Host-to-Pathogen Gene Transfer Facilitated Infection of Insects by a Pathogenic Fungus

Metarhizium robertsii is a plant root colonizing fungus that is also an insect pathogen. Its entomopathogenicity is a characteristic that was acquired during evolution from a plant endophyte ancestor. This transition provides a novel perspective on how new functional mechanisms important for host switching and virulence have evolved. From a random T-DNA insertion library, we obtained a pathogenicity defective mutant that resulted from the disruption of a sterol carrier gene (Mr-npc2a). Phylogenetic analysis revealed that Metarhizium acquired Mr-npc2a from an insect by horizontal gene transfer (HGT). Mr-NPC2a binds to cholesterol, an animal sterol, rather than the fungal sterol ergosterol, indicating it retains the specificity of insect NPC2 proteins. Mr-NPC2a is an intracellular protein and is exclusively expressed in the hemolymph of living insects. The disruption of Mr-npc2a reduced the amount of sterol in cell membranes of the yeast-like hyphal bodies that facilitate dispersal in the host body. These were consequently more susceptible to insect immune responses than the wild type. Transgenic expression of Mr-NPC2a increased the virulence of Beauveria bassiana, an endophytic insect-pathogenic fungus that lacks a Mr-NPC2a homolog.     

Host switching by an ambrosia beetle fungal mutualist: Mycangial colonization of indigenous beetles by the invasive laurel wilt fungal pathogen

Ambrosia beetles require their fungal symbiotic partner as their cultivated (farmed) food source in tree galleries. While most fungal-beetle partners do not kill the host trees they inhabit, since their introduction (invasion) into the United states around ~2002, the invasive beetle Xyleborus glabratus has vectored its mutualist partner (but plant pathogenic) fungus, Harringtonia lauricola, resulting in the deaths of over 300 million trees. Concerningly, indigenous beetles have been caught bearing H. lauricola. Here, we show colonization of the mycangia of the indigenous X. affinis ambrosia beetle by H. lauricola. Mycangial colonization occurred within 1 h of feeding, with similar levels seen for H. lauricola as found for the native X. affinis-R. arxii fungal partner. Fungal mycangial occupancy was stable over time and after removal of the fungal source, but showed rapid turnover when additional fungal cells were available. Microscopic visualization revealed two pre-oral mycangial pouches of ~100–200 × 25–50 μm/each, with narrow entry channels of 25–50 × 3–10 μm. Fungi within the mycangia underwent a dimorphic transition from filamentous/blastospore growth to yeast-like budding with alterations to membrane structures. These data identify the characteristics of ambrosia beetle mycangial colonization, implicating turnover as a mechanism for host switching of H. lauricola to other ambrosia beetle species.     

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.     

Stimulation of fermentation and yeast-like morphogenesis in Mucor rouxii by phenethyl alcohol

The germination of fungal spores into hyphae was inhibited by concentrations of phenethyl alcohol (PEA) from 0.05 to 0.3%. Spores of Mucor formed budding spherical cells instead of filaments. These cells were abundant in cultures of Mucor rouxii at 0.22% PEA, provided that the carbon source was a hexose at 2 to 5%. Morphology was filamentous with xylose, maltose, sucrose, or a mixture of amino acids. Removal of PEA resulted in the conversion of yeast-like cells into hyphae. PEA did not inhibit biosynthesis of cytochromes or oxygen uptake, but it stimulated CO₂ and ethyl alcohol production. PEA had no effect on the rate of oxygen uptake, but it inhibited the oxidative-phosphorylation activity of mitochondria. These results suggested that growth inhibition by PEA could result from uncoupling of oxidative phosphorylation and that, in Mucor, yeast-like morphology and fermentation were linked.     

Multifunctional role of a fungal pathogen-secreted laccase 2 in evasion of insect immune defense

Laccases are widely present in bacteria, fungi, plants and invertebrates and involved in a variety of physiological functions. Here, we report that Beauveria bassiana, an economic important entomopathogenic fungus, secretes a laccase 2 (BbLac2) during infection that detoxifies insect immune response-generated reactive oxygen species (ROS) and interferes with host immune phenoloxidase (PO) activation. BbLac2 is expressed in fungal cells during proliferation in the insect haemocoel and can be found to distribute on the surface of haemolymph-derived in vivo fungal hyphal bodies or be secreted. Targeted gene-knockout of BbLac2 increased fungal sensitivity to oxidative stress, decreased virulence to insect, and increased host PO activity. Strains overexpressing BbLac2 showed increased virulence, with reduced host PO activity and lowered ROS levels in infected insects. In vitro assays revealed that BbLac2 could eliminate ROS and oxidize PO substrates (phenols), verifying the enzymatic functioning of the protein in detoxification of cytotoxic ROS and interference with the PO cascade. Moreover, BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns (PAMPs), enabling the pathogen to evade immune recognition. Our data suggest a multifunctional role for fungal pathogen-secreted laccase 2 in evasion of insect immune defenses.     

Cryptococcus neoformans Hyperfilamentous Strain Is Hypervirulent in a Murine Model of Cryptococcal Meningoencephalitis

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.     

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.     

Conidiation Color Mutants of Aspergillus fumigatus Are Highly Pathogenic to the Heterologous Insect Host Galleria mellonella

The greater wax moth Galleria mellonella has been widely used as a heterologous host for a number of fungal pathogens including Candida albicans and Cryptococcus neoformans. A positive correlation in pathogenicity of these yeasts in this insect model and animal models has been observed. However, very few studies have evaluated the possibility of applying this heterologous insect model to investigate virulence traits of the filamentous fungal pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. Here, we have examined the impact of mutations in genes involved in melanin biosynthesis on the pathogenicity of A. fumigatus in the G. mellonella model. Melanization in A. fumigatus confers bluish-grey color to conidia and is a known virulence factor in mammal models. Surprisingly, conidial color mutants in B5233 background that have deletions in the defined six-gene cluster required for DHN-melanin biosynthesis caused enhanced insect mortality compared to the parent strain. To further examine and confirm the relationship between melanization defects and enhanced virulence in the wax moth model, we performed random insertional mutagenesis in the Af293 genetic background to isolate mutants producing altered conidia colors. Strains producing conidia of previously identified colors and of novel colors were isolated. Interestingly, these color mutants displayed a higher level of pathogenicity in the insect model compared to the wild type. Although some of the more virulent color mutants showed increased resistance to hydrogen peroxide, overall phenotypic characterizations including secondary metabolite production, metalloproteinase activity, and germination rate did not reveal a general mechanism accountable for the enhanced virulence of these color mutants observed in the insect model. Our observations indicate instead, that exacerbated immune response of the wax moth induced by increased exposure of PAMPs (pathogen-associated molecular patterns) may cause self-damage that results in increased mortality of larvae infected with the color mutants. The current study underscores the limitations of using this insect model for inferring the pathogenic potential of A. fumigatus strains in mammals, but also points to the importance of understanding the innate immunity of the insect host in providing insights into the pathogenicity level of different fungal strains in this model. Additionally, our observations that melanization defective color mutants demonstrate increased virulence in the insect wax moth, suggest the potential of using melanization defective mutants of native insect fungal pathogens in the biological control of insect populations.     

Roles of Cch1 and Mid1 in Morphogenesis, Oxidative Stress Response and Virulence in Candida albicans

Ca²⁺ channel Cch1, and its subunit Mid1, has been suggested as the protein complex responsible for mediating Ca²⁺ influx, which is often employed by fungal cells to maintain cell survival. The abilities of morphological switch and response to stress conditions are closely related to pathogenicity in Candida albicans. Cch1 and Mid1 activity are required for virulence of Cryptococcus neoformans and Claviceps purpurea, respectively. To investigate whether Cch1 and Mid1 also play a role in the virulence of C. albicans, we constructed cch1Δ/Δ and mid1Δ/Δ mutant strains for functional analysis of CCH1 and MID1. Although both of the mutants displayed the ability of yeast-to-hypha transition, they were defective in hyphae maintenance and invasive growth. Interestingly, deletion of CCH1 or MID1 in C. albicans led to an obvious defect phenotype in oxidative stress response. Moreover, the virulence of the mutants was reduced in a mouse model. Our results demonstrated that Cch1 and Mid1 activity are related to the virulence of C. albicans and may provide a new antifungal target.     

An alternative insect pathogenic strategy in an Aspergillus flavus auxotroph

In order to study fungal pathogen evolution, we used a model system whereby the opportunistic fungus Aspergillus flavus was serially propagated through the insect (Galleria mellonella) larvae, yielding a cysteine/methionine auxotroph of A. flavus with properties of an obligate insect pathogen. The auxotroph exhibited insect host restriction but did not show any difference in virulence when compared with the wild-type (Scully LR, Bidochka MJ, 2006. Microbiology 152, 223-232). Here, we report that on 1 % insect cuticle medium and synthetic Galleria medium, the auxotroph displayed increased extracellular protease production, a virulence factor necessary for insect pathogenesis. In the wild-type strain, protease production was deregulated during carbon (glucose), nitrogen (nitrate), or sulphate deprivation. If all three were present, protease production was vastly reduced. However, in the cysteine/methionine auxotroph, protease production was deregulated in complete medium. We suggest that the deficiency in sulphate assimilation in the auxotroph resulted in deregulation of protease production. The auxotroph exhibited delayed germination and slower hyphal growth when compared to the wild-type but there were no differences in virulence or cuticle penetration, suggesting a shift in pathogenic strategy that compensated decreased growth with increased virulence factor (extracellular protease) production. We concluded that the biosynthetic deficiency that mediated insect host restriction also increased protease production in the slow-growing auxotroph, resulting in an alternate, more host-specific pathogenic strategy. However, we argue that transmission is not necessarily correlated with virulence as competition bioassays in insect larvae showed that the wild-type generally out-competed the auxotroph by producing the majority of the conidia on the sporulating cadavers. This is one of the few examples that highlight the effect of genome decay on nutrition acquisition, virulence, and transmission in fungal pathogen evolution.     

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.     

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.     

十种虫生真菌液体培养芽生孢子的形态发生

芽生孢子是许多真菌通过出芽方式产生的无性孢子,在液体发酵中主要以这种形式大量繁殖。真菌杀虫剂活性成分分生孢子的大量生产使用液体发酵得到的芽生孢子作为接种物,另外,它也是当今虫生真菌遗传转化的重要受体,因而研究虫生真菌芽生孢子的形态和发生特点具有重要意义。本研究分别对液体发酵中的球孢白僵菌、粉棒束孢、蝉棒束孢、环链棒束孢、玫烟色棒束孢、细脚棒束孢、斜链棒束孢、金龟子绿僵菌、蝗绿僵菌和蜡蚧霉等10种常见虫生真菌芽生孢子的形成过程进行显微观察,了解其分生孢子产生过程的异同。结果表明,芽生孢子的产生方式有两种类型：1）蝉棒束孢在其整个生活史中以菌丝生长为主,芽生孢子产生的数量很少。2）其他各种真菌芽生孢子产生方式相似,在菌丝体形成后就开始大量以菌丝出芽或缢缩产生芽生孢子,接着还可通过芽生孢子的出芽或缢缩断裂产生新的芽生孢子。芽生孢子的产生分为3个时期：初期先由菌丝形成芽生孢子;指数期芽生孢子大量增殖,菌丝和芽生孢子都可产生芽生孢子;后期以芽生孢子产新芽生孢子为主要方式。     

Modified Adamek's medium renders high yields of Metarhizium robertsii blastospores that are desiccation tolerant and infective to cattle-tick larvae

Blastospores are yeast-like cells produced by entomopathogenic fungi that are infective to arthropods. The economical feasible production of blastospores of the insect killing fungus Metarhizium spp. must be optimized to increase yields. Moreover, stabilization process is imperative for blastospore formulation as a final product. In this sense, our goal was to increase blastospore production of two Metarhizium isolates (ESALQ1426 and ESALQ4676) in submerged liquid cultures. A modified Adamek's medium was supplemented with increased glucose concentrations and the fermentation time was accelerated by using a blastospore pre-culture as inoculum. Virulence of air-dried stable blastospores was compared with conidia toward larvae of the cattle tick, Rhipicephalus microplus. Our results revealed that blastospore production of Metarhizium is isolate- and species-dependent. Glucose-enriched cultures (140 g glucose/L) inoculated with pre-cultures improved yields with optimal growth conditions attained for Metarhizium robertsii ESALQ1426 that rendered as high as 5.9 × 10⁸ blastospores/mL within 2 d. Resultant air-dried blastospores of ESALQ1426 were firstly proved to infect and quickly kill cattle tick larvae with comparable efficiency to conidia. Altogether, we argue that both osmotic pressure, induced by high glucose titers, and isolate selection are critical to produce high yields of blastospores that hold promise to control cattle-tick larvae.     

Process of infection of armored scale insects (Diaspididae) by an entomopathogenic Cosmospora sp

Several species in the fungal genus Cosmospora (synonym Nectria) (anamorph Fusarium) are specialist entomopathogens of armored scale insects (Diaspididae), known to cause periodic epizootics in host populations. Inconsistent mortality rates recorded under laboratory conditions prompted a study into the process of infection of armored scale insects by this fungus. Scale insect mortality following exposure to a Cosmospora sp. (Culture Collection Number: CC89) from New Zealand was related to insect age, with reproductively mature insects having a significantly higher infection rate than immature insects. Examination using scanning electron microscopy found no evidence that the fungus penetrated directly through the wax test (cap) of the scale insect or through the un-lifted interface between the test and the substrate on which the insect resided. However, fungal hyphae were observed growing beneath the test when the test of the reproductively mature insect lifted away from the substrate for the purpose of releasing crawlers, the mobile pre-settled juveniles. Once the hyphae of CC89 advanced under the test, germ-tubes readily penetrated the insect body through a number of natural openings (e.g. spiracles, vulva, stylet), with mycosis observed within seven days after inoculation. Direct penetration through the cuticle of the scale insect was not observed.