Mucor circinelloides induces platelet aggregation through integrin αIIbß3 and FcγRIIA.

Thrombosis is a hallmark of the fatal fungal infection mucormycosis. Yet, the platelet activation pathway in response to mucormycetes is unknown. In this study we determined the platelet aggregation potential of Mucor circinelloides (M. circinelloides) NRRL3631, characterized the signaling pathway facilitating aggregation in response to fungal spores, and identified the influence of the spore developmental stage upon platelet aggregation potential. Using impedance and light-transmission aggregometry, we showed that M. circinelloides induced platelet aggregation in whole blood and in platelet-rich plasma, respectively. The formation of large spore-platelet aggregates was confirmed by light-sheet microscopy, which showed spores dispersed throughout the aggregate. Aggregation potential was dependent on the spore's developmental stage, with the strongest platelet aggregation by spores in mid-germination. Inhibitor studies revealed platelet aggregation was mediated by the low affinity IgG receptor FcγRIIA and integrin αIIbß3; Src and Syk tyrosine kinase signaling; and the secondary mediators TxA2 and ADP. Flow cytometry of antibody stained platelets showed that interaction with spores increased expression of platelet surface integrin αIIbß3 and the platelet activation marker CD62P. Together, this is the first elucidation of the signaling pathways underlying thrombosis formation during a fungal infection, highlighting targets for therapeutic intervention.

Spore Germination of Pathogenic Filamentous Fungi.

Fungi, algae, plants, protozoa, and bacteria are all known to form spores, especially hardy and ubiquitous propagation structures that are also often the infectious agents of diseases. Spores can survive for thousands of years, frozen in the permafrost (Kochkina et al., 2012), with the oldest viable spores extracted after 250 million years from salt crystals (Vreeland, Rosenzweig, & Powers, 2000). Their resistance to high levels of UV, desiccation, pressure, heat, and cold enables the survival of spores in the harshest conditions (Setlow, 2016). For example, Bacillus subtilis spores can survive and remain viable after experiencing conditions similar to those on Mars (Horneck et al., 2012). Spores are disseminated through environmental factors. Wind, water, or animal carriage allow spores to be spread ubiquitously throughout the environment. Spores will break dormancy and begin to germinate once exposed to favorable conditions. Germination is the mechanism that converts the spore from a dormant biological organism to one that grows vegetatively and is capable of either sexual or asexual reproduction. The process of germination has been well studied in plants, moss, bacteria, and many fungi (Hohe & Reski, 2005; Huang & Hull, 2017; Vesty et al., 2016). Unfortunately, information on the complex signaling involved in the regulation of germination, particularly in fungi remains lacking. This chapter will discuss germination of fungal spores covering our current understanding of the regulation, signaling, outcomes, and implications of germination of pathogenic fungal spores. Owing to the morphological similarities between the spore-hyphal and yeast-hyphal transition and their relevance for disease progression, relevant aspects of fungal dimorphism will be discussed alongside spore germination in this chapter.

Cryptococcus gattii VGIII isolates causing infections in HIV/AIDS patients in Southern California: identification of the local environmental source as arboreal.

Ongoing Cryptococcus gattii outbreaks in the Western United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving emergence and expansion of microbial pathogens. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no evidence of nuclear genetic exchange, indicating these represent distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS patients in Southern California. VGI, VGII, and VGIII have been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only two environmental isolates of C. gattii have ever been reported from California: CBS7750 (VGII) and WM161 (VGIII). The incongruence of frequent clinical presence and uncommon environmental isolation suggests an unknown C. gattii reservoir in California. Here we report frequent isolation of C. gattii VGIII MATα and MATa isolates and infrequent isolation of VGI MATα from environmental sources in Southern California. VGIII isolates were obtained from soil debris associated with tree species not previously reported as hosts from sites near residences of infected patients. These isolates are fertile under laboratory conditions, produce abundant spores, and are part of both locally and more distantly recombining populations. MLST and whole genome sequence analysis provide compelling evidence that these environmental isolates are the source of human infections. Isolates displayed wide-ranging virulence in macrophage and animal models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less virulent. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 cryptococcal infections occurring annually for which the causative isolate is rarely assigned species status. Thus, the C. gattii global health burden could be more substantial than currently appreciated.

Transmission of Hypervirulence traits via sexual reproduction within and between lineages of the human fungal pathogen cryptococcus gattii.

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal.

pH manipulation as a novel strategy for treating mucormycosis.

Mucormycosis is a fatal fungal disease caused by several organisms within the order Mucorales. In recent years, traumatic injury has emerged as a novel risk factor for mucormycosis. Current antifungal therapy is ineffective, expensive, and typically requires extensive surgical debridement. There is thus a pressing need for safe prophylactic treatment that can be rapidly and easily applied to high-risk patients, such as those with major trauma injuries. Acetic acid has been used as a topical treatment for burn wounds for centuries and has proven activity against Gram-negative bacteria. Here, we demonstrate that acetic acid is also highly effective against major pathogenic groups of Mucorales, even at very low concentrations (0.3%). This antifungal effect is not seen with other acids, such as hydrochloric and lactic acid, suggesting that acetic acid activity against Mucorales spores is not solely evoked by low environmental pH. In agreement with this, we demonstrate that the antifungal activity of acetic acid arises from a combination of its ability to potently lower intracellular pH and from pH-independent toxicity. Thus, dilute acetic acid may offer a low-cost, safe, prophylactic treatment for patients at risk of invasive mucormycosis following traumatic injury.

A diverse population of Cryptococcus gattii molecular type VGIII in southern Californian HIV/AIDS patients.

Cryptococcus gattii infections in southern California have been reported in patients with HIV/AIDS. In this study, we examined the molecular epidemiology, population structure, and virulence attributes of isolates collected from HIV/AIDS patients in Los Angeles County, California. We show that these isolates consist almost exclusively of VGIII molecular type, in contrast to the VGII molecular type isolates causing the North American Pacific Northwest outbreak. The global VGIII population structure can be divided into two molecular groups, VGIIIa and VGIIIb. Isolates from the Californian patients are virulent in murine and macrophage models of infection, with VGIIIa significantly more virulent than VGIIIb. Several VGIII isolates are highly fertile and produce abundant sexual spores that may serve as infectious propagules. The a and α VGIII MAT locus alleles are largely syntenic with limited rearrangements compared to the known VGI (a/α) and VGII (α) MAT loci, but each has unique characteristics including a distinct deletion flanking the 5' VGIII MATa alleles and the α allele is more heterogeneous than the a allele. Our studies indicate that C. gattii VGIII is endemic in southern California, with other isolates originating from the neighboring regions of Mexico, and in rarer cases from Oregon and Washington state. Given that >1,000,000 cases of cryptococcal infection and >620,000 attributable mortalities occur annually in the context of the global AIDS pandemic, our findings suggest a significant burden of C. gattii may be unrecognized, with potential prognostic and therapeutic implications. These results signify the need to classify pathogenic Cryptococcus cases and highlight possible host differences among the C. gattii molecular types influencing infection of immunocompetent (VGI/VGII) vs. immunocompromised (VGIII/VGIV) hosts.

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

A bacterial endosymbiont of the fungus Rhizopus microsporus drives phagocyte evasion and opportunistic virulence.

Opportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ubiquitous in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this and the evolutionary drivers underlying the acquisition of virulence mechanisms are poorly understood. Here, we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice and that this endosymbiosis enables the secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum, as well as their ability to engulf and kill other microbes. As amoebas are natural environmental predators of both bacteria and fungi, we propose that this tri-kingdom interaction contributes to establishing endosymbiosis and the acquisition of anti-phagocyte activity. Importantly, we show that this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a new role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals and suggest a mechanism of virulence acquisition through environmental interactions with amoebas.

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.

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).

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.

Pathways of Pathogenicity: Transcriptional Stages of Germination in the Fatal Fungal Pathogen Rhizopus delemar.

Rhizopus delemar is an invasive fungal pathogen responsible for the frequently fatal disease mucormycosis. Germination, a crucial mechanism by which infectious spores of Rhizopus delemar cause disease, is a key developmental process that transforms the dormant spore state into a vegetative one. The molecular mechanisms that underpin this transformation may be key to controlling mucormycosis; however, the regulation of germination remains poorly understood. This study describes the phenotypic and transcriptional changes that take place over the course of germination. This process is characterized by four distinct stages: dormancy, isotropic swelling, germ tube emergence, and hyphal growth. Dormant spores are shown to be transcriptionally unique, expressing a subset of transcripts absent in later developmental stages. A large shift in the expression profile is prompted by the initiation of germination, with genes involved in respiration, chitin, cytoskeleton, and actin regulation appearing to be important for this transition. A period of transcriptional consistency can be seen throughout isotropic swelling, before the transcriptional landscape shifts again at the onset of hyphal growth. This study provides a greater understanding of the regulation of germination and highlights processes involved in transforming Rhizopus delemar from a single-cellular to multicellular organism.IMPORTANCE Germination is key to the growth of many organisms, including fungal spores. Mucormycete spores exist abundantly within the environment and germinate to form hyphae. These spores are capable of infecting immunocompromised individuals, causing the disease mucormycosis. Germination from spore to hyphae within patients leads to angioinvasion, tissue necrosis, and often fatal infections. This study advances our understanding of how spore germination occurs in the mucormycetes, identifying processes we may be able to inhibit to help prevent or treat mucormycosis.

Correction: Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation in Vibrio cholerae and downregulate the expression of virulence genes.

[This corrects the article DOI: 10.1039/C7SC00615B.].

cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization.

The bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes.IMPORTANCE Cholera is an infectious disease that is caused by the bacterium Vibrio cholerae Best known for causing disease in humans, the bacterium is most commonly found in aquatic ecosystems. Hence, humans acquire cholera following ingestion of food or water contaminated with V. cholerae Transition between an aquatic environment and a human host triggers a lifestyle switch that involves reprogramming of V. cholerae gene expression patterns. This process is controlled by a network of transcription factors. In this paper, we show that the cAMP receptor protein (CRP) is a key regulator of V. cholerae gene expression in response to lifestyle changes.

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.

Gene flow contributes to diversification of the major fungal pathogen Candida albicans.

Elucidating population structure and levels of genetic diversity and recombination is necessary to understand the evolution and adaptation of species. Candida albicans is the second most frequent agent of human fungal infections worldwide, causing high-mortality rates. Here we present the genomic sequences of 182 C. albicans isolates collected worldwide, including commensal isolates, as well as ones responsible for superficial and invasive infections, constituting the largest dataset to date for this major fungal pathogen. Although, C. albicans shows a predominantly clonal population structure, we find evidence of gene flow between previously known and newly identified genetic clusters, supporting the occurrence of (para)sexuality in nature. A highly clonal lineage, which experimentally shows reduced fitness, has undergone pseudogenization in genes required for virulence and morphogenesis, which may explain its niche restriction. Candida albicans thus takes advantage of both clonality and gene flow to diversify.

Innate and Adaptive Immunity to Mucorales.

Mucormycosis is an invasive fungal infection characterised by rapid filamentous growth, which leads to angioinvasion, thrombosis, and tissue necrosis. The high mortality rates (50-100%) associated with mucormycosis are reflective of not only the aggressive nature of the infection and the poor therapeutics currently employed, but also the failure of the human immune system to successfully clear the infection. Immune effector interaction with Mucorales is influenced by the developmental stage of the mucormycete spore. In a healthy immune environment, resting spores are resistant to phagocytic killing. Contrarily, swollen spores and hyphae are susceptible to damage and degradation by macrophages and neutrophils. Under the effects of immune suppression, the recruitment and efficacy of macrophage and neutrophil activity against mucormycetes is considerably reduced. Following penetration of the endothelial lining, Mucorales encounter platelets. Platelets adhere to both mucormycete spores and hyphae, and exhibit germination suppression and hyphal damage capacity in vitro. Dendritic cells are activated in response to Mucorales hyphae only, and induce adaptive immunity. It is crucial to further knowledge regarding our immune system's failure to eradicate resting spores under intact immunity and inhibit fungal growth under immunocompromised conditions, in order to understand mucormycosis pathogenicity and enhance therapeutic strategies for mucormycosis.

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.