In Vivo Yeast Cell Morphogenesis Is Regulated by a p21-Activated Kinase in the Human Pathogen Penicillium marneffei

Pathogens have developed diverse strategies to infect their hosts and evade the host defense systems. Many pathogens reside within host phagocytic cells, thus evading much of the host immune system. For dimorphic fungal pathogens which grow in a multicellular hyphal form, a central attribute which facilitates growth inside host cells without rapid killing is the capacity to switch from the hyphal growth form to a unicellular yeast form. Blocking this transition abolishes or severely reduces pathogenicity. Host body temperature (37°C) is the most common inducer of the hyphal to yeast transition in vitro for many dimorphic fungi, and it is often assumed that this is the inducer in vivo. This work describes the identification and analysis of a new pathway involved in sensing the environment inside a host cell by a dimorphic fungal pathogen, Penicillium marneffei. The pakB gene, encoding a p21-activated kinase, defines this pathway and operates independently of known effectors in P. marneffei. Expression of pakB is upregulated in P. marneffei yeast cells isolated from macrophages but absent from in vitro cultured yeast cells produced at 37°C. Deletion of pakB leads to a failure to produce yeast cells inside macrophages but no effect in vitro at 37°C. Loss of pakB also leads to the inappropriate production of yeast cells at 25°C in vitro, and the mechanism underlying this requires the activity of the central regulator of asexual development. The data shows that this new pathway is central to eliciting the appropriate morphogenetic response by the pathogen to the host environment independently of the common temperature signal, thus clearly separating the temperature- and intracellular-dependent signaling systems.     

AreA controls nitrogen source utilisation during both growth programs of the dimorphic fungus Penicillium marneffei

The opportunistic pathogen Penicillium marneffei displays a temperature-dependent dimorphic switching program with saprophytic hyphal growth at 25 °C and yeast growth at 37 °C. The areA gene of P. marneffei has been isolated and found to be required for the utilisation of nonpreferred nitrogen sources during both growth programs of P. marneffei, albeit to differing degrees. Based on this functional characterisation and high degree of sequence conservation with other fungal GATA factors, P. marneffei areA represents an orthologue of Aspergillus nidulans areA and Neurospora crassa NIT2. Based on this study it is proposed that AreA is likely to contribute to the pathogenicity of P. marneffei by facilitating growth in the host environment and regulating the expression of potential virulence factors such as extracellular proteases.     

Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth

Candida albicans is a human commensal and a clinically important fungal pathogen that grows in both yeast and hyphal forms during human infection. Although Candida can cause cutaneous and mucosal disease, systemic infections cause the greatest mortality in hospitals. Candidemia occurs primarily in immunocompromised patients, for whom the innate immune system plays a paramount role in immunity. We have developed a novel transparent vertebrate model of candidemia to probe the molecular nature of Candida-innate immune system interactions in an intact host. Our zebrafish infection model results in a lethal disseminated disease that shares important traits with disseminated candidiasis in mammals, including dimorphic fungal growth, dependence on hyphal growth for virulence, and dependence on the phagocyte NADPH oxidase for immunity. Dual imaging of fluorescently marked immune cells and fungi revealed that phagocytosed yeast cells can remain viable and even divide within macrophages without germinating. Similarly, although we observed apparently killed yeast cells within neutrophils, most yeast cells within these innate immune cells were viable. Exploiting this model, we combined intravital imaging with gene knockdown to show for the first time that NADPH oxidase is required for regulation of C. albicans filamentation in vivo. The transparent and easily manipulated larval zebrafish model promises to provide a unique tool for dissecting the molecular basis of phagocyte NADPH oxidase-mediated limitation of filamentous growth in vivo.     

Regulation of hyphal morphogenesis by Ras and Rho small GTPases

The fungal kingdom is extremely diverse – comprised of over 1.5 million species including yeasts, molds and mushrooms. Essentially, all fungi have cell walls that contain chitin and the cells of most fungi grow as tube-like filaments called hyphae. These filamentous fungi, such as the mold Neurospora crassa, develop branched radial networks of hyphae referred to as mycelium. In contrast, non-filamentous fungi do not form radial mycelia, but grow as single cells, which reproduce by either budding or fission such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, respectively. Finally, there are fungi that are capable of switching between single cell, yeast form growth and filamentous growth such as Candida albicans. The switch from yeast to filamentous growth in these so-called dimorphic fungi is a virulence trait in many human and plant pathogens. Highly conserved master regulators of all three fungal growth modes – filamentous, non-filamentous and dimorphic – are the Ras and Rho small GTPases, which spatially and temporally control cell polarity establishment and maintenance. This review summarizes the key roles of the Ras and Rho GTPases during hyphal morphogenesis in a range of fungi.     

Key thermally dimorphic fungal pathogens: shaping host immunity

Exposure to fungal pathogens from the environment is inevitable and with the number of at-risk populations increasing, the prevalence of invasive fungal infection is on the rise. An interesting group of fungal organisms known as thermally dimorphic fungi predominantly infects immunocompromised individuals. These potential pathogens are intriguing in that they survive in the environment in one form, mycelial phase, but when entering the host, they are triggered by the change in temperature to switch to a new pathogenic form. Considering the growing prevalence of infection and the need for improved diagnostic and treatment approaches, studies identifying key components of fungal recognition and the innate immune response to these pathogens will significantly contribute to our understanding of disease progression. This review focuses on key endemic dimorphic fungal pathogens that significantly contribute to disease, including Histoplasma, Coccidioides and Talaromyces species. We briefly describe their prevalence, route of infection and clinical presentation. Importantly, we have reviewed the major fungal cell wall components of these dimorphic fungi, the host pattern recognition receptors responsible for recognition and important innate immune responses supporting adaptive immunity and fungal clearance or the failure thereof.     

Differentially regulated high‐affinity iron assimilation systems support growth of the various cell types in the dimorphic pathogen Talaromyces marneffei

Iron is a key trace element important for many biochemical processes and its availability varies with the environment. For human pathogenic fungi iron acquisition can be particularly problematical because host cells sequester free iron as part of the acute‐phase response to infection. Fungi rely on high‐affinity iron uptake systems, such as reductive iron assimilation (RIA) and siderophore‐mediated iron uptake (non‐RIA). These have been extensively studied in pathogenic fungi that exist outside of host cells, but much less is known for intracellular fungal pathogens. Talaromyces marneffei is a dimorphic fungal pathogen endemic to Southeast Asia. In the host T. marneffei resides within macrophages where it grows as a fission yeast. T. marneffei has genes of both iron assimilation systems as well as a paralogue of the siderophore biosynthetic gene sidA, designated sidX. Unlike other fungi, deletion of sidA or sidX resulted in cell type‐specific effects. Mutant analysis showed that T. marneffei yeast cells also employ RIA for iron acquisition, providing an additional system in this cell type that differs substantially from hyphal cells. These data illustrate the specialized iron acquisition systems used by the different cell types of a dimorphic fungal pathogen and highlight the complexity in siderophore‐biosynthetic pathways amongst fungi.     

The crucial role of the Aspergillus fumigatus siderophore system in interaction with alveolar macrophages

Iron plays a central role in manifestation of infections for a variety of pathogens. To ensure an adequate supply with iron, Aspergillus fumigatus employs extra- and intracellular siderophores (low-molecular mass iron chelators), which are of importance for fungal growth in particular during iron starvation. Here we show that the lack of extracellular siderophores, and especially, the lack of the entire siderophore system cause in immunosuppressed mice in vivo (i) a reduced extracellular growth rate, (ii) a reduced intracellular growth rate in alveolar macrophages, and (iii) an increased susceptibility to conidial growth inhibition by alveolar macrophages. These data underline the crucial role of the fungal siderophore system not only for extracellular growth but also in the interaction with the host immune cells. Moreover, the hyphal growth rate within alveolar macrophages compared to extracellular lavage fluid was significantly decreased indicating that, besides elimination of fungal conidia, inhibition of pathogenic growth is a function of macrophages.     

N-acetylglucosamine (GlcNAc) Triggers a Rapid,Temperature-Responsive Morphogenetic Program in Thermally Dimorphic Fungi

The monosaccharide N-acetylglucosamine (GlcNAc) is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc stimulates developmental pathways in the fungal pathogen Candida albicans, which is a commensal organism that colonizes the mammalian gut and causes disease in the setting of host immunodeficiency. Here we investigate GlcNAc signaling in thermally dimorphic human fungal pathogens, a group of fungi that are highly evolutionarily diverged from C. albicans and cause disease even in healthy individuals. These soil organisms grow as polarized, multicellular hyphal filaments that transition into a unicellular, pathogenic yeast form when inhaled by a human host. Temperature is the primary environmental cue that promotes reversible cellular differentiation into either yeast or filaments; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. We found GlcNAc to be a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. In addition to increasing the rate of filamentous growth, micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift that was independent of GlcNAc catabolism, indicating that fungal cells sense GlcNAc to promote filamentation. Whole-genome expression profiling to identify candidate genes involved in establishing the filamentous growth program uncovered two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes.     

NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.     

Map kinases in fungal pathogens

MAP kinases in eukaryotic cells are well known for transducing a variety of extracellular signals to regulate cell growth and differentiation. Recently, MAP kinases homologous to the yeast Fus3/Kss1 MAP kinases have been identified in several fungal pathogens and found to be important for appressorium formation, invasive hyphal growth, and fungal pathogenesis. This MAP kinase pathway also controls diverse growth or differentiation processes, including conidiation, conidial germination, and female fertility. MAP kinases homologous to yeast Slt2 and Hog1 have also been characterized in Candida albicans and Magnaporthe grisea. Mutants disrupted of the Slt2 homologues have weak cell walls, altered hyphal growth, and reduced virulence. The Hog1 homologues are dispensable for growth but are essential for regulating responses to hyperosmotic stress in C. albicans and M. grisea. Overall, recent studies have indicated that MAP kinase pathways may play important roles in regulating growth, differentiation, survival, and pathogenesis in fungal pathogens.     

Profiling a killer, the development of Cryptococcus neoformans

The ability of fungi to transition between unicellular and multicellular growth has a profound impact on our health and the economy. Many important fungal pathogens of humans, animals, and plants are dimorphic, and the ability to switch between morphological states has been associated with their virulence. Cryptococcus neoformans is a human fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised and, in some cases, immunocompetent hosts. Cryptococcus neoformans grows vegetatively as a budding yeast and switches to hyphal growth during the sexual cycle, which is important in the study of cryptococcal pathogenicity because spores resulting from sexual development are infectious propagules and can colonize the lungs of a host. In addition, sexual reproduction contributes to the genotypic variability of Cryptococcus species, which may lead to increased fitness and virulence. Despite significant advances in our understanding of the mechanisms behind the development of C. neoformans, our knowledge is still incomplete. Recent studies have led to the emergence of many intriguing questions and hypotheses. In this review, we describe and discuss the most interesting aspects of C. neoformans development and address their impact on pathogenicity.     

Polarized hyphal growth in Candida albicans requires the Wiskott-Aldrich Syndrome protein homolog Wal1p

The yeast-to-hypha transition is a key feature in the cell biology of the dimorphic human fungal pathogen Candida albicans. Reorganization of the actin cytoskeleton is required for this dimorphic switch in Candida. We show that C. albicans WAL1 mutants with both copies of the Wiskott-Aldrich syndrome protein (WASP) homolog deleted do not form hyphae under all inducing conditions tested. Growth of the wild-type and wal1 mutant strains was monitored by in vivo time-lapse microscopy both during yeast-like growth and under hypha-inducing conditions. Isotropic bud growth produced round wal1 cells and unusual mother cell growth. Defects in the organization of the actin cytoskeleton resulted in the random localization of actin patches. Furthermore, wal1 cells exhibited defects in the endocytosis of the lipophilic dye FM4-64, contained increased numbers of vacuoles compared to the wild type, and showed defects in bud site selection. Under hypha-inducing conditions wal1 cells were able to initiate polarized morphogenesis, which, however, resulted in the formation of pseudohyphal cells. Green fluorescent protein (GFP)-tagged Wal1p showed patch-like localization in emerging daughter cells during the yeast growth phase and at the hyphal tips under hypha-inducing conditions. Wal1p-GFP localization largely overlapped with that of actin. Our results demonstrate that Wal1p is required for the organization of the actin cytoskeleton and hyphal morphogenesis in C. albicans as well as for endocytosis and vacuole morphology.     

The interface between virulence and host response to the pathogenic fungus Histoplasma capsulatum

Intracellular pathogens have evolved machinery to evade the immune response in order to survive within a host. Histoplasma capsulatum, one of the intracellular pathogens, is a dimorphic fungus that dodges innate and adaptive immunity; it escapes immunity presumably through virulence factors that permit fungal survival and replication within the host. This review discusses immune factors that contribute to the control of H. capsulatum infection, several host-survival mechanisms H. capsulatum uses, and new techniques that have led to the identification of several H. capsulatum virulence factors, which will most likely aid in the discovery of many more.     

Comparison of morphogenetic networks of filamentous fungi and yeast.

Fungi generally display either of two growth modes, yeast-like or filamentous, whereas dimorphic fungi, upon environmental stimuli, are able to switch between the yeast-like and the filamentous growth mode. Signal transduction pathways have been elucidated in the budding yeast Saccharomyces cerevisiae, establishing a morphogenetic network that links cell-cycle events with cellular morphogenesis. Recent molecular genetic studies in several filamentous fungal model systems revealed key components required for distinct steps from fungal spore germination to the maintenance of polar hyphal growth, mycelium formation, and nuclear division. This allows a mechanistic comparison of yeast-like and hyphal growth and the establishment of a core model morphogenetic network for filamentous growth including signaling via the cAMP pathway, Rho modules, and cell cycle kinases. Appreciating similarities between morphogenetic networks of the unicellular yeasts and the multicellular filamentous fungi will open new research directions, help in isolating the central network components, and ultimately pave the way to elucidate the central differences (of many) that distinguish, e.g., the growth mode of filamentous fungi from that of their yeast-like relatives, the role of cAMP signaling, and nuclear division.     

The Swi/Snf chromatin remodeling complex is essential for hyphal development in Candida albicans

The ability of dimorphic transition between yeast and hyphal forms in Candida albicans is one of the vital determinants for its pathogenicity and virulence. We isolated C. albicans SWI1 as a suppressor of the invasive growth defect in a Saccharomyces cerevisiae mutant. Expression of C. albicans SWI1 in S. cerevisiae partially complemented the growth defect of a swi1 mutant in the utilization of glycerol. Swi1 is in a complex with Snf2 in C. albicans, and both proteins are localized in the nucleus independent of the growth form. Deleting SWI1 or SNF2 in C. albicans prevented true hyphal formation and resulted in constitutive pseudohypha-like growth in all media examined. Furthermore, swi1/swi1 mutant was defective in hypha-specific gene expression and avirulent in a mouse model of systemic infection. These data strongly suggest the conserved Swi/Snf complex in C. albicans is required for hyphal development and pathogenicity.