Virulence Of Fungal Pathogens Research Articles

Ergosterol Biosynthesis and Regulation Impact the Antifungal Resistance and Virulence of Candida spp.

Ergosterol is a key fungal sterol that is mainly found in the plasma membrane and is responsible for the proper membrane structure, rigidity, permeability and activity of membrane proteins. Ergosterol plays a crucial role in the ability of fungi to adapt to environmental stresses. The biosynthesis of ergosterol is also intimately connected with the antifungal resistance and virulence of pathogenic fungi. The most common etiological agents of life-threatening fungal infections are yeasts belonging to the genus Candida. The antifungal agents mostly used to treat Candida spp. infections are azoles, which act as competitive inhibitors of sterol demethylase, a key enzyme in the fungal ergosterol biosynthetic pathway. Although most studies on ergosterol biosynthesis, its regulation and the uptake of sterols are from the baker’s yeast Saccharomyces cerevisiae, the study of ergosterol biosynthesis and its relationship to antifungal drug resistance and virulence in pathogenic fungi is of utmost importance. The increasing antifungal drug resistance of Candida spp. and the limited armamentarium of antimycotics pose a challenge in the development of new therapeutic approaches. This review summarizes the available data on ergosterol biosynthesis and related phenomena in Candida albicans and non-albicans Candida species (Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida auris) with special emphasis on C. albicans and C. glabrata as the most common etiological agents of systemic candidiasis.

A Review on Molecular Mechanism of Plant Immunity against Fungal Pathogens

The molecular mechanisms of plant immunity, with a particular focus on how plants defend themselves against fungal pathogens. Plant immunity is a complex, multi-layered system involving pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), which together form a robust defense against a wide array of pathogens. Advances in genomics and transcriptomics have significantly enhanced our understanding of these immune mechanisms by identifying key resistance (R) genes and uncovering the transcriptional networks that regulate immune responses. Proteomics and metabolomics further elucidate the functional aspects of immunity, revealing how proteins and metabolites are mobilized during pathogen attack. The advent of gene editing technologies, particularly CRISPR-Cas9, has opened new avenues for enhancing plant immunity by enabling precise modifications of genes associated with disease resistance. The ever-evolving nature of fungal pathogens, driven by genetic diversity and environmental changes, poses ongoing challenges. Emerging pathogens and the breakdown of existing resistance in crops underscore the need for durable resistance strategies, which can be achieved through the pyramiding of multiple R genes, susceptibility gene knockouts, and the harnessing of beneficial plant microbiomes. As climate change exacerbates the spread and virulence of fungal pathogens, developing climate-resilient crops that can withstand both abiotic stresses and pathogen pressures is becoming increasingly important. Future research should prioritize understanding the molecular dynamics of plant-pathogen interactions, leveraging new technologies for crop improvement, and fostering interdisciplinary collaboration to address these challenges. Ultimately, translating these scientific advances into practical applications will be crucial for ensuring global food security and sustainable agricultural systems in the face of mounting environmental and biological threats.

Microsporidian coinfection reduces fitness of a fungal pathogen due to rapid host mortality.

Infection outcomes can be strongly context dependent, shifting a host-symbiont relationship along a parasitism-mutualism continuum. Numerous studies show that under stressful conditions, symbionts that are typically mutualistic can become parasitic. The reverse possibility, a parasite becoming mutualistic, has received much less study. We investigated whether the parasitic microsporidium Ordospora pajunii can become beneficial for its host Daphnia dentifera in the presence of the more virulent fungal pathogen Metschnikowia bicuspidata. We found that, even though infection with O. pajunii reduces the frequency of penetration of M. bicuspidata spores into the host body cavity, it does not improve the survival or reproduction of the host; conversely, coinfection increased the mortality of Daphnia. This shorter lifespan of coinfected hosts disrupted the life cycle of M. bicuspidata, greatly reducing its fitness. Thus, coinfection with both pathogens was detrimental to the host at the individual level but might be beneficial for the host population as a result of greatly reduced production of M. bicuspidata spores. If so, this would mean that O. pajunii outbreaks should delay or prevent M. bicuspidata outbreaks. In support of this, in an analysis of dynamics of naturally occurring outbreaks in two lakes where these pathogens co-occur, we found a time lag in occurrence between O. pajunii and M. bicuspidata, with M. bicuspidata epidemics only occurring after the collapse of O. pajunii epidemics. Thus, these results suggest that the interaction between co-occurring symbionts, and the net impact of a symbiont on a host, might be qualitatively different at different scales.IMPORTANCEUnderstanding the factors that modify infection probability and virulence is crucial for identifying the drivers of infection outbreaks and modeling disease epidemic progression, and increases our ability to control diseases and reduce the harm they cause. One factor that can strongly influence infection probability and virulence is the presence of other pathogens. However, while coexposures and coinfections are incredibly common, we still have only a limited understanding of how pathogen interactions alter infection outcomes or whether their impacts are scale dependent. We used a system of one host and two pathogens to show that sequential coinfection can have a tremendous impact on the host and the infecting pathogens and that the outcome of (co-)infection can be negative or positive depending on the focal organization level.

Hormesis in Pathogenic and Biocontrol Fungi: From Inhibition to Stimulation

Hormesis, a biphasic response where low doses of stressors stimulate growth and high doses inhibit it, has significant implications for agricultural chemical use. This review explores the impact of low-dose fungicides or pesticides on pathogenic and biocontrol fungi. This study highlights how sublethal dose exposure can enhance growth and virulence of fungal pathogens, while also potentially increasing their stress tolerance at higher doses. This phenomenon complicates fungicide resistance management strategies. The review focuses on specific fungicides such as dimethachlone, prochloraz, carbendazim, and others, and their hormetic effects on fungal pathogens. Furthermore, the effects of low-dose pesticides and fungicides on beneficial fungi like entomopathogenic and arbuscular mycorrhizal fungi (AMF) are discussed. Here, low-dose exposure can stimulate the germination of entomopathogenic fungal conidia and enhance AMF root colonization and phosphorus uptake. However, hormesis can have trade-offs, potentially leading to unforeseen consequences for the organism. A thorough understanding of hormesis and dose-dependent fungicide effects is crucial for optimizing disease management and agricultural practices. This knowledge can inform strategies to minimize pathogen resistance and promote sustainable approaches. Implementing integrated disease management that combines fungicides with different modes of action alongside other control methods can be effective. Careful consideration of fungicide doses and potential hormesis effects is paramount for ensuring sustainable plant disease control and food security.

Divergent mitochondrial responses and metabolic signal pathways secure the azole resistance in Crabtree-positive and negative Candida species.

Azole drugs are the main therapeutic drugs for invasive fungal infections. However, azole-resistant strains appear repeatedly in the environment, posing a major threat to human health. Several reports have shown that mitochondria are associated with the virulence of pathogenic fungi. However, there are few studies on the mechanisms of mitochondria-mediated azoles resistance. Here, we first performed mitochondrial proteomic analysis on multiple Candida species (Candida albicans, Nakaseomyces glabrata, Pichia kudriavzevii, and Candida auris) and analyzed the differentially expressed mitochondrial proteins (DEMPs) between azole-sensitive and azole-resistant Candida species. Subsequently, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene ontology analysis, and protein-protein interaction network analysis of DEMPs. Our results showed that a total of 417, 165, and 25 DEMPs were identified in resistant C. albicans, N. glabrata, and C. auris, respectively. These DEMPs were enriched in ribosomal biogenesis at cytosol and mitochondria, tricarboxylic acid cycle, glycolysis, transporters, ergosterol, and cell wall mannan biosynthesis. The high activations of these cellular activities, found in C. albicans and C. auris (at low scale), were mostly opposite to those observed in two fermenter species-N. glabrata and P. kudriavzevii. Several transcription factors including Rtg3 were highly produced in resistant C. albicans that experienced a complex I activation of mitochondrial electron transport chain (ETC). The reduction of mitochondrial-related activities and complex IV/V of ETC in N. glabrata and P. kudriavzevii was companying with the reduced proteins of Tor1, Hog1, and Snf1/Snf4.IMPORTANCECandida spp. are common organisms that cause a variety of invasive diseases. However, Candida spp. are resistant to azoles, which hinders antifungal therapy. Exploring the drug-resistance mechanism of pathogenic Candida spp. will help improve the prevention and control strategy and discover new targets. Mitochondria, as an important organelle in eukaryotic cells, are closely related to a variety of cellular activities. However, the role of mitochondrial proteins in mediating azole resistance in Candida spp. has not been elucidated. Here, we analyzed the mitochondrial proteins and signaling pathways that mediate azole resistance in Candida spp. to provide ideas and references for solving the problem of azole resistance. Our work may offer new insights into the connection between mitochondria and azoles resistance in pathogenic fungi and highlight the potential clinical value of mitochondrial proteins in the treatment of invasive fungal infections.

The basic leucine zipper transcription factor MeaB is critical for biofilm formation, cell wall integrity, and virulence in Aspergillus fumigatus.

The regulation of fungal cell wall biosynthesis is crucial for cell wall integrity maintenance and directly impacts fungal pathogen virulence. Although numerous genes are involved in fungal cell wall polysaccharide biosynthesis through multiple pathways, the underlying regulatory mechanism is still not fully understood. In this study, we identified and functionally characterized a direct downstream target of SomA, the basic-region leucine zipper transcription factor MeaB, playing a certain role in Aspergillus fumigatus cell wall integrity. Loss of meaB reduces hyphal growth, causes severe defects in galactosaminogalactan-mediated biofilm formation, and attenuates virulence in a Galleria mellonella infection model. Furthermore, the meaB null mutant strain exhibited hypersensitivity to cell wall-perturbing agents and significantly alters the cell wall structure. Transcriptional profile analysis revealed that MeaB positively regulates the expression of the galactosaminogalactan biosynthesis and β-1,3-glucanosyltransferase genes uge3, agd3, and sph3 and gel1, gel5, and gel7, respectively, as well as genes involved in amino sugar and nucleotide sugar metabolism. Further study demonstrated that MeaB could respond to cell wall stress and contribute to the proper expression of mitogen-activated protein kinase genes mpkA and mpkC in the presence of different concentrations of congo red. In conclusion, A. fumigatus MeaB plays a critical role in cell wall integrity by governing the expression of genes encoding cell wall-related proteins, thus impacting the virulence of this fungus.IMPORTANCEAspergillus fumigatus is a common opportunistic mold that causes life-threatening infections in immunosuppressed patients. The fungal cell wall is a complex and dynamic organelle essential for the development of pathogenic fungi. Genes involved in cell wall polysaccharide biosynthesis and remodeling are crucial for fungal pathogen virulence. However, the potential regulatory mechanism for cell wall integrity remains to be fully defined in A. fumigatus. In the present study, we identify basic-region leucine zipper transcription factor MeaB as an important regulator of cell wall galactosaminogalactan biosynthesis and β-1,3-glucan remodeling that consequently impacts stress response and virulence of fungal pathogens. Thus, we illuminate a mechanism of transcriptional control fungal cell wall polysaccharide biosynthesis and stress response. As these cell wall components are promising therapeutic targets for fungal infections, understanding the regulatory mechanism of such polysaccharides will provide new therapeutic opportunities.

The influence of amoeba metal homeostasis on antifungal activity against Cryptococcus gattii.

Free-living amoebas are natural predators of fungi, including human pathogens of the Cryptococcus genus. To survive and proliferate inside phagocytes, cryptococcal cells must acquire several nutrients. Zinc is fundamental for all life forms and develops a crucial role in the virulence of fungal pathogens, phagocytes reduce the availability of this metal to reduce the development of infection. The Acanthamoeba castellanii ACA1_271600 gene codes a metal transporter that is possibly associated with such antifungal strategy. Here, we evaluated the impact of A. castellanii metal homeostasis on C. gattii survival. Gene silencing of ACA1_271600 was performed and the interaction outcome of amoeba cells with both WT and zinc homeostasis-impaired mutant cryptococcal cells was evaluated. Decreased levels of ACA1_271600 in silenced amoeba cells led to higher proliferation of such cryptococcal strains. This effect was more pronounced in the zip1 mutant of C. gattii, suggesting that ACA1_271600 gene product modulates metal availability in Cryptococcus-infected amoebae. In addition, a systems biology analysis allowed us to infer that ACA1_271600 may also be involved in other biological processes that could compromise amoebae activity over cryptococcal cells. These results support the hypothesis that A. castellanii can apply nutritional immunity to hamper cryptococcal survival.

Fus3 regulates asexual development and trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora

Mitogen-activated protein kinase (MAPK) Fus3 is an essential regulator of cell differentiation and virulence in fungal pathogens of plants and animals. However, the function and regulatory mechanism of MAPK signaling in nematode-trapping (NT) fungi remain largely unknown. NT fungi can specialize in the formation of "traps", an important indicator of transition from a saprophytic to a predatory lifestyle. Here, we characterized an orthologous Fus3 in a typical NT fungus Arthrobotrys oligospora using multi-phenotypic analysis and multi-omics approaches. Our results showed that Fus3 plays an important role in asexual growth and development, conidiation, stress response, DNA damage, autophagy, and secondary metabolism. Importantly, Fus3 plays an indispensable role in hyphal fusion, trap morphogenesis, and nematode predation. Moreover, we constructed the regulatory networks of Fus3 by means of transcriptomic and yeast two-hybrid techniques. This study provides insights into the mechanism of MAPK signaling in asexual development and pathogenicity of NT fungi.

Genetic polymorphism and plant growth promotion traits of potent fungal entomopathogens of rice leaf folder.

Entomopathogenic fungal biocides are preferred for environment friendly sustainable management of insect pests due to their host specificity and harmlessness to non-target insects. Plant growth promotion (PGP) functions of the entomofungi are also important attributes but hitherto insignificantly explored. Therefore, virulence of 17 natural fungal entomocides (Cordyceps, Beauveria, Metarhizium, Nomuraea, Fusarium, Verticillium, Trichoderma and Paecilomyces spp.) were evaluated for pathogenicity against five rice pests (brown plant hopper (Nilaparvata lugens) and green leaf hopper (Nephotettix virescens) nymphs, leaf folder (Cnaphalocrosis medinalis) and yellow stem borer (Scirpophaga incertulas) larvae and swarming caterpillar (Spodoptera mauritia), respectively), and PGP traits of the potent leaf folder pathogens. Among the fungi, only the leaf folder pathogens (3 isolates each of Beauveria and Metarhizium spp.) infected > 50% (80-90%) larvae but other fungi were ineffective as infected < 50% (0-47%) insects. Besides, the leaf folder pathogens exhibited diverse PGP traits such as organic/inorganic phosphate solubilization (104.7-236.4µg/ml), and siderophore, ammonia, hydrogen cyanide (HCN), indole production etc. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequences repeat (SSR) and internal transcribed spacers (ITS) analysis ascertained strain identity and genetic (inter and intra-specific) diversity among the potent biocides Beauveria and Metarhizium spp. The virulent natural fungal pathogens of rice pests with polyvalent PGP traits may be prospected for rice growth promotion and biocontrol of leaf folder.

The Candida glabrata Parent Strain Trap: How Phenotypic Diversity Affects Metabolic Fitness and Host Interactions.

Reference strains improve reproducibility by standardizing observations and methodology, which has ultimately led to important insights into fungal pathogenesis. However, recent investigations have highlighted significant genotypic and phenotypic heterogeneity across isolates that influence genetic circuitry and virulence within a species. Candida glabrata is the second leading cause of candidiasis, a life-threatening infection, and undergoes extensive karyotype and phenotypic changes in response to stress. Much of the work conducted on this pathogen has focused on two sequenced strains, CBS138 (ATCC 2001) and BG2. Few studies have compared these strains in detail, but key differences include mating type and altered patterns of expression of EPA adhesins. In fact, most C. glabrata isolates and BG2 are MATa, while CBS138 is MATα. However, it is not known if other phenotypic differences between these strains play a role in our understanding of C. glabrata pathogenesis. Thus, we set out to characterize metabolic, cell wall, and host-interaction attributes for CBS138 and BG2. We found that BG2 utilized a broader range of nitrogen sources and had reduced cell wall size and carbohydrate exposure than CBS138, which we hypothesized results in differences in innate immune interactions and virulence. We observed that, although both strains were phagocytosed to a similar extent, BG2 replicated to higher numbers in macrophages and was more virulent during Galleria mellonella infection than CBS138 in a dose-dependent manner. Interestingly, deletion of SNF3, a major nutrient sensor, did not affect virulence in G. mellonella for BG2, but significantly enhanced larval killing in the CBS138 background compared to the parent strain. Understanding these fundamental differences in metabolism and host interactions will allow more robust conclusions to be drawn in future studies of C. glabrata pathogenesis. IMPORTANCE Reference strains provide essential insights into the mechanisms underlying virulence in fungal pathogens. However, recent studies in Candida albicans and other species have revealed significant genotypic and phenotypic diversity within clinical isolates that are challenging paradigms regarding key virulence factors and their regulation. Candida glabrata is the second leading cause of candidiasis, and many studies use BG2 or CBS138 for their investigations. Therefore, we aimed to characterize important virulence-related phenotypes for both strains that might alter conclusions about C. glabrata pathogenesis. Our study provides context for metabolic and cell wall changes and how these may influence host interaction phenotypes. Understanding these differences is necessary to support robust conclusions about how virulence factors may function in these and other very different strain backgrounds.

Роль фитогормонов в регуляции экспрессии генов эффекторов и специфических факторов транскрипции патогена Stagonospora nodorum в культуре

The causative agent of wheat blotch the pathogenic fungus Stagonospora nodorum Berk. is one of the most harmful wheat pathogens. Necrotrophic effectors (NE) have been identified in the pathogen, which ensure the virulence of S. nodorum strains to the host plant, in the genome of which the dominant susceptibility gene corresponding to the effector is present. Currently, the study of the regulation of the genes expression responsible for virulence is becoming an increasingly important objective. Recently, the role of various transcription factors (TFs) in the regulation of effector genes expression in the fungal pathogens has been established. However, new data are emerging on the effect of hormones on the growth, development, and virulence of pathogenic fungi. Therefore, the aim of our research was to study the effect of cytokinins (CK) and abscisic acid (ABA) on the expression of the NE SnToxA, SnTox1, SnTox3, and TF SnStuA, SnPf2, and SnCon7 genes in various isolates of the pathogen S. nodorum SnB, Sn4VD, Sn9MN-3A, and Sn1SP, which differ in virulence. Our results showed that the intensity of NE and TF gene expression was associated with the aggressiveness of the pathogen isolate, and TF gene expression correlated with NE gene expression. The highest expression of NE genes was found in SnB and Sn1SP isolates. When the hormones ABA or CK were added to the cultivation medium, they mainly had a positive effect on the expression of the NE and TF genes. ABA treatment increased the expression of all three NE genes SnToxA, SnTox3, and SnTox1. Treatment with CK had a positive effect only on the expression of the SnTox3 gene. Based on the results, an assumption was made about the indirect effect of hormones on virulence factors through the regulation of growth and metabolism of fungi.

Calcineurin Inhibitor CN585 Exhibits Off-Target Effects in the Human Fungal Pathogen Aspergillus fumigatus.

Calcineurin (CN) is an attractive antifungal target as it is critical for growth, stress response, drug resistance, and virulence in fungal pathogens. The immunosuppressive drugs, tacrolimus (FK506) and cyclosporin A (CsA), are fungistatic and specifically inhibit CN through binding to their respective immunophilins, FK506-binding protein (FKBP12), and cyclophilin (CypA). We are focused on CN structure-based approaches for the development of non-immunosuppressive FK506 analogs as antifungal therapeutics. Here, we examined the effect of the novel CN inhibitor, CN585, on the growth of the human pathogen Aspergillus fumigatus, the most common cause of invasive aspergillosis. Unexpectedly, in contrast to FK506, CN585 exhibited off-target effect on A. fumigatus wild-type and the azole- and echinocandin-resistant strains. Unlike with FK506 and CsA, the A. fumigatus CN, FKBP12, CypA mutants (ΔcnaA, Δfkbp12, ΔcypA) and various FK506-resistant mutants were all sensitive to CN585. Furthermore, in contrast to FK506 the cytosolic to nuclear translocation of the CN-dependent transcription factor (CrzA-GFP) was not inhibited by CN585. Molecular docking of CN585 onto human and A. fumigatus CN complexes revealed differential potential binding sites between human CN versus A. fumigatus CN. Our results indicate CN585 may be a non-specific inhibitor of CN with a yet undefined antifungal mechanism of activity.

The Tricalbin-Family Endoplasmic Reticulum-Plasma Membrane Tethering Proteins Attenuate ROS-Involved Caspofungin Sensitivity in Candida albicans.

The endoplasmic reticulum-plasma membrane (ER-PM) contacts are one kind of important membrane contact structures in eukaryotic cells, which mediate material and message exchange between the ER and the PM. However, the specific types and functions of ER-PM tethering proteins are poorly understood in the human fungal pathogen Candida albicans. In this study, we observed that the two tricalbin-family proteins, i.e., Tcb1 and Tcb3, were colocalized with the ER-PM contacts in C. albicans. Deletion of the tricalbin-encoding genes TCB1 and TCB3 remarkably reduced ER-PM contacts, suggesting that tricalbins are ER-PM tethering proteins of C. albicans. Stress sensitivity assays showed that the TCB-deleted strains, including tcb1Δ/Δ, tcb3Δ/Δ, and tcb1Δ/Δ tcb3Δ/Δ, exhibited hypersensitivity to cell wall stress induced by caspofungin. Further investigation revealed that caspofungin induced drastic reactive oxygen species (ROS) accumulation in the mutants, which was attributed to enhanced oxidation of Ero1 in the ER lumen. Removal of intracellular ROS by the ROS scavenger vitamin C rescued the growth of the mutants under caspofungin treatment, indicating that Ero1 oxidation-related ROS accumulation was involved in caspofungin hypersensitivity of the mutants. Moreover, deletion of the TCB genes decreased secretion of extracellular aspartyl proteinases, reduced transport of the cell wall protein Hwp1 from the cytoplasm to the cell wall, and attenuated virulence of the fungal pathogen. This study sheds a light on the role of ER-PM tethering proteins in maintenance of cell wall integrity and virulence in fungal pathogens. IMPORTANCE The endoplasmic reticulum-plasma membrane contacts are important membrane contact structures in eukaryotic cells, functioning in material and message exchange between the ER and the PM. We observed that the two tricalbin-family endoplasmic reticulum-plasma membrane contact proteins are required for tolerance to caspofungin-induced cell wall stress in the pathogenic fungus Candida albicans. The tricalbin mutants exhibited hypersensitivity to cell wall stress induced by caspofungin. Further investigation revealed that Ero1 oxidation-related reactive species oxygen accumulation was involved in caspofungin hypersensitivity of the tricalbin mutants. Moreover, loss of tricalbins reduced secretion of extracellular aspartyl proteinases, decreased transport of the cell wall proteins from the cytoplasm to the cell wall, and attenuated virulence of the fungal pathogen. This study uncovers the role of ER-PM tethering proteins in sustaining protein secretion, maintenance of cell wall integrity and virulence in fungal pathogens.

Multiple F-Box Proteins Collectively Regulate Cell Development and Pathogenesis in the Human Pathogen Cryptococcus neoformans.

The ubiquitin-proteasome system (UPS) mediates intracellular proteins degradation that influences various cellular functions in eukaryotic cells. The UPS is also involved in the development and virulence of pathogenic fungi. F-box proteins, which are part of the SCF (Skp1-Cullin-F-box protein) ligase, are a key component of UPS and are essential for the recognition of specific substrates. In this study, we identified 20 F-box proteins in C. neoformans and obtained deletion mutants for 19 of them. A comprehensive phenotypic analysis of these mutants revealed the diverse function of F-box proteins in stress response, cell size regulation, sexual reproduction, antifungal drug resistance, and fungal virulence in C. neoformans. The importance of three F-box proteins: Fbp4, Fbp8, and Fbp11, in these cellular functions were characterized in detail. This study provides an overall view of the F-box gene family in C. neoformans, which will lead to a better understanding of the function of fungal SCF E3 ligase-mediated UPS in fungal development and pathogenesis.

Associations of Rap1 with Cell Wall Integrity, Biofilm Formation, and Virulence in Candida albicans.

Rap1 (repressor activator protein 1) is a multifunctional protein, playing important roles in telomeric and nontelomeric functions in many eukaryotes. Candida albicans Rap1 has been previously shown to be involved in telomeric regulation, but its other functions are still mostly unknown. In this study, we found that the deletion of the RAP1 gene altered cell wall properties, composition, and gene expression. In addition, deletion of RAP1 affected C. albicans biofilm formation and modulated phagocytosis and cytokine release by host immune cells. Finally, the RAP1 gene deletion mutant showed attenuation of C. albicans virulence in a Galleria mellonella infection model. Therefore, these findings provide new insights into Rap1 functions that are particularly relevant to pathogenesis and virulence of C. albicans. IMPORTANCE C. albicans is an important fungal pathogen of humans. The cell wall is the outermost layer of C. albicans and is important for commensalism and infection by this pathogen. Moreover, the cell wall is also an important target for antifungals. Studies of how C. albicans maintains its cell wall integrity are critical for a better understanding of fungal pathogenesis and virulence. This work focuses on exploring unknown functions of C. albicans Rap1 and reveals its contribution to cell wall integrity, biofilm formation, and virulence. Notably, these findings will also improve our general understanding of complex machinery to control pathogenesis and virulence of fungal pathogens.

Transcription Factor VdCf2 Regulates Growth, Pathogenicity, and the Expression of a Putative Secondary Metabolism Gene Cluster in Verticillium dahliae.

Transcription factors (TFs) bind to the promoters of target genes to regulate gene expression in response to different stimuli. The functions and regulatory mechanisms of transcription factors (TFs) in Verticillium dahliae are, however, still largely unclear. This study showed that a C2H2-type zinc finger TF, VdCf2 (V. dahliae chorion transcription factor 2), plays key roles in V. dahliae growth, melanin production, and virulence. Transcriptome sequencing analysis showed that VdCf2 was involved in the regulation of expression of genes encoding secreted proteins, pathogen-host interaction (PHI) homologs, TFs, and G protein-coupled receptors (GPCRs). Furthermore, VdCf2 positively regulated the expression of VdPevD1 (VDAG_02735), a previously reported virulence factor. VdCf2 thus regulates the expression of several pathogenicity-related genes that also contribute to virulence in V. dahliae. VdCf2 also inhibited the transcription of the Vd276-280 gene cluster and interacted with two members encoding proteins (VDAG_07276 and VDAG_07278) in the gene cluster. IMPORTANCE Verticillium dahliae is an important soilborne phytopathogen which can ruinously attack numerous host plants and cause significant economic losses. Transcription factors (TFs) were reported to be involved in various biological processes, such as hyphal growth and virulence of pathogenic fungi. However, the functions and regulatory mechanisms of TFs in V. dahliae remain largely unclear. In this study, we identified a new transcription factor, VdCf2 (V. dahliae chorion transcription factor 2), based on previous transcriptome data, which participates in growth, melanin production, and virulence of V. dahliae. We provide evidence that VdCf2 regulates the expression of the pathogenicity-related gene VdPevD1 (VDAG_02735) and Vd276-280 gene cluster. VdCf2 also interacts with VDAG_07276 and VDAG_07278 in this gene cluster based on a yeast two-hybrid and bimolecular fluorescence complementation assay. These results revealed the regulatory mechanisms of a pivotal pathogenicity-related transcription factor, VdCf2 in V. dahliae.

Overexpression of salicylic acid methyltransferase reduces salicylic acid-mediated pathogen resistance in poplar

Salicylic acid (SA) is generally considered to be a critical signal transduction factor in plant defenses against pathogens. It could be converted to methyl salicylate (MeSA) for remote signals by salicylic acid methyltransferase (SAMT) and converted back to SA by SA-binding protein 2 (SABP2). In order to verify the function of SAMT in poplar plants, we isolated the full-length cDNA sequence of PagSAMT from 84K poplar and cultivated PagSAMT overexpression lines (OE-2 isolate) to test its role in SA-mediated defenses against the virulent fungal pathogen Botryosphaeria dothidea. Our results showed that after inoculation with B. dothidea, OE-2 significantly increased MeSA content and reduced SA content which is associated with increased expression of SAMT in both infected and uninfected leaves, when compared against the wild type (WT). Additionally, SAMT overexpression plant lines (OE-2) exhibited higher expression of pathogenesis-related genes PR-1 and PR-5, but were still susceptible to B. dothidea suggesting that in poplar SA might be responsible for resistance against this pathogen. This study expands the current understanding of joint regulation of SAMT and SABP2 and the balance between SA and MeSA in poplar responses to pathogen invasion.

S7.4a Vaccine-inducing lung resident CD4 + memory T cells are protective against Cryptococcus gattii infections

S7.4 Pathogenesis and host defense, September 23, 2022, 10:30 AM - 12:00 PM Cryptococccus gattii is a highly virulent fungal pathogen that can cause cryptococcosis in previously healthy individuals. It is not fully understood how innate and acquired immune responses cooperatively suppress C. gattii infection. Hitherto, we have reported the following findings, (1) Specific environment for exposure of dectin-1 and dectin-2 ligands in cryptococcal cells (PLOS ONE 2019, PMID 31398236), (2) CD11b-mediated immune recognition for C. gattii and capsule dependent immune evasion from CD11b recognition (Eur J Immunol 2021, PMID 33728652), (3) Dendritic cells (DCs)-based vaccine induced lung resident memory Th17 cells and ameliorates C. gattii infection (Mucosal Immunol 2018, PMID 30279512), and (4) Neutrophil and opsonization can mediate fungicidal activity for cryptococcal cells (Med Mycol 2019, PMID 30668754). Based on these findings, we have inferred that induction of lung resident memory T cells (lung TRM) and TRM-driven myeloid cells including granulocytes is necessary for controlling this infection. Currently, we are developing new intranasal (IN) vaccines using highly immunogenic whole cell antigens and characterizing protective lung TRM induced by the vaccines. Just as with component vaccines, whole cell vaccines are also featured for cryptococcal infection because these are highly cross-reactive and protective against each serotype (Biol Pharm Bull 2020, PMID 32009111). We developed two IN vaccines using whole cell antigen and adjuvant. Both vaccines induced IL-5-producing lung TRM and significantly reduced fungal burden in lungs and improved survival rates of mice following C. gattii infection, while not protective against C. neoformans infection. Although immune suppressive agent FTY720 can eliminate most circulating T cells and sustain tissue-resident subset, IN vaccines induced Th2-related immune response including eosinophils and IFN-γ independent granuloma, and reduced the fungal burden in lungs after the infection in the presence of FTY720. Vaccine-mediated protection was completely lost in the Rag-1 knockout mice deficient in T- and B-cells, while the fungal burden was reduced in the Rag-1 knockout mice after receiving the immunized CD4 + T cells in the adoptive transfer experiments. These results suggest that Th2-related lung TRM induced by IN vaccines are also protective against C. gattii infection as well as Th17-related counterparts induced by DC vaccine.

The Role of the Glutathione System in Stress Adaptation, Morphogenesis and Virulence of Pathogenic Fungi

Morphogenesis and stress adaptation are key attributes that allow fungal pathogens to thrive and infect human hosts. During infection, many fungal pathogens undergo morphological changes, and this ability is highly linked to virulence. Furthermore, pathogenic fungi have developed multiple antioxidant defenses to cope with the host-derived oxidative stress produced by phagocytes. Glutathione is a major antioxidant that can prevent cellular damage caused by various oxidative stressors. While the role of glutathione in stress detoxification is known, studies of the glutathione system in fungal morphological switching and virulence are lacking. This review explores the role of glutathione metabolism in fungal adaptation to stress, morphogenesis, and virulence. Our comprehensive analysis of the fungal glutathione metabolism reveals that the role of glutathione extends beyond stressful conditions. Collectively, glutathione and glutathione-related proteins are necessary for vitality, cellular development and pathogenesis.

The carbon catabolite repressor CreA is an essential virulence factor of Metarhizium acridum against Locusta migratoria.

CreA has been proved to be a core gene in asexual conidiation in Metarhizium acridum, which regulates the shift of normal conidiation and microcycle conidiation. At present, research on CreA in fungi has focused on carbon source metabolism. There is a lack of research on the effect of CreA in virulence of pathogenic fungi. The virulence of the MaCreA disrupted strain (ΔMaCreA) for Locusta migratoria was lost by topical inoculation bioassay. The formation rate and turgor pressure of the appressoria decreased. Growth of ΔMaCreA in host hemolymph was delayed, and the number of hyphal bodies was significantly reduced. The conidial cell wall of ΔMaCreA became thicker, the mannan content decreased, and the chitin content increased significantly, and it was more sensitive to calcofluor white and Congo Red. α-1,3-Glucan and β-1,3-glucan are more exposed on the surface of ΔMaCreA conidia than on the wild type. Lmspätzle and Lmcactus, the immune response genes in the host Toll pathway, showed stronger transcriptional activities at the early stage of ΔMaCreA invasion. The phenoloxidase activity assay also showed stronger immunostimulation by ΔMaCreA in vitro. The main reasons for the loss of virulence of ΔMaCreA in the topical inoculation were the reduced penetration ability of appressoria, limited growth in hemolymph and stronger insect immunostimulation of ΔMaCreA. © 2022 Society of Chemical Industry.