Media matters! Alterations in the loading and release of
            <scp>
              <i>Histoplasma capsulatum</i>
            </scp>
            extracellular vesicles in response to different nutritional milieus

Cleare, Levi G.; Zamith, Daniel; Heyman, Heino M.; Couvillion, Sneha; Nimrichter, Leonardo; Rodrigues, Márcio L.; Nakayasu, Ernesto; Nosanchuk, Joshua D.

doi:10.1111/cmi.13217

Cited by 53 publications

(73 citation statements)

References 36 publications

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“…Similarly, the protein composition of fungal EVs has been shown to vary depending on the morphology of the cells and nutrients available in the environment. For example, protein cargo was distinct when EVs from the biofilm and planktonic form of C. albicans were compared [ 52 ], while the nutrient composition of culture media was found to influence EV release and protein composition in H. capsulatum [ 53 ]. Z. tritici grows as blastospores, a yeast-like form, in vitro but switches to hyphal growth during colonisation of the wheat apoplast [ 54 ].…”

Section: Discussionmentioning

confidence: 99%

Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

Solomon

2020

Fungal Biol Biotechnol

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Background The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs)—secreted, membrane-bound nanoparticles containing molecular cargo—being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes. Results Zymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 and 250 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC–MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs. Conclusion We have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

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Section: Discussionmentioning

confidence: 99%

Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

Solomon

2020

Fungal Biol Biotechnol

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“…BHI medium consists of an infusion of brain-heart, sodium hydrogen phosphate, glucose, peptone, and sodium chloride. These components provide a suitable environment for the growth of fastidious and non-fastidious microorganisms ( Rosenow, 1919 ; Cleare et al, 2020 ). HAM-F12 medium is widely used for the cultivation and maintenance of mammalian cell cultures ( Ham, 1965 ; Cravchik et al, 1996 ).…”

Section: Discussionmentioning

confidence: 99%

Biofilm Formation by Histoplasma capsulatum in Different Culture Media and Oxygen Atmospheres

et al. 2020

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Histoplasma capsulatum is a dimorphic fungus that causes an important systemic mycosis called histoplasmosis. It is an infectious disease with high prevalence and morbidity that affects the general population. Recently, the ability of these fungi to form biofilms, a phenotype that can induce resistance and enhance virulence, has been described. Despite some efforts, data regarding the impact of nutrients and culture media that affect the H. capsulatum biofilm development in vitro are not yet available. This work aimed to study H. capsulatum biofilms, by checking the influence of different culture media and oxygen atmospheres in the development of these communities. The biofilm formation by two strains (EH-315 and G186A) was characterized under different culture media: [Brain and Heart Infusion (BHI), Roswell Park Memorial Institute (RPMI) with 2% glucose, Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and nutrient medium HAM-F12 (HAM-F12) supplemented with glucose (18.2 g/L), glutamic acid (1 g/L), HEPES (6 g/L) and L-cysteine (8.4 mg/L)] and oxygen atmospheres (aerobiosis and microaerophilia), using the XTT reduction assay to quantify metabolic activities, crystal violet staining for biomass, safranin staining for the quantification of polysaccharide material and scanning electron microscopy (SEM) for the observation of topographies. Results indicated that although all culture mediums have stimulated the maturation of the communities, HAM-F12 provided the best development of biomass and polysaccharide material when compared to others. Regarding the oxygen atmospheres, both stimulated an excellent development of the communities, however in low oxygen conditions an exuberant amount of extracellular matrix was observed when compared to biofilms formed in aerobiosis, mainly in the HAM-F12 media. SEM images showed yeasts embedded by an extracellular matrix in several points, corroborating the colorimetric assays. However, biofilms formed in BHI, RPMI, and DMEM significantly induced yeast to hyphae reversal, requiring further investigation. The results obtained so far contribute to in vitro study of biofilms formed by these fungi and show that nutrition promoted by different media modifies the development of these communities. These data represent advances in the field of biofilms and contribute to future studies that can prove the role of these communities in the fungi-host interaction.

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“…Similarly, the protein composition of fungal EVs has been shown to vary depending on the morphology of the cells and nutrients available in the environment. For example, protein cargo was distinct when EVs from the bio lm and planktonic form of C. albicans were compared [52], while the nutrient composition of culture media was found to in uence EV release and protein composition in H. capsulatum [53]. Z. tritici grows as blastospores, a yeast-like form, in vitro but switches to hyphal growth during colonisation of the wheat apoplast [54].…”

Section: Discussionmentioning

confidence: 99%

Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

Solomon

2020

Preprint

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Background: The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs) – secreted, membrane-bound nanoparticles containing molecular cargo – being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes.Results: Zymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 – 250 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC-MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs.Conclusion: We have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

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Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus

Cited by 53 publications

References 36 publications

Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

Biofilm Formation by Histoplasma capsulatum in Different Culture Media and Oxygen Atmospheres

Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

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