is the most aggressive pathogen of citrus fruits. Tryptoquialanines are major indole alkaloids produced by
It is unknown if tryptoquialanines are involved in the damage of citrus fruits caused by
To ...investigate the pathogenic roles of tryptoquialanines, we initially asked if tryptoquialanines could affect the germination of
seeds. Exposure of the citrus seeds to tryptoquialanine A resulted in a complete inhibition of germination and an altered metabolic response. Since this phytotoxic effect requires the extracellular export of tryptoquialanine A, we investigated the mechanisms of extracellular delivery of this alkaloid in
We detected extracellular vesicles (EVs) released by
both in culture and during infection of citrus fruits. Compositional analysis of EVs produced during infection revealed the presence of a complex cargo, which included tryptoquialanines and the mycotoxin fungisporin. The EVs also presented phytotoxicity activity
and caused damage to the tissues of citrus seeds. Through molecular networking, it was observed that the metabolites present in the
EVs are produced in all of its possible hosts. Our results reveal a novel phytopathogenic role of
EVs and tryptoquialanine A, implying that this alkaloid is exported in EVs during plant infection.
During the postharvest period, citrus fruits can be affected by phytopathogens such as
, which causes green mold disease and is responsible for up to 90% of total citrus losses. Chemical fungicides are widely used to prevent green mold disease, leading to concerns about environmental and health risks. To develop safer alternatives to control phytopathogens, it is necessary to understand the molecular basis of infection during the host-pathogen interaction. In the
model, the virulence strategies are poorly known. Here, we describe the production of phytotoxic extracellular vesicles (EVs) by
during the infection of citrus fruits. We also characterized the secondary metabolites in the cargo of EVs and found in this set of molecules an inhibitor of seed germination. Since EVs and secondary metabolites have been related to virulence mechanisms in other host-pathogen interactions, our data are important for the comprehension of how
causes damage to its primary hosts.
The human diseases caused by the fungal pathogens
and
are associated with high indices of mortality and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to ...combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anticryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anticryptococcal benzimidazole. Fenbendazole was inhibitory against 17 different isolates of
and
at a low concentration. The mechanism of anticryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anticryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mouse model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.
Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, ...virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre‐treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi‐antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen‐specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL‐12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL‐12p70, TGFβ, IL‐4 and IL‐10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, −20 or −80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.
The small molecule (molecular mass <900 Daltons) composition of extracellular vesicles (EVs) produced by the pathogenic fungus
is unknown, which limits the understanding of the functions of ...cryptococcal EVs. In this study, we analyzed the composition of small molecules in samples obtained from solid cultures of
by a combination of chromatographic and spectrometric approaches, and untargeted metabolomics. This analysis revealed previously unknown components of EVs, including small peptides with known biological functions in other models. The peptides found in
EVs had their chemical structure validated by chemical approaches and comparison with authentic standards, and their functions tested in a
model of cryptococcal infection. One of the vesicular peptides (isoleucine-proline-isoleucine, Ile-Pro-Ile) improved the survival of
lethally infected with
or
. These results indicate that small molecules exported in EVs are biologically active in
. Our study is the first to characterize a fungal EV molecule inducing protection, pointing to an immunological potential of extracellular peptides produced by
.
Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data ...regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii. Using cutting‐edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein‐based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analysing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane‐bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection.
Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, ...using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin‐like inhibitor of serine peptidases, ISP2. We generated ISP2‐null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add‐back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease‐activated receptor 2 (PAR2) or an inhibitor of Toll‐like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add‐back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add‐back lines. We propose that ISP2 contributes to protect T. cruzi from the anti‐microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.
Trypanosoma cruzi has a functional ecotin‐like inhibitor of serine peptidases called ISP2.
ISP2 null mutant is more susceptible to killing by human serum than control epimastigotes.
ISP2 null mutant trypomastigotes showed an increased infection capacity.
PAR2 and TLR4, and the serine protease TMPRSS2, mediate the enhanced infectivity of ISP2 null mutant parasites.
ISP2 null mutant T. cruzi is more inflammatory to mice.
There is an urgent unmet need for novel antifungals. In this study, we searched for novel antifungal activities in the Pandemic Response Box, a collection of 400 structurally diverse compounds in ...various phases of drug discovery. We identified five molecules which could control the growth of Cryptococcus neoformans, Cryptococcus deuterogattii, and the emerging global threat Candida auris. After eliminating compounds which demonstrated paradoxical antifungal effects or toxicity to mammalian macrophages, we selected compound MMV1593537 as a nontoxic, fungicidal molecule for further characterization of antifungal activity. Scanning electron microscopy revealed that MMV1593537 affected cellular division in all three pathogens. In Cryptococcus, MMV1593537 caused a reduction in capsular dimensions. Treatment with MMV1593537 resulted in increased detection of cell wall chitooligomers in these three species. Since chitooligomers are products of the enzymatic hydrolysis of chitin, we investigated whether surface chitinase activity was altered in response to MMV1593537 exposure. We observed peaks of enzyme activity in C. neoformans and C. deuterogattii in response to MMV1593537. We did not detect any surface chitinase activity in C. auris. Our results suggest that MMV1593537 is a promising, nontoxic fungicide whose mechanism of action, at least in Cryptococcus spp, requires chitinase-mediated hydrolysis of chitin.
The development of novel antifungals is a matter of urgency. In this study, we evaluated antifungal activities in a collection of 400 molecules, using highly lethal fungal pathogens as targets. One of these molecules, namely, MMV1593537, was not toxic to host cells and controlled the growth of isolates of Cryptococcus neoformans, C. deuterogattii, C. gattii, Candida auris, C. albicans, C. parapsilosis, and C. krusei. We tested the mechanisms of antifungal action of MMV1593537 in the Cryptococcus and C. auris models and concluded that the compound affects the cell wall, a structure which is essential for fungal life. At least in Cryptococcus, this effect involved chitinase, an enzyme which is required for remodeling the cell wall. Our results suggest that MMV1593537 is a candidate for future antifungal development.
Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known.
is a eukaryotic gene that is required for the activity of ...benzimidazoles against
. In this study, during the phenotypic characterization of
mutants expected to lack
expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing
otentially
ltered
ugar
ransport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a
model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in
. These results connect EV biogenesis, cargo, and cryptococcal virulence.
Pathogenic species of Cryptococcus kill approximately 200,000 people each year. The most important virulence mechanism of C. neoformans and C. gattii, the causative agents of human and animal ...cryptococcosis, is the ability to form a polysaccharide capsule. Acapsular mutants of C. neoformans are avirulent in mice models of infection, and extracellularly released capsular polysaccharides are deleterious to the immune system. The principal capsular component in the Cryptococcus genus is a complex mannan substituted with xylosyl and glucuronyl units, namely glucuronoxylomannan (GXM). The second most abundant component of the cryptococcal capsule is a galactan with multiple glucuronyl, xylosyl, and mannosyl substitutions, namely glucuronoxylomannogalactan (GXMGal). The literature about the structure and functions of these two polysaccharides is rich, and a number of comprehensive reviews on this topic are available. Here, we focus our discussion on the less explored glycan components associated with the cryptococcal capsule, including mannoproteins and chitin-derived molecules. These glycans were selected for discussion on the basis that i) they have been consistently detected not only in the cell wall but also within the cryptococcal capsular network and ii) they have functions that impact immunological and/or pathogenic mechanisms in the Cryptococcus genus. The reported functions of these molecules strongly indicate that the biological roles of the cryptococcal capsule go far beyond the well-known properties of GXM and GXMGal.
Golgi reassembly and stacking protein (GRASP) is required for polysaccharide secretion and virulence in
. In fungal species, extracellular vesicles (EVs) participate in the export of polysaccharides, ...proteins and RNA. In the present work, we investigated if EV-mediated RNA export is functionally connected with GRASP in
using a
Δ mutant. Since GRASP-mediated unconventional secretion involves autophagosome formation in yeast, we included the
Δ mutant with defective autophagic mechanisms in our analysis. All fungal strains exported EVs but deletion of
or
profoundly affected vesicular dimensions. The mRNA content of the
Δ EVs differed substantially from that of the other two strains. The transcripts associated to the endoplasmic reticulum were highly abundant transcripts in
Δ EVs. Among non-coding RNAs (ncRNAs), tRNA fragments were the most abundant in both mutant EVs but
Δ EVs alone concentrated 22 exclusive sequences. In general, our results showed that the EV RNA content from
Δ and WT were more related than the RNA content of
Δ, suggesting that GRASP, but not the autophagy regulator Atg7, is involved in the EV export of RNA. This is a previously unknown function for a key regulator of unconventional secretion in eukaryotic cells.
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