to host reactive oxygen species. These fungal cells then facilitate the rapid growth of neighbouring C. gattii cells with non-tubular mitochondria, allowing for effective establishment of the pathogen within a macrophage intracellular niche. Thus, host reactive oxygen species, an essential component of the innate immune response, act as major signalling molecules to trigger a 'division of labour' in the intracellular fungal population, leading to increased pathogenesis within this outbreak lineage.

Ancient dispersal of the human fungal pathogen Cryptococcus gattii from the Amazon rainforest.

Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

Cryptococcal interactions with the host immune system.

Opportunistic pathogens have become of increasing medical importance over the last decade due to the AIDS pandemic. Not only is cryptococcosis the fourth-most-common fatal infectious disease in sub-Saharan Africa, but also Cryptococcus is an emerging pathogen of immunocompetent individuals. The interaction between Cryptococcus and the host's immune system is a major determinant for the outcome of disease. Despite initial infection in early childhood with Cryptococcus neoformans and frequent exposure to C. neoformans within the environment, immunocompetent individuals are generally able to contain the fungus or maintain the yeast in a latent state. However, immune deficiencies lead to disseminating infections that are uniformly fatal without rapid clinical intervention. This review will discuss the innate and adaptive immune responses to Cryptococcus and cryptococcal strategies to evade the host's defense mechanisms. It will also address the importance of these strategies in pathogenesis and the potential of immunotherapy in cryptococcosis treatment.

Automated analysis of cryptococcal macrophage parasitism using GFP-tagged cryptococci.

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the macrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP-expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed.

Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans.

The pathogenic yeast Cryptococcus neoformans and C. gattii commonly cause severe infections of the central nervous system in patients with impaired immunity but also increasingly in immunocompetent individuals. Cryptococcus is phagocytosed by macrophages but can then survive and proliferate within the phagosomes of these infected host cells. Moreover, Cryptococcus is able to escape into the extracellular environment via a recently discovered nonlytic mechanism (termed expulsion or extrusion). Although it is well established that the host's cytokine profile dramatically affects the outcome of cryptococcal disease, the molecular basis for this effect is unclear. Here, we report a systematic analysis of the influence of Th1, Th2, and Th17 cytokines on the outcome of the interaction between macrophages and cryptococci. We show that Th1 and Th17 cytokines activate, whereas Th2 cytokines inhibit, anticryptococcal functions. Intracellular yeast proliferation was significantly lower after treatment with the Th1 cytokines gamma interferon and tumor necrosis factor alpha and the Th17 cytokine interleukin-17 (IL-17). Interestingly, however, the Th2 cytokines IL-4 and IL-13 significantly increased intracellular yeast proliferation while reducing the occurrence of pathogen expulsion. These results help explain the observed poor prognosis associated with the Th2 cytokine profile (e.g., in human immunodeficiency virus-infected patients).