Role of F-box Protein Cdc4 in Fungal Virulence and Sexual Reproduction of Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic yeast-like pathogen that mainly infects immunocompromised individuals and causes fatal meningitis. Sexual reproduction can promote the exchange of genetic material between different strains of C. neoformans, which is one of the reasons leading to the emergence of highly pathogenic and drug-resistant strains of C. neoformans. Although much research has been done on the regulation mechanism of Cryptococcus sexual reproduction, there are few studies on the sexual reproduction regulation of Cryptococcus by the ubiquitin-proteasome system. This study identified an F-box protein, Cdc4, which contains a putative F-box domain and eight WD40 domains. The expression pattern analysis showed that the CDC4 gene was expressed in various developmental stages of C. neoformans, and the Cdc4 protein was localized in the nucleus of cryptococcal cells. In vitro stress responses assays showed that the CDC4 overexpression strains are sensitive to SDS and MMS but not Congo red, implying that Cdc4 may regulate the cell membrane integrity and repair of DNA damage of C. neoformans. Fungal virulence assay showed that although the cdc4Δ mutant grows normally and can produce typical virulence factors such as capsule and melanin, the cdc4Δ mutant completely loses its pathogenicity in a mouse systemic-infection model. Fungal mating assays showed that Cdc4 is also essential for fungal sexual reproduction in C. neoformans. Although normal mating hyphae were observed during mating, the basidiospores’ production was blocked in bilateral mating between cdc4Δ mutants. Fungal nuclei development assay showed that the nuclei failed to undergo meiosis after fusion inside the basidia during the bilateral mating of cdc4Δ mutants, indicating that Cdc4 is critical to regulating meiosis during cryptococcal mating. In summary, our study revealed that the F-box protein Cdc4 is critical for fungal virulence and sexual reproduction in C. neoformans.

The Role of Candida albicans Virulence Factors in the Formation of Multispecies Biofilms With Bacterial Periodontal Pathogens

Periodontal disease depends on the presence of different microorganisms in the oral cavity that during the colonization of periodontal tissues form a multispecies biofilm community, thus allowing them to survive under adverse conditions or facilitate further colonization of host tissues. Not only numerous bacterial species participate in the development of biofilm complex structure but also fungi, especially Candida albicans, that often commensally inhabits the oral cavity. C. albicans employs an extensive armory of various virulence factors supporting its coexistence with bacteria resulting in successful host colonization and propagation of infection. In this article, we highlight various aspects of individual fungal virulence factors that may facilitate the collaboration with the associated bacterial representatives of the early colonizers of the oral cavity, the bridging species, and the late colonizers directly involved in the development of periodontitis, including the “red complex” species. In particular, we discuss the involvement of candidal cell surface proteins—typical fungal adhesins as well as originally cytosolic “moonlighting” proteins that perform a new function on the cell surface and are also present within the biofilm structures. Another group of virulence factors considered includes secreted aspartic proteases (Sap) and other secreted hydrolytic enzymes. The specific structure of the candidal cell wall, dynamically changing during morphological transitions of the fungus that favor the biofilm formation, is equally important and discussed. The non-protein biofilm-composing factors also show dynamic variability upon the contact with bacteria, and their biosynthesis processes could be involved in the stability of mixed biofilms. Biofilm-associated changes in the microbe communication system using different quorum sensing molecules of both fungal and bacterial cells are also emphasized in this review. All discussed virulence factors involved in the formation of mixed biofilm pose new challenges and influence the successful design of new diagnostic methods and the application of appropriate therapies in periodontal diseases.

Flavopiridol Protects Against Fungal Keratitis by Alleviating Inflammation Through the Promotion of Autophagy

Background: Fungal keratitis is a serious infectious keratopathy related to fungal virulence and excessive inflammatory responses. Autophagy exhibits a potent ability to resolve inflammation during fungal infection. This study aimed to investigate the protective function of flavopiridol in fungal keratitis and explore its effects on autophagy.Methods: A mouse model of fungal keratitis was established and then treated with 5 μM flavopiridol. RAW 264.7 cells were treated with 200 nM flavopiridol before fungal stimulation. The severity of corneal diseases was evaluated by slit-lamp microscopy. The expression levels of cytokines were detected by RT-PCR and ELISA. The protein levels of LC3, Beclin-1 and Atg7 were determined by western blot and immunofluorescence. A Cell Counting Kit-8 assay was used to test cell viability. Autolysosomes were detected by transmission electron microscopy (TEM). An inhibitor of autophagy, 3-methyladenine (3-MA), was used to pretreat RAW 264.7 cells. Phagocytosis of RAW 264.7 cells was evaluated by counting colony forming units. A. fumigatus was incubated with flavopiridol, and the hyphae were stained with calcofluor white. Absorbance assay, crystal violet staining and adherence assay were used to detect the antifungal activity of flavopiridol.Results: Flavopiridol treatment notably reduced corneal opacity and the clinical scores of infected corneas. Compared with DMSO treatment, flavopiridol treatment greatly downregulated IL-1β, IL-6 and TNF-a expression in infected corneas. In RAW 264.7 cells, flavopiridol treatment inhibited IL-1β, IL-6 and TNF-a expression but promoted IL-10 expression. TEM images showed that more autolysosomes were presented in infected corneas and RAW 264.7 cells after flavopiridol treatment than after DMSO treatment. Flavopiridol treatment notably upregulated the protein expression of LC3, Beclin-1 and Atg7 in infected corneas as well as in RAW 264.7 cells. 3-MA pretreatment counteracted the cytokine regulation induced by flavopiridol. Moreover, flavopiridol promoted the phagocytosis of RAW 264.7 cells. Flavopiridol also exhibited antifungal activity by restricting fungal growth and limiting fungal biofilm formation and conidial adhesion. Conclusions: Flavopiridol significantly alleviated the inflammation of fungal keratitis by activating autophagy. In addition, flavopiridol promoted the phagocytosis of RAW 264.7 cells and exhibited antifungal function, indicating the potential therapeutic role of flavopiridol in fungal keratitis.

Genome-Wide Identification of bZIP Transcription Factor Genes and Functional Analyses of Two Members in Cytospora chrysosperma

The basic leucine zipper (bZIP) transcription factor (TF) family, one of the largest and the most diverse TF families, is widely distributed across the eukaryotes. It has been described that the bZIP TFs play diverse roles in development, nutrient utilization, and various stress responses in fungi. However, little is known of the bZIP members in Cytospora chrysosperma, a notorious plant pathogenic fungus, which causes canker disease on over 80 woody plant species. In this study, 26 bZIP genes were systematically identified in the genome of C. chrysosperma, and two of them (named CcbZIP05 and CcbZIP23) significantly down-regulated in CcPmk1 deletion mutant (a pathogenicity-related mitogen-activated protein kinase) were selected for further analysis. Deletion of CcbZIP05 or CcbZIP23 displayed a dramatic reduction in fungal growth but showed increased hypha branching and resistance to cell wall inhibitors and abiotic stresses. The CcbZIP05 deletion mutants but not CcbZIP23 deletion mutants were more sensitive to the hydrogen peroxide compared to the wild-type and complemented strains. Additionally, the CcbZIP23 deletion mutants produced few pycnidia but more pigment. Remarkably, both CcbZIP05 and CcbZIP23 deletion mutants were significantly reduced in fungal virulence. Further analysis showed that CcbZIP05 and CcbZIP23 could regulate the expression of putative effector genes and chitin synthesis-related genes. Taken together, our results suggest that CcbZIP05 and CcbZIP23 play important roles in fungal growth, abiotic stresses response, and pathogenicity, which will provide comprehensive information on the CcbZIP genes and lay the foundation for further research on the bZIP members in C. chrysosperma.

The F-Box Protein Fbp1 Regulates Virulence of Cryptococcus neoformans Through the Putative Zinc-Binding Protein Zbp1

The ubiquitin-proteasome system (UPS) is the major protein turnover mechanism that plays an important role in regulating various cellular functions. F-box proteins are the key proteins of the UPS, responsible for the specific recognition and ubiquitination of downstream targets. Our previous studies showed that the F-box protein Fbp1 plays an essential role in the virulence of C. neoformans. However, the molecular mechanism of Fbp1 regulating the virulence of C. neoformans is still unclear. In this study, we analyzed the potential Fbp1 substrates using an iTRAQ-based proteomic approach and identified the zinc-binding protein Zbp1 as a substrate of Fbp1. Protein interaction and stability assays showed that Zbp1 interacts with Fbp1 and is a downstream target of Fbp1. Ubiquitination analysis in vivo showed that the ubiquitination of Zbp1 is dependent on Fbp1 in C. neoformans. Subcellular localization analysis revealed that the Zbp1 protein was localized in the nucleus of C. neoformans cells. In addition, both deletion and overexpression of the ZBP1 gene led to the reduced capsule size, while overexpression has a more significant impact on capsule size reduction. Fungal virulence assays showed that although the zbp1Δ mutants are virulent, virulence was significantly attenuated in the ZBP1 overexpression strains. Fungal load assay showed that the fungal burdens recovered from the mouse lungs decreased gradually after infection, while no yeast cells were recovered from the brains and spleens of the mice infected by ZBP1 overexpression strains. Thus, our results revealed a new determinant of fungal virulence involving the post-translational regulation of a zinc-binding protein.

Thromboxane Mobilizes Insect Blood Cells to Infection Foci

Innate immune responses are effective for insect survival to defend against entomopathogens including a fungal pathogen, Metarhizium rileyi, that infects a lepidopteran Spodoptera exigua. In particular, the fungal virulence was attenuated by cellular immune responses, in which the conidia were phagocytosed by hemocytes (insect blood cells) and hyphal growth was inhibited by hemocyte encapsulation. However, the chemokine signal to drive hemocytes to the infection foci was little understood. The hemocyte behaviors appeared to be guided by a Ca2+ signal stimulating cell aggregation to the infection foci. The induction of the Ca2+ signal was significantly inhibited by the cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A2 or B2 (TXA2 or TXB2) among COX products was the most effective to recover the Ca2+ signal and hemocyte aggregation. TXB2 alone induced a microaggregation behavior of hemocytes under in vitro conditions. Indeed, TXB2 titer was significantly increased in the plasma of the infected larvae. The elevated TXB2 level was further supported by the induction of phospholipase A2 (PLA2) activity in the hemocytes and subsequent up-regulation of COX-like peroxinectins (SePOX-F and SePOX-H) in response to the fungal infection. Finally, the expression of a thromboxane synthase (Se-TXAS) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited the Ca2+ signal and hemocyte aggregation around fungal hyphae, which were rescued by the addition of TXB2. Without any ortholog to mammalian thromboxane receptors, a prostaglandin receptor was essential to mediate TXB2 signal to elevate the Ca2+ signal and mediate hemocyte aggregation behavior. Specific inhibitor assays suggest that the downstream signal after binding TXB2 to the receptor follows the Ca2+-induced Ca2+ release pathway from the endoplasmic reticulum of the hemocytes. These results suggest that hemocyte aggregation induced by the fungal infection is triggered by TXB2via a Ca2+ signal through a PG receptor.

A systemic analysis of amino acid transporters identifies Gnp2 as the main proline permease in Candida albicans

The tight association of Candida albicans with the human host has driven the evolution of mechanisms that permit metabolic flexibility. Amino acids, present in free form or peptide bound, are an abundant carbon and nitrogen source in many host niches. Further,the capacity to sense and utilize certain amino acids, like proline, is directly linked to virulence. The C. albicans genome encodes for at least 24 amino acid permeases (AAPs), highlighting the importance of flexible amino acid uptake for fungal growth and virulence. Although the substrate specificity and role of certain AAPs has been investigated, a comprehensive characterization was missing. Therefore, we assembled a library of AAP deletion strains, which was tested for resistance to toxic amino acid analogs. Most striking was the specific resistance of gnp2Δ to the proline analog 3,4-dehydroproline. Subsequent tests validated that Gnp2 is a specific proline permease in C. albicans, which is contrary to the model yeast Saccharomyces cerevisiae where proline transport is mediated by four permeases. Furthermore, the induction of GNP2 appears to be independent of the SPS (Ssy1-Ptr3-Ssy5) regulatory pathway that controls proline utilization in the model yeast, pointing towards rewired proline uptake in C. albicans. Additionally, strains lacking GNP2were unable to respond to proline-induced filamentation, displayed decreased cytotoxicity to macrophages and showed increased sensitivity to oxidative stress, underlining the importance of proline uptake for fungal virulence. Taken together, the role of Gnp2-mediated proline uptake illustrates the importance of metabolism-driven virulence in C. albicans.

Short peptides derived from EntV inhibit virulence and biofilm formation in Candida albicans

Candida albicans exists as a member of the commensal flora of the skin and gut where many complex polymicrobial interactions occur with genera such as Pseudomonas, Staphylococcus, and Streptococcus. Some of these interactions potentiate or inhibit virulence. The bacterial gastrointestinal commensal speciesEnterococcus faecalisproduces a small peptide, EntV, that modulates C. albicans virulence. The active 68 amino acid EntV peptide inhibits biofilm formationin vitro; biofilm-related infections are difficult to treat with current therapeutics. EntV also attenuates fungal virulence in a Caenorhabditis elegansinfection model and a murine oral candidiasis model. We sought to identify the regions of EntV responsible for the anti-fungal activity, and based on structural information, we hypothesized that it could be localized to a single helix of the mature peptide. In this study, we report that smaller peptides derived from this helix ranging from 12 to 16 amino acids have equal to improved efficacy in inhibiting C. albicans virulence andbiofilm formation. These smaller peptides attenuate virulence in the C. elegans infection model, inhibit initial adhesion to abiotic surfaces, and reduce the size of mature biofilms measured by confocal microscopy. Further trimming of these peptides to fewer than 11 amino acids reduces and eventually eliminates activity. These data indicate that EntV-derived peptides warrant further investigation as potential non-fungicidal additives to medical devices and antifungal therapeutics.

Transcription factor lineages in plant-pathogenic fungi, connecting diversity with fungal virulence

Plant-pathogenic fungi span diverse taxonomic lineages. Their host-infection strategies are often specialised and require the coordinated regulation of molecular virulence factors. Transcription factors (TFs) are fundamental regulators of gene expression, controlling development and virulence in plant pathogenic fungi. Recent research has established regulatory roles for several taxonomically conserved fungal TFs, but the evolution of specific virulence regulators is not well understood. This study sought to explore the representation of TFs across a taxonomically-diverse range of fungi, with a focus on plant pathogens. A significant trend was observed among the obligate, host-associated pathogens, which possess a reduced overall TF repertoire, alluding to a lack of pressure for maintaining diversity. A novel orthology-based analysis is then presented that refined TF classifications, traditionally based on the nature of the DNA-binding domains. Using this analysis, cases of TF over/underrepresentation across fungal pathogen lineages are systematically highlighted. Specific examples are then explored and discussed that included the TF orthologues of Ste12, Pf2 and EBR1, plus phytotoxic secondary-metabolite cluster regulators, which all presented novel and distinct evolutionary insights. Ultimately, as the examples presented demonstrate, this resource can be interrogated to guide functional studies that seek to characterise virulence-specific regulators and shed light on the factors underpinning plant pathogenicity.

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.