RESUMO

This study explores the potential of geranium essential oil as a natural solution for combating marine biofouling, addressing the environmental concerns associated with commercial antifouling coatings. Compounds with bactericidal activities were identified by 13Carbon nuclear magnetic resonance (13C NMR). Thermogravimetric analysis (TGA) revealed minimal impact on film thermal stability, maintaining suitability for antifouling applications. The addition of essential oil induced changes in the morphology of the film and Fourier transform infrared spectroscopy (FTIR) analysis indicated that oil remained within the film. Optical microscopy showed an increase in coating porosity after immersion in a marine environment. A total of 18 bacterial colonies were isolated, with Psychrobacter adeliensis and Shewanella algidipiscicola being the predominant biofilm-forming species. The geranium essential oil-based coating demonstrated the ability to reduce the formation of Psychrobacter adeliensis biofilms and effectively inhibit macrofouling adhesion for a duration of 11 months.

Assuntos

Incrustação Biológica , Geranium , Óleos Voláteis , Psychrobacter , Biofilmes , Incrustação Biológica/prevenção & controle , Óleos Voláteis/farmacologia , Óleos de Silicone/farmacologia , Silicones

RESUMO

Cryptococcus gattii is one of the etiological agents of cryptococcosis. To achieve a successful infection, C. gattii cells must overcome the inhospitable host environment and deal with the highly specialized immune system and poor nutrients availability. Inside the host, C. gattii uses a diversified set of tools to maintain homeostasis and establish infection, such as the expression of remarkable and diverse heat shock proteins (Hsps). Grouped by molecular weight, little is known about the Hsp12 subset in pathogenic fungi. In this study, the function of the C. gattii HSP12.1 and HSP12.2 genes was characterized. Both genes were upregulated during murine infection and heat shock. The hsp12.1 Δ null mutant cells were sensitive to plasma membrane and oxidative stressors. Moreover, HSP12 deletion induced C. gattii reactive oxygen species (ROS) accumulation associated with a differential expression pattern of oxidative stress-responsive genes compared to the wild type strain. Apart from these findings, the deletion of the paralog gene HSP12.2 did not lead to any detectable phenotype. Additionally, the double-deletion mutant strain hsp12.1 Δ /hsp12.2 Δ presented a similar phenotype to the single-deletion mutant hsp12.1 Δ, suggesting a minor participation of Hsp12.2 in these processes. Furthermore, HSP12.1 disruption remarkably affected C. gattii virulence and phagocytosis by macrophages in an invertebrate model of infection, demonstrating its importance for C. gattii pathogenicity.

Assuntos

Criptococose , Cryptococcus gattii , Proteínas de Choque Térmico Pequenas , Animais , Camundongos , Criptococose/microbiologia , Cryptococcus gattii/genética , Proteínas de Choque Térmico Pequenas/metabolismo , Fagocitose , Virulência

RESUMO

Here, we report the occurrence and complete genome sequence of a novel victorivirus infecting Metarhizium anisopliae, named "Metarhizium anisopliae victorivirus 1" (MaVV1). The genome is 5353 bp in length and contains two open reading frames (ORFs), encoding a coat protein and an RNA-dependent RNA polymerase (RdRp), that overlap at the octanucleotide sequence AUGAGUAA. These ORFs showed sequence similarity to the corresponding ORFs of Ustilaginoidea virens RNA virus L (68.23%) and Ustilaginoidea virens RNA virus 13 (58.11%), respectively, both of which belong to the family Totiviridae. Phylogenetic analysis based on RdRp sequences revealed that MaVV1 clustered with members of the genus Victorivirus. This is the first genome sequence reported for a virus belonging to the genus Victorivirus infecting the entomopathogenic fungus M. anisopliae.

Assuntos

Genoma Viral , Metarhizium , Totiviridae , Genoma Viral/genética , Metarhizium/genética , Metarhizium/virologia , Fases de Leitura Aberta , Filogenia , RNA de Cadeia Dupla , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Totiviridae/genética

RESUMO

The genus Paracoccidioides comprises the species complex causing paracoccidioidomycoses (PCM). These fungi are a serious public health problem due to the long-term drug therapy, follow-up treatment, and frequent sequelae generated by the infection, such as pulmonary fibrosis. In this sense, the objective of this work was to generate bioluminescent reporter strains of Paracoccidioides spp. harboring a thermostable, red-shifted luciferase gene under the control of different constitutive promoters. The strains were generated by the integration of a species-specific codon-optimized luciferase gene upon actin or enolase promoter's control. The insertion of the constructs in Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells were performed through Agrobacterium tumefaciens-mediated transformation. The results demonstrated the presence of several transformants harboring the luciferase gene. These transformants were further confirmed by the expression of luciferase and by the presence of the hygromycin resistance gene. Moreover, the luciferase activity could be detected in in vitro bioluminescence assays and in vivo models of infection. In general, this work presents the methodology for the construction of bioluminescent strains of Paracoccidioides spp., highlighting potential promoters and proposing an in vivo model, in which those strains could be used for the systemic study of PCM.

Assuntos

Paracoccidioides , Paracoccidioidomicose , Actinas , Paracoccidioides/genética , Paracoccidioidomicose/microbiologia , Fosfopiruvato Hidratase

RESUMO

Filamentous fungi are the organisms of choice for most industrial biotechnology. Some species can produce a variety of secondary metabolites and enzymes of commercial interest, and the production of valuable molecules has been enhanced through different molecular tools. Methods for genetic manipulation and transformation have been essential for the optimization of these organisms. The genus Simplicillium has attracted increased attention given several potential biotechnological applications. The Simplicillium genus harbors several entomopathogenic species and some isolates have been explored for bioremediation of heavy metal contaminants. Furthermore, the myriad of secondary metabolites isolated from Simplicillium spp. render these organisms as ideal targets for deep exploration and further biotechnological mining possibilities. However, the lack of molecular tools hampered the exploration of this genus. Thus, an Agrobacterium tumefaciens-mediated transformation method was established for Simplicillium subtropicum, employing the far-red fluorescent protein TURBOFP635/Katushka, as a visual marker, and the selection marker SUR gene, that confers resistance to chlorimuron ethyl. Notably, one round of transformation using the established method yielded almost 400 chlorimuron resistant isolates. Furthermore, these transformants displayed mitotic stability for, at least, five generations. We anticipate that this method can be useful for deep molecular exploration and improvement of strains in the Simplicillium genus.

RESUMO

Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3ß was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen.

RESUMO

The first line of the Arthropods defense against infections is the hard-structured exoskeleton, a physical barrier, usually rich in insoluble chitin. For entomopathogenic fungi that actively penetrate the host body, an arsenal of hydrolytic enzymes (as chitinases and N-acetylglucosaminidases), that break down chitin, is essential. Notably, twenty-one putative chitinase genes have been identified in the genome of Metarhizium anisopliae, a generalist entomopathogenic fungus. As a multigenic family, with enzymes that, presumably, perform redundant functions, the main goal is to understand the singularity of each one of such genes and to discover their precise role in the fungal life cycle. Specially chitinases that can act as virulence determinants are of interest since these enzymes can lead to more efficient biocontrol agents. Here we explored a horizontally acquired chitinase from M. anisopliae, named chiMaD1. The deletion of this gene did not lead to phenotypic alterations or diminished supernatant's chitinolytic activity. Surprisingly, chiMaD1 deletion enhanced M. anisopliae virulence to the cattle tick (Rhipicephalus microplus) larvae and engorged females, while did not alter the virulence to the mealworm larvae (Tenebrio molitor). These results add up to recent reports of deleted genes that enhanced entomopathogenic virulence, showing the complexity of host-pathogen interactions.

Assuntos

Quitinases/genética , Proteínas Fúngicas/genética , Metarhizium/patogenicidade , Rhipicephalus/microbiologia , Animais , Quitina/metabolismo , Quitinases/metabolismo , Proteínas Fúngicas/metabolismo , Deleção de Genes , Transferência Genética Horizontal , Interações Hospedeiro-Patógeno , Larva/microbiologia , Metarhizium/classificação , Metarhizium/enzimologia , Metarhizium/genética , Controle Biológico de Vetores , Filogenia , Tenebrio/microbiologia , Virulência

RESUMO

Cryptococcus neoformans is the etiological agent of cryptococcal meningoencephalitis. The recommended available treatment has low efficiency, with high toxicity and resistance as recurrent problems. In the search of new treatment protocols, the proposal of new pharmacological approaches is considered an innovative strategy, mainly nanotechnological systems considering fungal diseases. The antiarrhythmic drug amiodarone has demonstrated antifungal activity against a range of fungi, including C. neoformans. Here, considering the importance of calcium storage mediated by transporters on cryptococcal virulence, we evaluated the use of the calcium channel blocker amiodarone as an alternative therapy for cryptococcosis. C. neoformans displayed high sensitivity to amiodarone, which was also synergistic with fluconazole. Amiodarone treatment influenced some virulence factors, interrupting the calcium-calcineurin signaling pathway. Experiments with murine cryptococcosis models revealed that amiodarone treatment increased the fungal burden in the lungs, while its combination with fluconazole did not improve treatment compared to fluconazole alone. In addition, we have developed different innovative nanotechnological formulations, one of which combining two drugs with different mechanisms of action. Lipid-core nanocapsules (LNC) loaded with amiodarone (LNCAMD), fluconazole (LNCFLU) and both (LNCAMD+FLU) were produced to achieve a better efficacy in vivo. In an intranasal model of treatment, all the LNC formulations had an antifungal effect. In an intraperitoneal treatment, LNCAMD showed an enhanced anticryptococcal effect compared to the free drug, whereas LNCFLU or LNCAMD+FLU displayed no differences from the free drugs. In this way, nanotechnology using amiodarone formulations could be an effective therapy for cryptococcal infections.

Assuntos

Amiodarona , Criptococose , Nanocápsulas , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Criptococose/tratamento farmacológico , Fluconazol/uso terapêutico , Lipídeos/uso terapêutico , Camundongos , Testes de Sensibilidade Microbiana , Nanocápsulas/uso terapêutico , Nanotecnologia

RESUMO

In nature, microorganisms often exhibit competitive behavior for nutrients and limited space, allowing them to alter the virulence determinants of pathogens. The human pathogenic yeast Cryptococcus neoformans can be found organized in biofilms, a complex community composed of an extracellular matrix which confers protection against predation. The aim of this study was to evaluate and characterize antagonistic interactions between two cohabiting microorganisms: C. neoformans and the bacteria Serratia marcescens. The interaction of S. marcescens with C. neoformans expressed a negative effect on biofilm formation, polysaccharide capsule, production of urease, and melanization of the yeast. These findings evidence that competition in mixed communities can result in dominance by one species, with direct impact on the physiological modulation of virulence determinants. Such an approach is key for understating the response of communities to the presence of competitors and, ultimately, rationally designing communities to prevent and treat certain diseases.

Assuntos

Biofilmes , Cryptococcus neoformans , Interações Microbianas , Serratia marcescens , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/fisiologia , Interações Microbianas/fisiologia , Serratia marcescens/patogenicidade , Serratia marcescens/fisiologia , Fatores de Virulência/metabolismo

RESUMO

Phenotypic heterogeneity is an important trait for the development and survival of many microorganisms including the yeast Cryptococcus spp., a deadly pathogen spread worldwide. Here, we have applied scanning electron microscopy (SEM) to define four Cryptococcus spp. capsule morphotypes, namely Regular, Spiky, Bald, and Phantom. These morphotypes were persistently observed in varying proportions among yeast isolates. To assess the distribution of such morphotypes we implemented an automated pipeline capable of (1) identifying potentially cell-associated objects in the SEM-derived images; (2) computing object-level features; and (3) classifying these objects into their corresponding classes. The machine learning approach used a Random Forest (RF) classifier whose overall accuracy reached 85% on the test dataset, with per-class specificity above 90%, and sensitivity between 66 and 94%. Additionally, the RF model indicates that structural and texture features, e.g., object area, eccentricity, and contrast, are most relevant for classification. The RF results agree with the observed variation in these features, consistently also with visual inspection of SEM images. Finally, our work introduces morphological variants of Cryptococcus spp. capsule. These can be promptly identified and characterized using computational models so that future work may unveil morphological associations with yeast virulence.

Assuntos

Variação Anatômica , Cryptococcus/ultraestrutura , Cápsulas Fúngicas/ultraestrutura , Aprendizado de Máquina , Microscopia Eletrônica de Varredura/métodos , Cryptococcus/genética , Fenótipo

RESUMO

Cryptococcus neoformans is an encapsulated yeast responsible for more than 180,000 deaths per year. The standard therapeutic approach against cryptococcosis is a combination of amphotericin B with flucytosine. In countries where cryptococcosis is most prevalent, 5-fluorocytosine is rarely available, and amphotericin B requires intravenous administration. C. neoformans biofilm formation is related to increased drug resistance, which is an important outcome for hospitalized patients. Here, we describe new molecules with anti-cryptococcal activity. A collection of 66 semisynthetic derivatives of ursolic acid and betulinic acid was tested against mature biofilms of C. neoformans at 25 µM. Out of these, eight derivatives including terpenes, benzazoles, flavonoids, and quinolines were able to cause damage and eradicate mature biofilms. Four terpene compounds demonstrated significative growth inhibition of C. neoformans. Our study identified a pentacyclic triterpenoid derived from betulinic acid (LAFIS13) as a potential drug for anti-cryptococcal treatment. This compound appears to be highly active with low toxicity at minimal inhibitory concentration and capable of biofilm eradication.

Assuntos

Biofilmes/efeitos dos fármacos , Criptococose/prevenção & controle , Cryptococcus neoformans/fisiologia , Triterpenos Pentacíclicos/farmacologia , Linhagem Celular , Criptococose/microbiologia , Cryptococcus neoformans/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Triterpenos Pentacíclicos/química , Triterpenos/química , Ácido Betulínico , Ácido Ursólico

RESUMO

BACKGROUND: The Metarhizium genus harbors important entomopathogenic fungi. These species have been widely explored as biological control agents, and strategies to improve the fungal virulence are under investigation. Thus, the interaction between Metarhizium species and susceptible hosts have been explored employing different methods in order to characterize putative virulence determinants. However, the impact of epigenetic modulation on the infection cycle of Metarhizium is still an open topic. Among the different epigenetic modifications, DNA methylation of cytosine bases is an important mechanism to control gene expression in several organisms. To better understand if DNA methylation can govern Metarhizium-host interactions, the genome-wide DNA methylation profile of Metarhizium anisopliae was explored in two conditions: tick mimicked infection and a saprophytic-like control. RESULTS: Using a genome wide DNA methylation profile based on bisulfite sequencing (BS-Seq), approximately 0.60% of the total cytosines were methylated in saprophytic-like condition, which was lower than the DNA methylation level (0.89%) in tick mimicked infection condition. A total of 670 mRNA genes were found to be putatively methylated, with 390 mRNA genes uniquely methylated in the tick mimicked infection condition. GO terms linked to response to stimuli, cell wall morphogenesis, cytoskeleton morphogenesis and secondary metabolism biosynthesis were over-represented in the tick mimicked infection condition, suggesting that energy metabolism is directed towards the regulation of genes associated with infection. However, recognized virulence determinants known to be expressed at distinct infection steps, such as the destruxin backbone gene and the collagen-like protein gene Mcl1, were found methylated, suggesting that a dynamic pattern of methylation could be found during the infectious process. These results were further endorsed employing RT-qPCR from cultures treated or not with the DNA methyltransferase inhibitor 5-Azacytidine. CONCLUSIONS: The set of genes here analyzed focused on secondary metabolites associated genes, known to be involved in several processes, including virulence. The BS-Seq pipeline and RT-qPCR analysis employing 5-Azacytidine led to identification of methylated virulence genes in M. anisopliae. The results provided evidences that DNA methylation in M. anisopliae comprises another layer of gene expression regulation, suggesting a main role of DNA methylation regulating putative virulence determinants during M. anisopliae infection cycle.

Assuntos

Metilação de DNA , Metarhizium/genética , Carrapatos/microbiologia , Animais , Genoma Fúngico , Metarhizium/metabolismo , Metarhizium/patogenicidade , RNA-Seq , Metabolismo Secundário , Virulência

RESUMO

Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces cerevisiae Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii EVs from a C. gattiiaim25Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release.IMPORTANCE Extracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid-translocating enzyme, in secretory processes of C. gattii.

Assuntos

Cryptococcus gattii/enzimologia , Vesículas Extracelulares/química , Polissacarídeos Fúngicos/química , Proteínas Fúngicas/genética , Micologia/métodos , Transporte Biológico , Cryptococcus gattii/genética , Cryptococcus neoformans/citologia , Cryptococcus neoformans/genética , Vesículas Extracelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Polissacarídeos/genética , Polissacarídeos/isolamento & purificação

RESUMO

The Pr1 family of serine endopeptidases plays an important role in pathogenicity and virulence of entomopathogens such as Metarhizium anisopliae (Ascomycota: Hypocreales). These virulence factors allow for the penetration of the host cuticle, a vital step in the infective process of this fungus, which possesses 11 Pr1 isoforms (Pr1A through Pr1K). The family is divided into two classes with Class II (proteinase K-like) comprising 10 isoforms further split into three subfamilies. It is believed that these isoforms act synergistically and with other virulence factors, allowing pathogenicity to multiple hosts. As virulence coevolves through reciprocal selection with hosts, positive selection may lead to the evolution of new protease families or isoforms of extant ones that can withstand host defenses. This work tests this hypothesis in Class II Pr1 proteins, focusing on M. anisopliae, employing different methods for phylogenetic inference in amino acid and nucleotide datasets in multiple arrangements for Metarhizium spp. and related species. Phylogenies depict groups that match the taxonomy of their respective organisms with high statistical support, with minor discrepancies. Positively selected sites were identified in six out of ten Pr1 isoforms, most of them located in the proteolytic domain and spatially close to the catalytic residues. Moreover, there was evidence of functional divergence in the majority of pairwise comparisons. These results imply the existence of differential selective pressure acting on Pr1 proteins and a potential new isoform, likely affecting host specificities, virulence, or even adapting the organism to different host-independent lifestyles.

Assuntos

Metarhizium/classificação , Metarhizium/patogenicidade , Serina Endopeptidases/química , Serina Endopeptidases/genética , Sítios de Ligação , Evolução Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Metarhizium/enzimologia , Família Multigênica , Filogenia , Domínios Proteicos , Seleção Genética , Fatores de Virulência/química , Fatores de Virulência/genética

RESUMO

Melanin formation is a promising target for antifungal development. We screened a collection of 727 compounds that were previously approved for clinical use in humans for inhibition of pigmentation in Cryptococcus gattii, a lethal fungal pathogen that causes damage to both immunocompetent and immunocompromised hosts. The pyrimidine analogues flucytosine (5-fluorocytosine [5-FC]), 5-fluorouracil (5-FU) and carmofur were identified as efficient inhibitors of pigmentation in the C. gattii model. Since melanin synthesis is enzymatically catalyzed by laccase in Cryptococcus, we investigated whether inhibition of pigmentation by the pyrimidine analogues was laccase-mediated. Enzyme activity and expression of LAC genes were not involved in the effects of the pyrimidine analogues, suggesting alternative cellular targets for inhibition of pigmentation. To address this hypothesis, we screened a collection of approximately 8000 mutants of C. gattii that were produced by insertional mutation after incubation with Agrobacterium tumefaciens and identified a gene product required for the anti-pigmentation activity of 5-FC as a beta-DNA polymerase. Reduced expression of this gene affected capsule formation and urease activity, suggesting essential roles in the cryptococcal physiology. These results demonstrate a previously unknown antifungal activity of 5-FC and reveal a promising target for the development of novel antifungals.

Assuntos

Antifúngicos/farmacologia , Cryptococcus gattii/efeitos dos fármacos , Melaninas/antagonistas & inibidores , Melaninas/biossíntese , Cryptococcus gattii/genética , Análise Mutacional de DNA , Avaliação Pré-Clínica de Medicamentos , Flucitosina/farmacologia , Fluoruracila/análogos & derivados , Fluoruracila/farmacologia , Testes Genéticos , Mutagênese Insercional

RESUMO

Cell walls are involved in manifold aspects of fungi maintenance. For several fungi, chitin synthesis, degradation and recycling are essential processes required for cell wall biogenesis; notably, the activity of ß-N-acetylglucosaminidases (NAGases) must be present for chitin utilization. For entomopathogenic fungi, such as Metarhizium anisopliae, chitin degradation is also used to breach the host cuticle during infection. In view of the putative role of NAGases as virulence factors, this study explored the transcriptional profile and evolution of putative GH20 NAGases (MaNAG1 and MaNAG2) and GH3 NAGases (MaNAG3 and MaNAG4) identified in M. anisopliae. While MaNAG2 orthologs are conserved in several ascomycetes, MaNAG1 clusters only with Aspergilllus sp. and entomopathogenic fungal species. By contrast, MaNAG3 and MaNAG4 were phylogenetically related with bacterial GH3 NAGases. The transcriptional profiles of M. anisopliae NAGase genes were evaluated in seven culture conditions showing no common regulatory patterns, suggesting that these enzymes may have specific roles during the Metarhizium life cycle. Moreover, the expression of MaNAG3 and MaNAG4 regulated by chitinous substrates is the first evidence of the involvement of putative GH3 NAGases in physiological cell processes in entomopathogens, indicating their potential influence on cell differentiation during the M. anisopliae life cycle.

RESUMO

The yeast-like pathogen Cryptococcus gattii is an etiological agent of cryptococcosis. The major cryptococcal virulence factor is the polysaccharide capsule, which is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs). The GXM and GalXM polysaccharides have been extensively characterized; however, there is little information about the role of mannoproteins in capsule assembly and their participation in yeast pathogenicity. The present study characterized the function of a predicted mannoprotein from C. gattii, designated Krp1. Loss-of-function and gain-of-function mutants were generated, and phenotypes associated with the capsular architecture were evaluated. The null mutant cells were more sensitive to a cell wall stressor that disrupts beta-glucan synthesis. Also, these cells displayed increased GXM release to the culture supernatant than the wild-type strain did. The loss of Krp1 influenced cell-associated cryptococcal polysaccharide thickness and phagocytosis by J774.A1 macrophages in the early hours of interaction, but no difference in virulence in a murine model of cryptococcosis was observed. In addition, recombinant Krp1 was antigenic and differentially recognized by serum from an individual with cryptococcosis, but not with serum from an individual with candidiasis. Taken together, these results indicate that C. gattii Krp1 is important for the cell wall structure, thereby influencing capsule assembly, but is not essential for virulence in vivoIMPORTANCECryptococcus gattii has the ability to escape from the host's immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in C. gattii pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of C. gattii main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in C. gattii cell wall.

Assuntos

Cryptococcus gattii/química , Cápsulas Fúngicas/química , Proteínas Fúngicas/química , Glicoproteínas de Membrana/química , Polissacarídeos/química , Fatores de Virulência/química , Animais , Linhagem Celular , Parede Celular/química , Criptococose/imunologia , Cryptococcus gattii/genética , Cryptococcus gattii/patogenicidade , Feminino , Proteínas Fúngicas/genética , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Mutação , Fagocitose , Fenótipo , Polissacarídeos/genética , Virulência , Fatores de Virulência/genética

RESUMO

Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 106, 107 and 108 conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT50 values obtained were 134.6, 27.2 and 24.8 days for concentrations of 106, 107 and 108 conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT50 values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 106, 107 and 108 conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.

Assuntos

Larva/microbiologia , Metarhizium/fisiologia , Controle Biológico de Vetores/métodos , Rhipicephalus/microbiologia , Controle de Ácaros e Carrapatos/métodos , Animais , Esporos Fúngicos/fisiologia

RESUMO

Colletotrichum musae is an important cosmopolitan pathogenic fungus that causes anthracnose in banana fruit. The entire genome of C. musae isolate GM20 (CMM 4420), originally isolated from infected banana fruit from Alagoas State, Brazil, was sequenced and annotated. The pathogen genomic DNA was sequenced on HiSeq Illumina platform. The C. musae GM20 genome has 50,635,197 bp with G + C content of 53.74% and in its present assembly has 2763 scaffolds, harboring 13,451 putative genes with an average length of 1626 bp. Gene prediction and annotation was performed by Funannotate pipeline, using a pattern for gene identification based on BUSCO.

RESUMO

The Cryptococcus gattii species complex harbors the main etiological agents of cryptococcosis in immunocompetent patients. C. gattii molecular type VGII predominates in the north and northeastern regions of Brazil, leading to high morbidity and mortality rates. C. gattii VGII isolates have a strong clinical relevance and phenotypic variations. These phenotypic variations among C. gattii species complex isolates suggest that some strains are more virulent than others, but little information is available related to the pathogenic properties of those strains. In this study, we analyzed some virulence determinants of C. gattii VGII strains (CG01, CG02, and CG03) isolated from patients in the state of Piauí, Brazil. The C. gattii R265 VGIIa strain, which was isolated from the Vancouver outbreak, differed from C. gattii CG01, CG02 and CG03 isolates (also classified as VGII) when analyzed the capsular dimensions, melanin production, urease activity, as well as the glucuronoxylomannan (GXM) secretion. Those differences directly reflected in their virulence potential. In addition, CG02 displayed higher virulence compared to R265 (VGIIa) strain in a cryptococcal murine model of infection. Lastly, we examined the genotypic diversity of these strains through Multilocus Sequence Type (MLST) and one new subtype was described for the CG02 isolate. This study confirms the presence and the phenotypic and genotypic diversity of highly virulent strains in the Northeast region of Brazil.