Molecular characterization of a novel victorivirus (order Ghabrivirales, family Totiviridae) infecting Metarhizium anisopliae.

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.

Intra-hemocoel injection of pseurotin A from Metarhizium anisopliae, induces dose-dependent reversible paralysis in the Greater Wax Moth (Galleria mellonella).

Species from the Metarhizium genus are the causal agents of the green muscardine disease of insects. These fungi have been successfully employed for the biological control of pests over decades. Besides the biocontrol applications, recent efforts for genome sequencing of species in this genus have revealed a great diversity of biosynthetic gene clusters potentially associated with secondary metabolite synthesis. Amongst such molecules are the pseurotins, compounds with several activities, as chitin synthase inhibitors, and immunoglobulin E suppressors. Here, we report, for the first time, the isolation of pseurotin A from the culture broth of M. anisopliae, as well as the characterization of the effects of this compound over the model-arthropod Galleria mellonella. Pseurotin A displayed dose-dependent reversible paralysis effects when injected into the larvae hemocoel. However, the posterior challenge of the treated insects with M. anisopliae conidia did not lead to increased mortality, suggesting that pseurotin A treatment did not increase larvae susceptibility to the green muscardine disease. Although apparent insecticidal effects were not observed for pseurotin A, the paralysis effect observed can be important in M. anisopliae infection development.

An efficient Agrobacterium tumefaciens-mediated transformation method for Simplicillium subtropicum (Hypocreales: Cordycipitaceae).

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.

The genetic diversity of "papillomavirome" in bovine teat papilloma lesions.

BACKGROUND: Papillomaviruses are small nonenveloped, circular double-stranded DNA viruses that belong to the Papillomaviridae family. To date, 29 Bos taurus papillomavirus (BPV) types have been described. Studies involving mixed BPV infections have rarely been reported in contrast to human papillomavirus (HPV), which is commonly described in numerous studies showing coinfections. Moreover, previous studies had shown that HPV coinfections increase the risk of carcinogenesis. In the present study, we used rolling-circle amplification followed by a high-throughput sequencing (RCA-HTS) approach in 23 teat papillomas from southern Brazil. RESULTS: Eleven well-characterized BPV types and 14 putative new BPV types were genetically characterized into the Xi, Epsilon and Dyoxipapillomavirus genera according to phylogenetic analysis of the L1 gene, which expands the previous 29 BPV types to 43. Moreover, BPV coinfections were detected in the majority (56.3%) of the papilloma lesions analyzed, suggesting a genetic diverse "papillomavirome" in bovine teat warts. CONCLUSIONS: The data generated in this study support the possibility that a wide range of BPV is probably underdetected by conventional molecular detection tools, and that BPV coinfections are underestimated and probably genetic diverse. Additionally, 14 new BPV types were characterized, increasing the knowledge regarding BPV genetic diversity.

The deletion of chiMaD1, a horizontally acquired chitinase of Metarhizium anisopliae, led to higher virulence towards the cattle tick (Rhipicephalus microplus).

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.

Polyketides produced by the entomopathogenic fungus Metarhizium anisopliae induce Candida albicans growth.

Metarhizium anisopliae is an important entomopathogenic species and model for arthropod-fungus interaction studies. This fungus harbors a diverse arsenal of unexplored secondary metabolite biosynthetic gene clusters, which are suggested to perform diverse roles during host interaction and soil subsistence as a saprophytic species. Here we explored an unusual carnitine acyltransferase domain-containing highly reducing polyketide synthase found in the genome of M. anisopliae. Employing heterologous expression in Aspergillus nidulans, two new polyketides were obtained, named BAA and BAB, as well as one known polyketide [(2Z,4E,6E)-octa-2,4,6-trienedioic acid]. Intra-hemocoel injection of the most abundant compound (BAA) in the model-arthropod Galleria mellonella larvae did not induce mortality or noticeable alterations, suggesting that this compound may not harbor insecticidal activity. Also, the potential role of such molecules in polymicrobial interactions was evaluated. Determination of minimum inhibitory concentration assays using distinct fungal species revealed that BAA and BAB did not alter Cryptococcus neoformans growth, while BAA exhibited weak antifungal activity against Saccharomyces cerevisiae. Unexpectedly, these compounds increased Candida albicans growth compared to control conditions. Furthermore, BAA can mitigate the fungicidal effects of fluconazole over C. albicans. Although the exact role of these compounds on the M. anisopliae life cycle is elusive, the described results add up to the complexity of secondary metabolites produced by Metarhizium spp. Moreover, up to our knowledge, these are the first polyketides isolated from filamentous fungi that can boost the growth of another fungal species.

Analysis of tRNA-derived RNA fragments (tRFs) in Cryptococcus spp.: RNAi-independent generation and possible compensatory effects in a RNAi-deficient genotype.

Small RNAs (sRNAs) are key factors in the regulation of gene expression. Recently, a new class of regulatory sRNAs derived from tRNAs was described, the tRNA-derived RNA fragments (tRFs). Such RNAs range in length from 14 to 30 nucleotides and are produced from both mature and primary tRNA transcripts, with very specific cleavage sites along the tRNA sequence. Although several mechanisms have been proposed for how tRFs mediate regulation of gene expression, the exact mechanism of tRF biogenesis and its dependency upon the RNAi pathway remain unclear. Cryptococcus gattii and Cryptococcus neoformans are basidiomycetous yeasts and important human pathogens. While C. neoformans is RNAi proficient, C. gattii VGII has lost essential RNAi genes. Here, we sought to identify the tRF production profile in C. gattii VGII and C. neoformans in order to assess the RNAi-dependency of tRF production in these fungal species. We developed a RNA-sequencing-based tRF prediction workflow designed to improve the currently available prediction tools. Using this methodology, we were able to identify tRFs in both organisms. Despite the loss of the RNAi pathway, C. gattii VGII displayed a number of identified tRFs that did not differ significantly from those observed in C. neoformans. The analysis of predicted tRF targets revealed that a higher number of targets was found for C. gattii VGII tRFs compared to C. neoformans tRFs. These results support the idea that tRFs are at least partially independent of the canonical RNAi machinery, raising questions about possible compensatory roles of alternative regulatory RNAs in the absence of a functional RNAi pathway.

New nanotechnological formulation based on amiodarone-loaded lipid core nanocapsules displays anticryptococcal effect.

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.

Application of an optimized annotation pipeline to the Cryptococcus deuterogattii genome reveals dynamic primary metabolic gene clusters and genomic impact of RNAi loss.

Evaluating the quality of a de novo annotation of a complex fungal genome based on RNA-seq data remains a challenge. In this study, we sequentially optimized a Cufflinks-CodingQuary-based bioinformatics pipeline fed with RNA-seq data using the manually annotated model pathogenic yeasts Cryptococcus neoformans and Cryptococcus deneoformans as test cases. Our results show that the quality of the annotation is sensitive to the quantity of RNA-seq data used and that the best quality is obtained with 5-10 million reads per RNA-seq replicate. We also showed that the number of introns predicted is an excellent a priori indicator of the quality of the final de novo annotation. We then used this pipeline to annotate the genome of the RNAi-deficient species Cryptococcus deuterogattii strain R265 using RNA-seq data. Dynamic transcriptome analysis revealed that intron retention is more prominent in C. deuterogattii than in the other RNAi-proficient species C. neoformans and C. deneoformans. In contrast, we observed that antisense transcription was not higher in C. deuterogattii than in the two other Cryptococcus species. Comparative gene content analysis identified 21 clusters enriched in transcription factors and transporters that have been lost. Interestingly, analysis of the subtelomeric regions in these three annotated species identified a similar gene enrichment, reminiscent of the structure of primary metabolic clusters. Our data suggest that there is active exchange between subtelomeric regions, and that other chromosomal regions might participate in adaptive diversification of Cryptococcus metabolite assimilation potential.

Zrg1, a cryptococcal protein associated with regulation of growth in nutrient deprivation conditions.

Cryptococcus gattii is one of the causes of cryptococcosis, a life-threatening disease generally characterized by pneumonia and/or meningitis. Zinc is an essential element for life, being required for the activity of many proteins with catalytic and structural roles. Here, we characterize ZRG1 (zinc-related gene 1), which codes a product involved in zinc metabolism. Transcriptional profiling revealed that zinc availability regulated the expression of ZRG1, and its null mutants demonstrated impaired growth in zinc- and nitrogen-limiting conditions. Moreover, zrg1 strains displayed alterations in the expression of the zinc homeostasis-related genes ZAP1 and ZIP1. Notably, cryptococcal cells lacking Zrg1 displayed upregulation of autophagy-like phenotypes. Despite no differences were detected in the classical virulence-associated traits; cryptococcal cells lacking ZRG1 displayed decreased capacity for survival inside macrophages and attenuated virulence in an invertebrate model. Together, these results indicate that ZRG1 plays an important role in proper zinc metabolism, and is necessary for cryptococcal fitness and virulence.

Calcium Binding Protein Ncs1 Is Calcineurin Regulated in Cryptococcus neoformans and Essential for Cell Division and Virulence.

Intracellular calcium (Ca2+) is crucial for signal transduction in Cryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signaling cascade implicated in cryptococcal stress adaptation and virulence, with Ca2+ binding mediated by the EF-hand domains of the Ca2+ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca2+ homeostasis under stress and nonstress conditions, as the ncs1Δ mutant is sensitive to a high Ca2+ concentration and has an elevated basal Ca2+ level. Furthermore, NCS1 expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to the case with Saccharomyces cerevisiae, NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1Δ mutant shares a high temperature and high Ca2+ sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ and cam1Δ), and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1 deficiency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data show that Ncs1 has a significant role as a Ca2+ sensor in C. neoformans, working with calcineurin to regulate Ca2+ homeostasis and, consequently, promote fungal growth and virulence.IMPORTANCECryptococcus neoformans is the major cause of fungal meningitis in HIV-infected patients. Several studies have highlighted the important contributions of Ca2+ signaling and homeostasis to the virulence of C. neoformans Here, we identify the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) and demonstrate its role in Ca2+ homeostasis, bud emergence, cell cycle progression, and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signaling cascade. Our work provides evidence of a link between Ca2+ homeostasis and cell cycle progression in C. neoformans.

Participation of Zip3, a ZIP domain-containing protein, in stress response and virulence in Cryptococcus gattii.

Cryptococcus gattii is an etiologic agent of cryptococcosis, a potentially fatal disease that affects humans and animals. The successful infection of mammalian hosts by cryptococcal cells relies on their ability to infect and survive in macrophages. Such phagocytic cells present a hostile environment to intracellular pathogens via the production of reactive nitrogen and oxygen species, as well as low pH and reduced nutrient bioavailability. To overcome the low-metal environment found during infection, fungal pathogens express high-affinity transporters, including members of the ZIP family. Previously, we determined that functional zinc uptake driven by Zip1 and Zip2 is necessary for full C.gattiivirulence. Here, we characterized the ZIP3 gene of C. gattii, an ortholog of the Saccharomyces cerevisiae ATX2, which codes a manganese transporter localized to the membrane of the Golgi apparatus. Cryptococcal cells lacking Zip3 were tolerant to toxic concentrations of manganese and had imbalanced expression of intracellular metal transporters, such as the vacuolar Pmc1 and Vcx1, as well as the Golgi Pmr1. Moreover, null mutants of the ZIP3 gene displayed higher sensitivity to reactive oxygen species (ROS) and substantial alteration in the expression of ROS-detoxifying enzyme-coding genes. In line with these phenotypes, cryptococcal cells displayed decreased virulence in a non-vertebrate model of cryptococcosis. Furthermore, we found that the ZIP3 null mutant strain displayed decreased melanization and secretion of the major capsular component glucuronoxylomannan, as well as an altered extracellular vesicle dimensions profile. Collectively, our data suggest that Zip3 activity impacts the physiology, and consequently, several virulence traits of C. gattii.

Red pepper peptide coatings control Staphylococcus epidermidis adhesion and biofilm formation.

Medical devices (indwelling) have greatly improved healthcare. Nevertheless, infections related to the use of these apparatuses continue to be a major clinical concern. Biofilms form on surfaces after bacterial adhesion, and they function as bacterial reservoirs and as resistance and tolerance factors against antibiotics and the host immune response. Technological strategies to control biofilms and bacterial adhesion, such as the use of surface coatings, are being explored more frequently, and natural peptides may promote their development. In this study, we purified and identified antibiofilm peptides from Capsicum baccatum (red pepper) using chromatography-tandem mass spectrometry, MALDI-MS, MS/MS and bioinformatics. These peptides strongly controlled biofilm formation by Staphylococcus epidermidis, the most prevalent pathogen in device-related infections, without any antibiotic activity. Furthermore, natural peptide-coated surfaces dislayed effective antiadhesive proprieties and showed no cytotoxic effects against different representative human cell lines. Finally, we determined the lead peptide predicted by Mascot and identified CSP37, which may be useful as a prime structure for the design of new antibiofilm agents. Together, these results shed light on natural Capsicum peptides as a possible antiadhesive coat to prevent medical device colonization.

Genome-wide DNA methylation analysis of Metarhizium anisopliae during tick mimicked infection condition.

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.

Molecular evolution and transcriptional profile of GH3 and GH20 ß-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae.

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.

A Predicted Mannoprotein Participates in Cryptococcus gattii Capsular Structure.

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.

Draft genome assembly of Colletotrichum musae, the pathogen of banana fruit.

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.

Transcriptional Analysis Allows Genome Reannotation and Reveals that Cryptococcus gattii VGII Undergoes Nutrient Restriction during Infection.

Cryptococcus gattii is a human and animal pathogen that infects healthy hosts and caused the Pacific Northwest outbreak of cryptococcosis. The inhalation of infectious propagules can lead to internalization of cryptococcal cells by alveolar macrophages, a niche in which C. gattii cells can survive and proliferate. Although the nutrient composition of macrophages is relatively unknown, the high induction of amino acid transporter genes inside the phagosome indicates a preference for amino acid uptake instead of synthesis. However, the presence of countable errors in the R265 genome annotation indicates significant inhibition of transcriptomic analysis in this hypervirulent strain. Thus, we analyzed RNA-Seq data from in vivo and in vitro cultures of C. gattii R265 to perform the reannotation of the genome. In addition, based on in vivo transcriptomic data, we identified highly expressed genes and pathways of amino acid metabolism that would enable C. gattii to survive and proliferate in vivo. Importantly, we identified high expression in three APC amino acid transporters as well as the GABA permease. The use of amino acids as carbon and nitrogen sources, releasing ammonium and generating carbohydrate metabolism intermediaries, also explains the high expression of components of several degradative pathways, since glucose starvation is an important host defense mechanism.

Alterations of zinc homeostasis in response to Cryptococcus neoformans in a murine macrophage cell line.

AIM: To evaluate alterations of zinc homeostasis in macrophages exposed to Cryptococcus neoformans. Materials & methods: Using a fluorescent zinc probe-based flow cytometry and atomic absorption spectrometry, zinc levels were evaluated in J774.A1 cell lines exposed to C. neoformans H99 cells. The transcription profile of macrophage zinc related homeostasis genes - metallothioneins and zinc transporters (ZnTs) of the SLC30 and SLC39 (Zrt-Irt-protein) families - was analyzed by quantitative PCR. RESULTS: Macrophage intracellular labile zinc levels decreased following exposure to C. neoformans. A significant decrease in transcription levels was detected in specific ZnTs from both the Zrt-Irt-protein and ZnT families, especially 24 h after infection. CONCLUSION: These findings suggest that macrophages may exhibit zinc depletion in response to C. neoformans infection.

sRNAs as possible regulators of retrotransposon activity in Cryptococcus gattii VGII.

BACKGROUND: The absence of Argonaute genes in the fungal pathogen Cryptococcus gattii R265 and other VGII strains indicates that yeasts of this genotype cannot have a functional RNAi pathway, an evolutionarily conserved gene silencing mechanism performed by small RNAs. The success of the R265 strain as a pathogen that caused the Pacific Northwest and Vancouver Island outbreaks may imply that RNAi machinery loss could be beneficial under certain circumstances during evolution. As a result, a hypermutant phenotype would be created with high rates of genome retrotransposition, for instance. This study therefore aimed to evaluate in silicio the effect of retrotransposons and their control mechanisms by small RNAs on genomic stability and synteny loss of C. gattii R265 through retrotransposons sequence comparison and orthology analysis with other 16 C. gattii genomic sequences available. RESULTS: Retrotransposon mining identified a higher sequence count to VGI genotype compared to VGII, VGIII, and VGIV. However, despite the lower retrotransposon number, VGII exhibited increased synteny loss and genome rearrangement events. RNA-Seq analysis indicated highly expressed retrotransposons as well as sRNA production. CONCLUSIONS: Genome rearrangement and synteny loss may suggest a greater retrotransposon mobilization caused by RNAi pathway absence, but the effective presence of sRNAs that matches retrotransposon sequences means that an alternative retrotransposon silencing mechanism could be active in genomic integrity maintenance of C. gattii VGII strains.