Proteinases in fungal morphogenesis

Fungal morphogenesis is a regulated series of events, leading to changes from one state to another, in which proteolysis could be regarded as one of the controlling functions. Proteinases are essential for the supply of amino acids, selective inactivation of specific growth phase proteins not required during development and for the activation and modification of the enzymes involved in cell wall synthesis. A critical evaluation of the role of proteinases as a biochemical correlate in fungal morphogenesis is discussed.     

Yeast and fungal morphogenesis from an evolutionary perspective

Cellular morphogenesis is a complex process and molecular studies in the last few decades have amassed a large amount of information that is difficult to grasp in any completeness. Fungal systems, in particular the budding and fission yeasts, have been important players in unravelling the basic structural and regulatory elements involved in a wide array of cellular processes. In this article, we address the design principles underlying the various processes of yeast and fungal morphogenesis. We attempt to explain the apparent molecular complexity from the perspective of the evolutionary theory of "facilitated variation". Following a summary of some of the most studied morphogenetic phenomena, we discuss, using recent examples, the underlying core processes and their associated "weak" regulatory linkages that bring about variation in morphogenetic phenotypes.     

Motors in fungal morphogenesis: cooperation versus competition

Fungal tip growth underlies substrate invasion and is essential for fungal virulence. It requires the activity of molecular motors that deliver secretory vesicles to the growth region or which mediate bi-directional motility of early endosomes. Visualizing motors and their cargo in living fungal cells revealed unexpected cooperation between motors in membrane trafficking: (1) Class V chitin synthase, which has a class 17 myosin motor domain, moves bi-directionally, with myosin-5 and kinesin-1 cooperating in delivery to the growth region, and dynein taking it back to the cell centre. The myosin-17 motor domain competes with dynein by tethering the chitin synthase to the plasma membrane before exocytosis; (2) Long-range endosome motility is based on a cooperation of kinesin-3 and dynein, but towards the microtubule plus-end dynein competes with kinesin-3 to prevent the organelles from 'falling off the track'. These results reveal a fine-balanced network of cooperative and competitive motor activity, required for fungal morphogenesis.     

Manganese and fungal pathogens: Metabolism and potential association with virulence

Nutritional immunity is one of the strategies employed by the host to combat invading pathogens. It consists of actively controlling micronutrient bioavailability in the site of infection to hinder microbial growth. The role of manganese in cell biology and nutritional immunity for bacterial pathogens is well understood, but data regarding fungi are still limited. Fungi have evolved complex regulatory systems to acquire, distribute, and utilize manganese. Therefore, the disruption of manganese homeostasis in pathogenic fungi may lead to severe phenotypes and impact virulence. Because the host presents tools for manganese sequestration, and this condition can reduce the growth of important fungal pathogens such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, it is feasible to suppose that manganese nutritional immunity could play an important role in fungal infections. However, direct evidence is still lacking, and little is known about manganese homeostasis, nutritional immunity, and specific adaptations in individual species of fungal pathogens. In this opinion, we present the current body of knowledge about these subjects, arguing about manganese importance in host–pathogen interactions.     

MAP kinase pathways as regulators of fungal virulence

MAP kinases are dual phosphorylated protein kinases, present in eukaryotes, which mediate differentiation programs and immune responses in mammalian cells. In pathogenic fungi, MAP kinases are key elements that control adaptation to environmental stress. Recent studies have shown that these pathways have an essential role in the control of essential virulence factors such as capsule biogenesis in Cryptococcus neoformans or morphogenesis, invasion and oxidative stress in Candida albicans. Although MAP kinases sense different activating signals, there is a considerable degree of crosstalk and/or overlap, which enables them to integrate, amplify and modulate the appropriate protective and adaptive response. MAP kinases behave as a 'functional nervous system' that controls virulence and influences the progression of the disease.     

Enzymes that counteract nitrosative stress promote fungal virulence

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS(-/-) animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.     

Interaction of sertraline with Candida species selectively attenuates fungal virulence in vitro

This study investigated whether the interaction between isolates of Candida albicans (n=7), Candida parapsilosis (n=3), Candida krusei (n=2), Candida dubliniensis (n=1) and sertraline, a typical selective serotonin reuptake inhibitor, alters candidal virulence. Sertraline treatment of Candida spp. significantly (P<0.05) affected hyphal elongation, phospholipase activity, production of secreted aspartyl proteinases and fungal viability. In addition, monocyte-derived macrophages (MDMs) treated with sertraline reduced inhibition of blastoconidia germination in comparison to MDMs alone. In conclusion, our findings suggest that the interaction between sertraline and Candida spp. may also diminish the virulence properties of this fungal pathogen in vivo.     

Intersection of fungal fitness and virulence in Cryptococcus neoformans

The use of insertional mutagenesis to discover genes that impact laccase activity has resulted in the identification of multiple cellular processes that affect the fitness of Cryptococcus neoformans. Fitness has been defined as the ability of an organism to propagate and evolve within a given environment. Because the human host is an evolutionary dead-end for an opportunistic pathogen, we have defined pathogenic fitness here as the capability to successfully propagate within the stressful environment of the host, causing disease by expression of virulence traits that damage the host. In this review, laccase-deficient insertional mutants will be highlighted in terms of the basic biological processes in which they are involved. The impact of laccase-associated cellular functions on fitness and virulence will be discussed, as will the mutants' potential as therapeutic targets. Vacuolar function, copper homeostasis, mitochondrial function and carbon repression are covered.     

Conserved cAMP signaling cascades regulate fungal development and virulence

Two well characterized signal transduction cascades regulating fungal development and virulence are the MAP kinase and cAMP signaling cascades. Here we review the current state of knowledge on cAMP signaling cascades in fungi. While the processes regulated by cAMP signaling in fungi are as diverse as the fungi themselves, the components involved in signal transduction are remarkably conserved. Fungal cAMP signaling cascades are also quite versatile, which is apparent from the differential regulation of similar biological processes. In this review we compare and contrast cAMP signaling pathways that regulate development in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and differentiation and virulence in the human pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis. We also present examples of interaction between the cAMP and MAP kinase signaling cascades in the regulation of fungal development and virulence.     

Signal transduction cascades regulating fungal development and virulence.

Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.     

Using non-mammalian hosts to study fungal virulence and host defense

Non-mammalian hosts have been used to study host-fungal interactions. Hosts such as Drosophila melanogaster, Caenorhabditis elegans, Acathamoeba castellanii, Dictyostelium discoideum, and Galleria mellonella have provided means to examine the physical barriers, cellular mechanisms and molecular elements of the host response. The Drosophila host-response to fungi is mediated through the Toll pathway, whereas in C. elegans the host-response is TIR-1-dependent. Virulence traits that are involved in mammalian infection are important for the interaction of fungi with these hosts. Screening of fungal virulence traits using mutagenized fungi to determine changes in fungal infectivity of non-mammalian hosts has been used to identify novel virulence proteins used to infect C. elegans such as Kin1 (a serine/threonine protein kinase) and Rom2 (a Rho1 guanyl-nucleotide exchange factor) from Cryptococcus neoformans. These heterologous non-mammalian hosts highlight the similarities and differences between different hosts in fungal pathogenesis and they complement studies in mammalian systems and those using other genetic approaches.     

NMR structure of a fungal virulence factor reveals structural homology with mammalian saposin B

The fungal protein CBP ( c alcium b inding p rotein) is a known virulence factor with an unknown virulence mechanism. The protein was identified based on its ability to bind calcium and its prevalence as Histoplasma capsulatum 's most abundant secreted protein. However, CBP has no sequence homology with other CBPs and contains no known calcium binding motifs. Here, the NMR structure of CBP reveals a highly intertwined homodimer and represents the first atomic level NMR model of any fungal virulence factor. Each CBP monomer is comprised of four α-helices that adopt the saposin fold, characteristic of a protein family that binds to membranes and lipids. This structural homology suggests that CBP functions as a lipid binding protein, potentially interacting with host glycolipids in the phagolysosome of host cells.     

Lipid biology in fungal stress and virulence: Entomopathogenic fungi

Broad host range insect pathogenic fungi penetrate through the host cuticle, necessitating an ability to confront and overcome surface lipids and other molecules that often include antimicrobial compounds. In this context, induction of lipid assimilatory pathways by exogenous substrates is crucial for successful infection to occur, and lipid growth substrates can have significant effects on the virulence of fungal infectious propagules, e.g. conidia. The production of lipases is a critical part of the cuticle-degrading repertoire and pathways involved in triglyceride metabolism and phospholipid homeostasis have been shown to contribute to host invasion. Mobilization of endogenous lipid stores via the activities of the caleosin and perilipin lipid storage-turnover proteins, have been linked to diverse processes including formation of penetration structures, e.g. germ tubes and appressoria, spore properties and dispersal, and the ability to respond to lipid growth substrates and virulence. Here, we summarize recent advances in our understanding of lipid assimilation and mobilization pathways in the ability of entomogenous fungi to infect and use host substrates. Host surface and internal lipids can alternatively act as antifungal barriers, inducers of pathogenesis-related pathways, and/or as fungal growth substrates. Lipids and lipid assimilation can be considered as forming a co-evolutionary web between the insect host and entomogenous fungi.     

Anastomosis is required for virulence of the fungal necrotroph Alternaria brassicicola

A fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogen Alternaria brassicicola and two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of the A. brassicicola isolates tested, as well as A. mimicula and A. japonica, were capable of self-fusion, with two isolates of A. brassicicola being capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) in A. brassicicola resulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenic A. brassicicola mutant defective for a mitogen-activated protein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity in A. brassicicola.     

Gene disruption to evaluate the role of fungal candidate virulence genes

Gene disruption is a powerful genetic tool that can define pathogenic or virulence factors. In the past two years gene disruption approaches have been used to identify fungal virulence genes. The capsule genes, an alpha subunit of G protein and certain kinases of Cryptococcus neoformans have clearly been demonstrated to be associated with pathogenicity. In Candida albicans at least four genes involved in hyphal formation have been disrupted and tested for virulence. In other fungi, such as Histoplasma capsulatum, however, more efficient gene disruption methods need to be developed before such approaches can be regularly used for identifying virulence genes.