Comprehensive genetic analysis of adhesin proteins and their role in virulence of Candida albicans

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.     

Independent Synchronized Control and Visualization of Interactions between Living Cells and Organisms

To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematode’s surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion.     

Control of Root-Knot Nematodes on Tomato by the Endoparasitic Fungus Meria coniospora

The endoparasitic nematophagous fungus Meria coniospora reduced root-knot nematode galling on tomatoes in greenhouse pot trials. The fungus was introduced to pots by addition of conidia at several inoculum levels directly to the soil or addition of nematodes infected with M. coniospora to the soil; both methods reduced root galling by root-knot nematodes. These studies represent a part of a recently initiated effort to evaluate the potential of endoparasitic nematophagous fungi for biocontrol of nematodes.     

Independent Synchronized Control and Visualization of Interactions between Living Cells and Organisms

To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematode’s surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion.     

The pseudokinase NIPI-4 is a novel regulator of antimicrobial peptide gene expression

Hosts have developed diverse mechanisms to counter the pathogens they face in their natural environment. Throughout the plant and animal kingdoms, the up-regulation of antimicrobial peptides is a common response to infection. In C. elegans, infection with the natural pathogen Drechmeria coniospora leads to rapid induction of antimicrobial peptide gene expression in the epidermis. Through a large genetic screen we have isolated many new mutants that are incapable of upregulating the antimicrobial peptide nlp-29 in response to infection (i.e. with a Nipi or ‘no induction of peptide after infection’ phenotype). More than half of the newly isolated Nipi mutants do not correspond to genes previously associated with the regulation of antimicrobial peptides. One of these, nipi-4, encodes a member of a nematode-specific kinase family. NIPI-4 is predicted to be catalytically inactive, thus to be a pseudokinase. It acts in the epidermis downstream of the PKC∂ TPA-1, as a positive regulator of nlp antimicrobial peptide gene expression after infection. It also controls the constitutive expression of antimicrobial peptide genes of the cnc family that are targets of TGFß regulation. Our results open the way for a more detailed understanding of how host defense pathways can be molded by environmental pathogens.     

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response

Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed “aspergilloses,” in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host–pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients’ lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host–pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.

This study reveals that the post-translational modification persulfidation is important for both fungal virulence and the host antifungal response. The level of persulfidation in the host, which correlates with its antifungal potency, impacts the level required in the fungus to counteract host attack, reflecting a functional correlation. Thus modulating persulfidation may be a promising strategy to target both pathogens and immune responses.     

Susceptibility of Neoaplectana spp. and Heterorhabditis heliothidis to the Endoparasitic Fungus Drechmeria coniospora

Adhesive conidia of the nematophagous fungus, Drechmeria coniospora (Drechsler) W. Gams and Jansson (Moniliales: Deuteromycetes), would occasionally attach but never penetrate the infective stages of insect parasitic Neoaplectana carpocapsae, N. glaseri, N. bibionis, N. intermedia, and Heterorhabditis helfothidis (Rhabditida). However, adult and pre-infective stages of Neoaplectana spp. became infected by the fungus.     

The NADPH oxidase AoNoxA in <Emphasis Type="Italic">Arthrobotrys oligospora</Emphasis> functions as an initial factor in the infection of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Reactive oxygen species (ROS) produced by NADPH oxidases can serve as signaling molecules to regulate a variety of physiological processes in multi-cellular organisms. In the nematophagous fungus Arthrobotrys oligospora, we found that ROS were produced during conidial germination, hyphal extension, and trap formation in the presence of nematodes. Generation of an AoNoxA knockout strain demonstrated the crucial role of NADPH oxidase in the production of ROS in A. oligospora, with trap formation impaired in the AoNoxA mutant, even in the presence of the nematode host. In addition, the expression of virulence factor serine protease P186 was up-regulated in the wild-type strain, but not in the mutant strain, in the presence of Caenorhabditis elegans. These results indicate that ROS derived from AoNoxA are essential for full virulence of A. oligospora in nematodes.     

HYR1-mediated detoxification of reactive oxygen species is required for full virulence in the rice blast fungus

During plant-pathogen interactions, the plant may mount several types of defense responses to either block the pathogen completely or ameliorate the amount of disease. Such responses include release of reactive oxygen species (ROS) to attack the pathogen, as well as formation of cell wall appositions (CWAs) to physically block pathogen penetration. A successful pathogen will likely have its own ROS detoxification mechanisms to cope with this inhospitable environment. Here, we report one such candidate mechanism in the rice blast fungus, Magnaporthe oryzae, governed by a gene we refer to as MoHYR1. This gene (MGG_07460) encodes a glutathione peroxidase (GSHPx) domain, and its homologue in yeast was reported to specifically detoxify phospholipid peroxides. To characterize this gene in M. oryzae, we generated a deletion mutantΔhyr1 which showed growth inhibition with increased amounts of hydrogen peroxide (H₂O₂). Moreover, we observed that the fungal mutants had a decreased ability to tolerate ROS generated by a susceptible plant, including ROS found associated with CWAs. Ultimately, this resulted in significantly smaller lesion sizes on both barley and rice. In order to determine how this gene interacts with other (ROS) scavenging-related genes in M. oryzae, we compared expression levels of ten genes in mutant versus wild type with and without H₂O₂. Our results indicated that the HYR1 gene was important for allowing the fungus to tolerate H₂O₂ in vitro and in planta and that this ability was directly related to fungal virulence.     

Host–pathogen interaction after infection of Galleria mellonella with the filamentous fungus Beauveria bassiana

The filamentous fungus Beauveria bassiana is a natural pathogen of the greater wax moth Galleria mellonella. Infection with this fungus triggered systemic immune response in G. mellonella; nevertheless, the infection was lethal if spores entered the insect hemocel. We observed melanin deposition in the insect cuticle and walls of air bags, while the invading fungus interrupted tissue continuity. We have shown colonization of muscles, air bags, and finally colonization and complete destruction of the fat body—the main organ responsible for the synthesis of defense molecules in response to infection. This destruction was probably not caused by simple fungal growth, because the fat body was not destroyed during colonization with a human opportunistic pathogen Candida albicans. This may mean that the infecting fungus is able to destroy actively the insect's fat body as part of its virulence mechanism. Finally, we were unable to reduce the extremely high virulence of B. bassiana against G. mellonella by priming of larvae with thermally inactivated fungal spores.     

Involvement of UDP-glucose pyrophosphorylase from Verticillium dahliae in cell morphogenesis,stress responses,and host infection

UDP-glucose pyrophosphorylase (UGP, EC 2.7.7.9) is an essential enzyme involved in carbohydrate metabolism. In Saccharomyces cerevisiae and other fungi, the UGP gene is indispensable for normal cell development, polysaccharide synthesis, and stress response. However, the function of the UGP homolog in plant pathogenic fungi has been rarely explored during pathogenesis. In this study, we characterize a UGP homolog named VdUGP from Verticillium dahliae, a soil-borne fungus that causes plant vascular wilt. In comparison with wild-type strain V07DF2 and complementation strains, the VdUGP knocked down mutant 24C9 exhibited sensitivity to sodium dodecyl sulfate (perturbing membrane integrity) and high sodium chloride concentration (high osmotic pressure stress). More than 25 % of the conidia of the mutant developed into short and swollen hypha and formed hyperbranching and compact colonies. The mutant exhibited decreased virulence on cotton and tobacco seedlings. Further investigation determined that the germination of the mutant spores was significantly delayed compared with the wild-type strain on the host roots. RNA-seq analysis revealed that a considerable number of genes encoding secreted proteins and carbohydrate-active enzymes were significantly downregulated in the mutant at an early stage of infection compared with those of the wild-type strain. RNA-seq data indicated that mutation affected many Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways both in the pathogen and in the inoculated plants at the infection stage. These alterations of the mutant in cultural phenotypes, virulence, and gene expression profiles clearly indicated that VdUGP played important roles in fungal cell morphogenesis, stress responses, and host infection.     

Real-Time PCR Detection of Dogwood Anthracnose Fungus in Historical Herbarium Specimens from Asia

Cornus species (dogwoods) are popular ornamental trees and important understory plants in natural forests of northern hemisphere. Dogwood anthracnose, one of the major diseases affecting the native North American Cornus species, such as C. florida, is caused by the fungal pathogen Discula destructiva. The origin of this fungus is not known, but it is hypothesized that it was imported to North America with its host plants from Asia. In this study, a TaqMan real-time PCR assay was used to detect D. destructiva in dried herbarium and fresh Cornus samples. Several herbarium specimens from Japan and China were detected positive for D. destructiva, some of which were collected before the first report of the dogwood anthracnose in North America. Our findings further support that D. destructiva was introduced to North America from Asia where the fungus likely does not cause severe disease.     

Cryptococcus neoformans: A sugar-coated killer with designer genes

Cryptococcus neoformans has become a common central nervous system pathogen as the immunocompromised populations enlarge world-wide. This encapsulated yeast has significant advantages for the study of fungal pathogenesis and these include: (1) a clinically important human pathogen; (2) a tractable genetic system; (3) advanced molecular biology foundation; (4) understanding of several virulence phenotypes; (5) well-studied pathophysiology; and (6) robust animal models. With the use of a sequenced genome and site-directed mutagenesis to produce specific null mutants, the virulence composite of C. neoformans has begun to be identified one gene at a time. Studies into capsule production, melanin synthesis, high temperature growth, metabolic pathways and a variety of signaling pathways have led to understandings of what makes this yeast a pathogen at the molecular level. Multiple principles of molecular pathogenesis have been demonstrated in virulence studies with C. neoformans. These include evolutionary differences between the varieties of C. neoformans in their genes for virulence, quantitative impact of genes on the virulence composite, species and site-specific importance of a virulence gene, gene expression correlation with its functional importance or phenotype and the impact of a pathogenesis gene on the host immune response. C. neoformans has now become a primary model to study molecular fungal pathogenesis with the goal of identifying drug targets or vaccine strategies.     

Increased Insect Virulence in Beauveria bassiana Strains Overexpressing an Engineered Chitinase

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.     

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.     

Development,Distribution, and Host Studies of the Fungus Macrobiotophthoira vermicola (Entomophthorales)

The life cycle and host range of Macrobiotophthora vermicola were studied. Secondary spores produced from forcibly ejected primary spores adhered to the cuticle of Cruznema tripartitum, germinated, and penetrated the cuticle within 30 minutes. New primary spores were produced within 24 hours of initial spore adhesion. In a host range study, species of Rhabditidae, Diplogasteridae, and Aphelenchoidea were hosts, but not species of Bunonematidae, Tripylidae, Cephalobida, or Tylenchina. Numbers of second-stage Meloidogyne incognita juveniles were not decreased when added to soil seeded with infected C. tripartitum. In six Tennessee soybean fields, Macrobiotophthora vermicola was the most commonly encountered nematode-destroying fungus, followed by a sterile, nonseptate fungus and Arthrobotrys conoides. Nematophagous fungi were isolated more frequently from silt loam soils than from clay soils. Addition of C. tripartitum to soil extract plates as a bait nematode did not increase isolations of nematophagous fungi.