Hyphal Wall Research Articles

Single-Molecule Localization Microscopy to Study Protein Organization in the Filamentous Fungus Trichoderma atroviride.

Single-molecule localization microscopy has boosted our understanding of biological samples by offering access to subdiffraction resolution using fluorescence microscopy methods. While in standard mammalian cells this approach has found wide-spread use, its application to filamentous fungi has been scarce. This is mainly due to experimental challenges that lead to high amounts of background signal because of ample autofluorescence. Here, we report the optimization of labeling, imaging and data analysis protocols to yield the first single-molecule localization microscopy images of the filamentous fungus Trichoderma atroviride. As an example, we show the spatial distribution of the Sur7 tetraspanin-family protein Sfp2 required for hyphal growth and cell wall stability in this mycoparasitic fungus.

Comparative Analysis of Virulence Factors of Homozygous and Heterozygous Strains of Candida albicans Vaginal Isolates.

Although the epidemiology of pathogenic Candida species is changing due to invasive diseases, Candida albicans has become the common cause of human infections worldwide. Candida albicans is a diploid yeast with a mostly clonal mode of reproduction and without known complete sexual cycle. This species has two heterozygous and homozygous strains at hyphal wall protein 1 gene locus (hwp1). Little is known about virulence factors of these strains. The aim of this study was to evaluate the exoenzyme activity of heterozygous and homozygous C. albicans strains. A total of 60 stock Candida albicans species isolates, which consisted of 30 homozygous and 30 heterozygous strains, were used for exoenzyme activities. We used egg yolk agar, Sabouraud blood agar, and bovine serum albumin agar for evaluation of phospholipase, hemolysin, and proteinase activity, respectively. Homozygous strains of Candida albicans had more phospholipase and proteinase activity than heterozygous strains. However, there were no significant statistical differences between the two strains in the severity of exoenzymes production. Beta hemolysin activity was seen in 100% and 96.7% of the homozygous and heterozygous strains, respectively. The results of this study indicated that both of the strains exhibited exoenzyme activities in different ranges. There were no significant statistical differences in virulence factors between the homozygous and heterozygous strains.

Subcellular localization of Sur7 and its pleiotropic effect on cell wall integrity, multiple stress responses, and virulence of Beauveria bassiana.

Sur7 is one of multiple proteins constituting MCC (membrane compartment of Can1 acting as an arginine/H+ symporter), a crucial membrane domain that can form punctuate eisosome spots on the plasma membrane and execute diverse functions in model yeast but remains poorly understood in filamentous fungi. Here, a Sur7 homolog bearing a typical SUR7 domain and four transmembrane domains was shown to localize in the conidial vesicles and enter vacuoles and appear sporadically on the periphery membrane during hyphal growth in the insect-pathogenic fungus Beauveria bassiana, implicating an involvement of Sur7 in cellular events linked to both plasma membrane and vacuoles. Deletion of sur7 resulted in reduced conidiation capacity and impaired conidial quality, which was featured by slower germination, attenuated virulence, and reduced carbohydrate epitopes (β-N-acetylglucosamine and sialic acids). Also, the hyphal cell walls of the deletion mutant were severely impaired due to ~ 70% reductions in chitin and neutral carbohydrate contents and a moderate increase in alkali-soluble carbohydrate content. Consequently, the deletion mutant became more sensitive to three cell wall perturbing chemicals (Congo red, calcofluor white, and SDS) and an antifungal drug (caspofungin) and surprisingly showed a hypersensitivity to oxidative stress of H2O2 and an increased sensitivity to osmotic stress of NaCl or sorbitol. Its hypersensitivity to H2O2 was associated with transcriptional repression of critical catalase genes required for H2O2 decomposition. These findings unveil that Sur7 takes part in both MCC/eisosome and vacuolar events and hence acts as a sustainer of conidiation capacity, cell wall integrity, multiple stress tolerance, and virulence in B. bassiana. Key points • Sur7 is a component of the crucial membrane domain MCC in Beauveria bassiana. • Sur7 localizes mainly in the vacuoles and sporadically on the periphery membrane. • Sur7 is required for cell wall integrity and has a pleiotropic effect on B. bassiana.

A novel study on the inhibitory effect of marine macroalgal extracts on hyphal growth and biofilm formation of candidemia isolates

Biofilm formation and hyphal growth are considered to be the most serious virulence factors of Candida species in blood causing candidemia infections, which are difficult to treat due to the spread of resistant Candida isolates to most antifungal drugs. Therefore, in this study, we investigated the effect of different types and concentrations of selected macroalgal extracts from Cladostephus spongiosus (Phaeophyta), Laurencia papillosa (Rhodophyta), and Codium arabicum (Chlorophyta) in inhibiting those virulence factors of the isolated Candida. Acetone extract of C. spongiosus (AECS) showed a stronger anticandidal activity against the selected strains than ethanol extract. Candida krusei was the highest biofilm producer among the selected isolates. AECS showed an inhibition of C. krusei biofilm formation as well as a reduction in the viability of preformed biofilms. Also, AECS reduced various sugars in the candidal exo-polysaccaride layer (EPS). Scanning electron microscopy (SEM) and light microscopic images revealed an absence of hyphae and an alteration in the morphology of biofilm cells when treated with AECS. Moreover, AECS downregulated the expression of hyphal specific genes, hyphal wall protein 1 (HWP1), Agglutinin-like protein 1 (ALS1) and fourth secreted aspartyl proteinase (SAP4), which confirmed the inhibitory effect of AECS on hyphal growth and biofilm formation. Gas chromatography-mass spectrophotometer (GC-MS) analysis of AECS showed three major compounds, which were non-existent in the ethanol extract, and might be responsible for the anticandidal activity; these revealed compounds were 4-hydroxy-4-methyl-2-pentanone, n-hexadecenoic acid, and phenol, 2-methoxy-4-(2-propenyl). These active compounds of AECS may be promising for future pharmaceutical applications in the treatment of candidemia.

Antifungal Potential of Nanostructured Crystalline Copper and Its Oxide Forms.

Copper has been used as an antimicrobial agent for over a century and is now being added to commercial fungicides. Nanomaterials have attracted much attention due to the special properties they have over their bulk form. We studied nanostructured copper (Cu-NPs), investigating the potential for improved antifungal properties derived from its special properties and studied any effect that the oxidation of copper (CuO-NPs) may have. We conducted this research against Colletotrichum gloeoesporioides, a devastating pathogen to plants/crops worldwide. Research on the effects of copper on this fungus are limited. Our studies showed that nanoforms of copper had significant antifungal activities, with Cu-NPs offering the most sustainable efficacy and was more effective than its oxidative form (CuO-NPs). Scanning Electron Microscopy (SEM) images of the treated pathogen show that the hyphae had a swollen appearance, lost their filamentous structure, and the mycelia had a powder-like structure, indicating the probable destruction of the hyphal tubular cell wall. X-ray Difractogram (XRD) outputs showed substantial changes in the physical characteristics of the Cu-NPs after interaction with the fungus. This is the first report to demonstrate chemo-physical changes in the metal compounds, opening new insights for further studies on the mechanism of copper’s antifungal properties.

Inhibition of Fungal Growth and Induction of a Novel Volatilome in Response to Chromobacterium vaccinii Volatile Organic Compounds.

The study of chemical bioactivity in the rhizosphere has recently broadened to include microbial metabolites, and their roles in niche construction and competition via growth promotion, growth inhibition, and toxicity. Several prior studies have identified bacteria that produce volatile organic compounds (VOCs) with antifungal activities, indicating their potential use as biocontrol organisms to suppress phytopathogenic fungi and reduce agricultural losses. We sought to expand the roster of soil bacteria with known antifungal VOCs by testing bacterial isolates from wild and cultivated cranberry bog soils for VOCs that inhibit the growth of four common fungal and oomycete plant pathogens, and Trichoderma sp. Twenty one of the screened isolates inhibited the growth of at least one fungus by the production of VOCs, and isolates of Chromobacterium vaccinii had broad antifungal VOC activity, with growth inhibition over 90% for some fungi. Fungi exposed to C. vaccinii VOCs had extensive morphological abnormalities such as swollen hyphal cells, vacuolar depositions, and cell wall alterations. Quorum-insensitive cviR− mutants of C. vaccinii were significantly less fungistatic, indicating a role for quorum regulation in the production of antifungal VOCs. We collected and characterized VOCs from co-cultivation assays of Phoma sp. exposed to wild-type C. vaccinii MWU328, and its cviR− mutant using stir bar sorptive extraction and comprehensive two-dimensional gas chromatography—time-of-flight mass spectrometry (SBSE-GC × GC-TOFMS). We detected 53 VOCs that differ significantly in abundance between microbial cultures and media controls, including four candidate quorum-regulated fungistatic VOCs produced by C. vaccinii. Importantly, the metabolomes of the bacterial-fungal co-cultures were not the sum of the monoculture VOCs, an emergent property of their VOC-mediated interactions. These data suggest semiochemical feedback loops between microbes that have co-evolved for sensing and responding to exogenous VOCs.

Palmitic Acid Inhibits the Virulence Factors of Candida tropicalis: Biofilms, Cell Surface Hydrophobicity, Ergosterol Biosynthesis, and Enzymatic Activity.

Biofilm is the fortitude of Candida species infections which eventually causes candidiasis in human. C. tropicalis is one of the predominant Candida species commonly found in systemic infections, next to C. albicans. In Candida species, biofilm maturity initiates irreversible surface attachment of cells and barricades the penetration of conventional antifungals. Hence, the current study investigated the antifungal and antivirulence potency of palmitic acid (PA) against C. tropicalis mature biofilm and its associated virulence factors. In vitro results revealed an effective inhibition of biofilm in PA-treated C. tropicalis, compared to C. albicans and C. glabrata. Also, PA reduced C. tropicalis mature biofilm at various time points. Further, PA treatment triggered apoptosis in C. tropicalis through ROS mediated mitochondrial dysfunction as demonstrated by confocal microscopic observation of PI, DAPI and DCFDA staining. PA regulated other virulence factors such as cell surface hydrophobicity, ergosterol biosynthesis, protease and lipase after 48 h of treatment. Downregulation of ERG11 (Lanosterol 14-alpha demethylase) was contributed to the reduction of ergosterol in PA-treated C. tropicalis. However, enhanced hyphal growth was observed in PA-treated C. tropicalis through upregulation HWP1 (Hyphal wall protein) and EFG1 (Enhanced filamentous growth). This study highlighted the antibiofilm and antivirulence potency of PA against C. tropicalis. Hence, PA could be applied synergistically with other antifungal agents to increase the efficacy for regulating NCAC infections.

The functionally conserved effector Sta1 is a fungal cell wall protein required for virulence in Ustilago maydis.

The biotrophic fungus Ustilago maydis causes the smut disease of maize. The interaction with its host and induction of characteristic tumors are governed largely by secreted effectors whose function is mostly unknown. To identify effectors with a prominent role in virulence, we used RNA sequencing and found that the gene sta1 is upregulated during early stages of infection. We characterized Sta1 by comparative genomics, reverse genetics, protein localization, stress assays, and microscopy. sta1 mutants show a dramatic reduction of virulence and show altered colonization of tissue neighboring the vascular bundles. Functional orthologues of Sta1 are found in related smut pathogens infecting monocot and dicot plants. Sta1 is secreted by budding cells but is attached to the cell wall of filamentous hyphae. Upon constitutive expression of Sta1, fungal filaments become susceptible to Congo red, β-glucanase, and chitinase, suggesting that Sta1 alters the structure of the fungal cell wall. Constitutive or delayed expression of sta1 during plant colonization negatively impacts on virulence. Our results suggest that Sta1 is a novel kind of effector, which needs to modify the hyphal cell wall to allow hyphae to be accommodated in tissue next to the vascular bundles.

Low prevalence of antifungal resistant Candida africana, in the C. albicans complex causing vulvovaginal candidiasis

The Candida (C.) albicans complex includes C. albicans, C. dubliniensis, C. stellatoidea, and C. africana, with the last mentioned as an important emerging agent of vulvovaginal candidiasis (VVC). The aim of the study was to identify C. africana and C. dubliniensis and assess their drug susceptibility in vaginitis. One-hundred Candida isolates of the C. albicans complex from women diagnosed with vaginitis and from vaginal samples in the culture collection of a medical mycology laboratory were examined. Species of the C. albicans complex were identified with conventional and molecular methods using polymerase chain reaction (PCR) for amplification and sequencing of the internal transcribed spacer (ITS) region, PCR for partial amplification of hyphal wall protein 1 (HWP1) gene and duplex PCR. The effects of antifungal drugs were evaluated according to standard broth microdilution protocols.Ninety-seven C. albicans (97%) and three C. africana (3%) isolates were identified. Results of susceptibility testing revealed one isolate of C. africana to be resistant to both clotrimazole and fluconazole, and one showed reduced susceptibility to itraconazole.Identification of Candida species especially C. africana in vaginitis is crucial, there are varying levels of resistance to antifungal drugs.

Pathways That Synthesize Phosphatidylethanolamine Impact Candida albicans Hyphal Length and Cell Wall Composition through Transcriptional and Posttranscriptional Mechanisms.

Candida albicans is a leading cause of systemic bloodstream infections, and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce PE by the Kennedy pathway, exhibits decreased kidney fungal burden in systemically infected mice. Conversely, overexpression of EPT1 results in a hypervirulent phenotype in this model. Thus, mutations that increase PE synthesis increase virulence, and mutations that decrease PE synthesis decrease virulence. However, the mechanism by which virulence is regulated by PE synthesis is only partially understood. RNA sequencing was performed on strains with deficient or excessive PE biosynthesis to elucidate the mechanism. Decreased PE synthesis from loss of EPT1 or PSD1 and PSD2 leads to downregulation of genes that impact mitochondrial function. Losses of PSD1 and PSD2, but not EPT1, cause significant increases in transcription of glycosylation genes, which may reflect the substantial cell wall defects in the psd1Δ/Δ psd2Δ/Δ mutant. These accumulated defects could contribute to the decreased virulence observed for mutants with deficient PE synthesis. In contrast to mutants with decreased PE synthesis, there were no transcriptional differences between the EPT1 overexpression strain and the wild type, indicating that the hypervirulent phenotype is a consequence of posttranscriptional changes. It was found that overexpression of EPT1 causes increased chitin content and increased hyphal length. These phenotypes may help to explain the previously observed hypervirulence in the EPT1 overexpressor.

Analysis of the Bioactive Metabolites of the Endangered Mexican Lost Fungi Campanophyllum – A Report from India

Meghalaya, (in India), in the region of the mega-biodiversity hotspots, is home to a plethora of wild mushrooms. The present study concerns the exploration of the order Agaricales, which includes rare gilled mushrooms considered endangered under IUCN A4c criteria, due to the declining habitat. Electron microscopy of the gill sections revealed an abundance of clamp connections, hyphal cell walls, cystidia, and basidia. This rare species which belongs to the family Cyphellaceae, exhibits morphological and molecular differences from the Cyphella spp. Phylogenetic analysis revealed that it formed a clade under the genus Campanophyllum of the order Agaricales, confirmed by both Neighbor Joining (NJ) and Bayesian phylogenetic analysis. Being nutritionally potent along with its efficient antioxidant value, the fungal extract shows significant rise of two-fold in the antimicrobial activity along with the commercial antibiotics. The compound, Phenol, 2, 4-bis (1, 1-Dimethylethyl) (2, 4-DTBP) showed in ample range in the fungal extract along with aliphatic hydrocarbons, terpene, alcohol and volatile organic compounds on further characterization in GCMS. The present study indicates the endangered Campanophyllum proboscideum could be a rich source of natural antioxidants and an effective pharmaceutical agent.

Candida africana and Candida dubliniensis as causes of pediatric candiduria: A study using HWP1 gene size polymorphism.

BackgroundCandida species cause a wide spectrum of disease entities. Candida africana and Candida dubliniensis–members of Candida albicans complex-are currently gaining both clinical and epidemiologic significance.Materials and methodsTotally, 150 pediatric isolates that had previously been identified as C. albicans species complex based on a positive germ tube test were included. The isolates were cultured on CHROMagar Candida medium to ensure their purity and the results of germ tube test. For definitive speciation, PCR amplification and size polymorphism of the hyphal wall protein 1 (HWP1) gene was used. The results of HWP1-PCR were confirmed by sequencing the amplified fragments for randomly selected isolates of C. africana and C. dubliniensis.ResultsAll 150 isolates included in this study were reconfirmed as C. albicans complex on chromogenic media. Based on the HWP1 gene size polymorphism, 141 (94%) isolates were identified as C. albicans, 2 (1.33%) as C. africana, and 1 (0.67%) as C. dubliniensis. The remaining 6 (4%) C. albicans complex isolates were a mix of C. albicans and C. africana. All isolates of C. africana and C. dubliniensis have been recovered from cases of candiduria.ConclusionC. africana, either alone or mixed with C. albicans, could be a cause of candiduria among pediatric patients and should not be ignored.

To Be or Not to Be a Pathogen: Candida albicans and Celiac Disease.

Celiac disease (CD) is an immune-mediated disorder triggered by the ingestion of gluten and characterized by reversible small-bowel mucosal atrophy in genetically predisposed subjects. Although the prevalence of CD has increased, many aspects of this pathology are still unrecognized. Candida albicans, a commensal of the human gastrointestinal tract, has been linked to CD for a long time based, among others, upon the observation of similarity between the fungal wall component, hyphal wall protein 1, and CD-related gliadin T-cell epitopes. We have recently demonstrated that Candida may switch from commensal to pathogen contingent upon several players, including mast cells, key sentinels of the immune system at the interface between the environment and the host, and the pleiotropic cytokine IL-9. However, other factors are likely to play a role by altering the balance between inflammation and tolerance. In this regard, tryptophan and its metabolites are increasingly being recognized in promoting mucosal homeostasis by balancing the immune response to external cues. Based on these premises, we will discuss how the output of Candida colonization in the gut is highly contextual, being determined at the intersection of many immunological (IL-9/mast cells) and metabolic (tryptophan) pathways that ultimately dictate the Candida commensalism vs. pathogenicity in CD, thus paving the way for novel therapeutic opportunities in CD.

Production of melanin pigments in saprophytic fungi in vitro and during infection.

Melanins are one of the great natural pigments produced by a wide variety of fungal species that promote fitness and cell survival in diverse hostile environments, including during mammalian infection. In this study, we sought to demonstrate the production of melanin in the conidia and hyphae of saprophytic fungi, including dematiaceous and hyaline fungi. We showed that a melanin-specific monoclonal antibody (MAb) avidly labeled the cell walls of hyphae and conidia, consistent with the presence of melanin in these structures, in 14 diverse fungal species. The conidia of saprophytic fungi were treated with proteolytic enzymes, denaturant, and concentrated hot acid to yield dark particles, which were shown to be stable free radicals, consistent with their identification as melanins. Samples obtained from patients with fungal keratitis due to Fusarium falciforme, Aspergillus fumigatus, Aspergillus flavus, Curvularia lunata, Exserohilum rostratum, or Fonsecaea pedrosoi were found to be intensely labeled by the melanin-specific MAb at the fungal hyphal cell walls. These results support the hypothesis that melanin is a common component that promotes survival under harsh conditions and facilitates fungal virulence. Increased understanding of the processes of melanization and the development of methods to interfere with pigment formation may lead to novel approaches to combat these complex pathogens that are associated with high rates of morbidity and mortality.

Candida albicans cell wall integrity transcription factors regulate polymicrobial biofilm formation with Streptococcus gordonii.

Polymicrobial biofilms play important roles in oral and systemic infections. The oral plaque bacterium Streptococcus gordonii is known to attach to the hyphal cell wall of the fungus Candida albicans to form corn-cob like structures in biofilms. However, the role of C. albicans in formation of polymicrobial biofilms is not completely understood. The objective of this study was to determine the role of C. albicans transcription factors in regulation of polymicrobial biofilms and antibiotic tolerance of S. gordonii. The proteins secreted by C. albicans and S. gordonii in mixed planktonic cultures were determined using mass spectrometry. Antibiotic tolerance of S. gordonii to ampicillin and erythromycin was determined in mixed cultures and mixed biofilms with C. albicans. Additionally, biofilm formation of S. gordonii with C. albicans knock-out mutants of 45 transcription factors that affect cell wall integrity, filamentous growth and biofilm formation was determined. Furthermore, these mutants were also screened for antibiotic tolerance in mixed biofilms with S. gordonii. Analysis of secreted proteomes resulted in the identification of proteins being secreted exclusively in mixed cultures. Antibiotic testing showed that S. gordonii had significantly increased survival in mixed planktonic cultures with antibiotics as compared to single cultures. C. albicans mutants of transcription factors Sfl2, Brg1, Leu3, Cas5, Cta4, Tec1, Tup1, Rim101 and Efg1 were significantly affected in mixed biofilm formation. Also mixed biofilms of S. gordonii with mutants of C. albicans transcription factors, Tec1 and Sfl2, had significantly reduced antibiotic tolerance as compared to control cultures. Our data indicates that C. albicans may have an important role in mixed biofilm formation as well as antibiotic tolerance of S. gordonii in polymicrobial biofilms. C. albicans may play a facilitating role than being just an innocent bystander in oral biofilms and infections.

Direct and indirect influence of arbuscular mycorrhizae on enhancing metal tolerance of plants.

The acerbic elevation of toxic metal ions in arable lands, enhance the risk of their accumulation and biomagnification in crops as well as in humans. Phytoremediation is an eco-friendly approach to clear metal-contaminated lands by making use of metal accumulation potential of plants; which are referred to as hyperaccumulators. This phytoremediation potential can be enhanced with the symbiotic association between the root of hyperaccumulators and arbuscular mycorrhizae. Modification of root morphology, enhancement of antioxidants biosynthesis, and the increase in shoot biomass are the changes observed in plants as a result of indirect influence of arbuscular mycorrhizae. Direct influence of arbuscular mycorrhizae on enhancing metal tolerance of plants includes immobilization strategies, adsorption of metals on to the hyphal wall and glomalin exudation. Furthermore, we have discussed arbuscular mycorrhizal induced increment in the metal tolerance potential of plants through the alteration in various metabolic processes with special emphasis to the phenylpropanoid pathway.

Role of glomalin in soil carbon storage and its variation across land uses in temperate Himalayan regime

Abstract Root symbiont arbuscular mycorrhizal fungi (AMF) are ubiquitous in soils of terrestrial ecosystems and play a major role in determining the soil structure dynamics. Glomalin related soil protein (GRSP) produced in the hyphal cell walls is a store house of carbon that influences aggregate formation, stabilization and contributes in soil carbon sequestration. In this work, the variation in the glomalin concentrations was studied across soil of temperate Himalayan regime, its potential role in carbon storage and soil aggregation under different cropping systems. Aggregate stability showed a positive correlation with soil organic carbon (r = 0.966; p

Evaluation of biofilm formation in the homozygous and heterozygous strains of vaginal Candida albicans isolates.

Background and Purpose: Candida albicans is one of the most opportunistic yeasts around the world. This species has two heterozygous and homozygous strains at hyphal wall protein 1 (hwp1) gene locus. A simple method for the discrimination of these two strains is the amplification of HWP1 gene. Regarding this, the aim of this study was to discriminate C. albicans heterozygous and homozygous strains via the amplification of hwp1 gene and evaluation of biofilm formation between the strains.Materials and Methods:A total of 60 homozygous (n=30) and heterozygous (n=30) strains were discriminated among 126 C. albicans vaginal isolates by the amplification of HWP1 gene, using specific primers. The evaluation of biofilm formation was accomplished using the visual method.Results:According to the results, the homozygous and heterozygous strains produced one and two DNA fragments, respectively. The frequency of homozygous strains among the C. albicans vaginal isolates was 76.2%. Biofilm formation activity in the heterozygous strains was more than that in the homozygous strains. However, statistical analysis showed no significant difference between the strains in terms of biofilm formation.Conclusion:As the findings indicated, the frequency of the heterozygous strains in C. albicans was lower than that of the homozygous strains. Both of the strains could form biofilm in the different ranges of severity. High activity of biofilm formation in heterozygous strains may set the ground for its pathogenicity.

Life at the edge – the cytology and physiology of the biotroph to necrotroph transition in Hymenoscyphus fraxineus during lesion formation in ash

The progress of colonization of ash stems from ascospore inocula of Hymenoscyphus fraxineus was examined by light and electron microscopy. The main aim of the study was to characterize the cytology of the biotroph to necrotroph transition during lesion formation. Following direct penetration into epidermal cells, the fungus produced intracellular hyphae that invaded up to five cells before plant cells died. A lack of close attachment between the hyphal cell wall and plant cell membrane was revealed by plasmolysis of epidermal cells. Plant cells died at the centre of the infection but hyphae at the edge were typically found in living plant cells even around large lesions. During biotrophic invasion, the cytoplasm of penetrated plant cells showed very little response despite the plant cell membrane being in direct contact with the fungal cell wall. Before plant cell death, dark staining of the cytoplasm and proliferation of small vesicles was noted, but organelles retained normal ultrastructure. Dead plant cells contained dark brown, osmiophilic droplets. Penetration between epidermal or collenchyma cells was usually targeted to shared pits and involved constriction of hyphae. The transition to necrotrophy was not associated with a clear change in hyphal morphology. Biotrophic intracellular hyphae contained dense cytoplasm but hyphae in dead plant cells were more vacuolated. Remarkably little plant cell wall degradation was observed despite the fungus penetrating up to 18 cells deep into stem tissue. Features of the development of the ash dieback fungus are compared with other hemibiotrophic pathogens.

Inhibition of Candida albicans morphogenesis by chitinase from Lactobacillus rhamnosus GG

Lactobacilli have been evaluated as probiotics against Candida infections in several clinical trials, but with variable results. Predicting and understanding the clinical efficacy of Lactobacillus strains is hampered by an overall lack of insights into their modes of action. In this study, we aimed to unravel molecular mechanisms underlying the inhibitory effects of lactobacilli on hyphal morphogenesis, which is a crucial step in C. albicans virulence. Based on a screening of different Lactobacillus strains, we found that the closely related taxa L. rhamnosus, L. casei and L. paracasei showed stronger activity against Candida hyphae formation compared to other Lactobacillus species tested. By exploring the activity of purified compounds and mutants of the model strain L. rhamnosus GG, the major peptidoglycan hydrolase Msp1, conserved in the three closely related taxa, was identified as a key effector molecule. We could show that this activity of Msp1 was due to its ability to break down chitin, the main polymer in the hyphal cell wall of C. albicans. This identification of a Lactobacillus-specific protein with chitinase activity having anti-hyphal activity will assist in better strain selection and improved application in future clinical trials for Lactobacillus-based Candida-management strategies.