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1.  Pathogenic diversity amongst serotype C VGIII and VGIV Cryptococcus gattii isolates 
Scientific Reports  2015;5:11717.
Cryptococcus gattii is one of the causative agents of human cryptococcosis. Highly virulent strains of serotype B C. gattii have been studied in detail, but little information is available on the pathogenic properties of serotype C isolates. In this study, we analyzed pathogenic determinants in three serotype C C. gattii isolates (106.97, ATCC 24066 and WM 779). Isolate ATCC 24066 (molecular type VGIII) differed from isolates WM 779 and 106.97 (both VGIV) in capsule dimensions, expression of CAP genes, chitooligomer distribution, and induction of host chitinase activity. Isolate WM 779 was more efficient than the others in producing pigments and all three isolates had distinct patterns of reactivity with antibodies to glucuronoxylomannan. This great phenotypic diversity reflected in differential pathogenicity. VGIV isolates WM 779 and 106.97 were similar in their ability to cause lethality and produced higher pulmonary fungal burden in a murine model of cryptococcosis, while isolate ATCC 24066 (VGIII) was unable to reach the brain and caused reduced lethality in intranasally infected mice. These results demonstrate a high diversity in the pathogenic potential of isolates of C. gattii belonging to the molecular types VGIII and VGIV.
PMCID: PMC4495446  PMID: 26153364
2.  Effects of zinc transporters on Cryptococcus gattii virulence 
Scientific Reports  2015;5:10104.
Zinc is an essential nutrient for all living organisms because it is a co-factor of several important proteins. Furthermore, zinc may play an essential role in the infectiousness of microorganisms. Previously, we determined that functional zinc metabolism is associated with Cryptococcus gattii virulence. Here, we characterized the ZIP zinc transporters in this human pathogen. Transcriptional profiling revealed that zinc levels regulated the expression of the ZIP1, ZIP2 and ZIP3 genes, although only the C. gattii zinc transporter Zip1 was required for yeast growth under zinc-limiting conditions. To associate zinc uptake defects with virulence, the most studied cryptococcal virulence factors (i.e., capsule, melanin and growth at 37 °C) were assessed in ZIP mutant strains; however, no differences were detected in these classical virulence-associated traits among the mutant and WT strains. Interestingly, higher levels of reactive oxygen species were detected in the zip1Δ and in the zip1Δ zip2Δ double mutants. In line with these phenotypic alterations, the zip1Δ zip2Δ double mutant displayed attenuated virulence in a murine model of cryptococcosis. Together, these results indicate that adequate zinc uptake is necessary for cryptococcal fitness and virulence.
PMCID: PMC4423424  PMID: 25951314
3.  Vesicular transport systems in fungi 
Future microbiology  2011;6(11):1371-1381.
Canonical and unconventional mechanisms of secretion in many eukaryotic cells are relatively well known. In contrast to the situation in animal cells, mechanisms of secretion in fungi must include the capacity for trans-cell wall passage of macromolecules to the extracellular space. Although these mechanisms remain somewhat elusive, several studies in recent years have suggested that vesicular transport is required for trans-cell wall secretion of large molecules. Several fungal molecules, including proteins, lipids, polysaccharides and pigments, are released to the extracellular space in vesicles. In pathogenic fungi, a number of these vesicular components are associated with fungal virulence. Indeed, extracellular vesicles produced by fungi can interfere with the immunomodulatory activity of host cells. Fungal vesicles share many functional aspects with mammalian exosomes and extracellular vesicles produced by bacteria, plants and protozoa, but their cellular origin remains unknown. Here, we discuss the involvement of vesicular transport systems in fungal physiology and pathogenesis, making parallels with the mammalian, bacterial, protozoan and plant cell literature.
PMCID: PMC4286297  PMID: 22082294
Cryptococcus neoformans; extracellular vesicles; fungal pathogens; secretion
5.  Comparative genome analysis of entomopathogenic fungi reveals a complex set of secreted proteins 
BMC Genomics  2014;15:822.
Metarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins.
We determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity.
The data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-822) contains supplementary material, which is available to authorized users.
PMCID: PMC4246632  PMID: 25263348
Genome sequence; Entomopathogenic fungi; Secretome; Phylogenomics
6.  Genomic Analyses and Transcriptional Profiles of the Glycoside Hydrolase Family 18 Genes of the Entomopathogenic Fungus Metarhizium anisopliae 
PLoS ONE  2014;9(9):e107864.
Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of β-1,4-linkages in chitin. This linear homopolymer of N-acetyl-β-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen Metarhizium anisopliae, chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the M. anisopliae genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named chit1, chi2 and chi3. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-N-acetyl-β-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different M. anisopliae developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of M. anisopliae chitinases and ENGases and a more detailed study concerning the enzymes’ roles in morphological or nutritional functions will allow comprehensive insights into the chitinolytic potential of this highly infective entomopathogenic fungus.
PMCID: PMC4169460  PMID: 25232743
7.  The vacuolar-sorting protein Snf7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex. 
Scientific Reports  2014;4:6198.
Fungal pathogenesis requires a number of extracellularly released virulence factors. Recent studies demonstrating that most fungal extracellular molecules lack secretory tags suggest that unconventional secretion mechanisms and fungal virulence are strictly connected. Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans. Snf7 is a key ESCRT operator required for unconventional secretion in Eukaryotes. In this study we generated snf7Δ mutant strains of C. neoformans and its sibling species C. gattii. Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation. This phenotype culminated with loss of virulence in an intranasal model of murine infection in both species. Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic. These results are in agreement with the observation that unconventional secretion is essential for cryptococcal pathogenesis and strongly suggest the occurrence of still obscure mechanisms of exportation of non-protein molecules in Eukaryotes.
PMCID: PMC4151102  PMID: 25178636
8.  Proteomic profiling of the influence of iron availability on Cryptococcus gattii 
Journal of Proteome Research  2011;11(1):189-205.
Iron is essential and ubiquitous in living organisms. The competition for this micronutrient between the host and its pathogens has been related to disease establishment. Cryptococcus gattii is an encapsulated yeast that causes cryptococcosis mainly in immunocompetent individuals. In this study, we analyzed the proteomic profile of the C. gattii R265 Vancouver Island isolate under iron-depleted and –replete conditions by Multidimensional Protein Identification Technology (MudPIT) and by 2D-GE. Proteins and key mechanisms affected by alteration of iron levels such as capsule production, cAMP-signaling pathway, response to stress, and metabolic pathways related to mitochondrial function were identified. Our results also show both proteomic methodologies employed to be complementary.
PMCID: PMC3253262  PMID: 21970549
Cryptococcus gattii R265; Proteomics; 2D-LC-MS/MS; MudPIT; 2D-GE; iron
9.  Fungal zinc metabolism and its connections to virulence 
Zinc is a ubiquitous metal in all life forms, as it is a structural component of the almost 10% of eukaryotic proteins, which are called zinc-binding proteins. In zinc-limiting conditions such as those found during infection, pathogenic fungi activate the expression of several systems to enhance the uptake of zinc. These systems include ZIP transporters (solute carrier 39 family) and secreted zincophores, which are proteins that are able to chelate zinc. The expression of some fungal zinc uptake systems are regulated by a master regulator (Zap1), first characterized in the yeast Saccharomyces cerevisiae. In this review, we highlight features of zinc uptake and metabolism in human fungal pathogens and aspects of the relationship between proper zinc metabolism and the expression of virulence factors and adaptation to the host habitat.
PMCID: PMC3796257  PMID: 24133658
zinc ZIP transporters; zinc metabolism; zinc deprivation; ZAP transcription factor; fungal virulence
10.  Zap1 Regulates Zinc Homeostasis and Modulates Virulence in Cryptococcus gattii 
PLoS ONE  2012;7(8):e43773.
Zinc homeostasis is essential for fungal growth, as this metal is a critical structural component of several proteins, including transcription factors. The fungal pathogen Cryptococcus gattii obtains zinc from the stringent zinc-limiting milieu of the host during the infection process. To characterize the zinc metabolism in C. gattii and its relationship to fungal virulence, the zinc finger protein Zap1 was functionally characterized. The C. gattii ZAP1 gene is an ortholog of the master regulatory genes zafA and ZAP1 that are found in Aspergillus fumigatus and Saccharomyces cerevisiae, respectively. There is some evidence to support an association between Zap1 and zinc metabolism in C. gattii: (i) ZAP1 expression is highly induced during zinc deprivation, (ii) ZAP1 knockouts demonstrate impaired growth in zinc-limiting conditions, (iii) Zap1 regulates the expression of ZIP zinc transporters and distinct zinc-binding proteins and (iv) Zap1 regulates the labile pool of intracellular zinc. In addition, the deletion of ZAP1 reduces C. gattii virulence in a murine model of cryptococcosis infection. Based on these observations, we postulate that proper zinc metabolism plays a crucial role in cryptococcal virulence.
PMCID: PMC3423376  PMID: 22916306
11.  Role for Golgi reassembly and stacking protein (GRASP) in polysaccharide secretion and fungal virulence 
Molecular microbiology  2011;81(1):206-218.
Secretion of virulence factors is a critical mechanism for the establishment of cryptococcosis, a disease caused by the yeast pathogen Cryptococcus neoformans. One key virulence strategy of C. neoformans is the release of glucuronoxylomannan (GXM), a capsule-associated immune-modulatory polysaccharide that reaches the extracellular space through secretory vesicles. Golgi reassembly and stacking protein (GRASP) is required for unconventional protein secretion mechanisms in different eukaryotic cells, but its role in polysaccharide secretion is unknown. This study demonstrates that a C. neoformans functional mutant of a GRASP ortholog had attenuated virulence in an animal model of cryptococcosis, in comparison to wild type (WT) and reconstituted cells. Mutant cells manifested altered Golgi morphology, failed to produce typical polysaccharide capsules and showed a reduced ability to secrete GXM both in vitro and during animal infection. Isolation of GXM from cultures of WT, reconstituted or mutant strains revealed that the GRASP ortholog mutant produced polysaccharides with reduced dimensions. The mutant was also more efficiently associated to and killed by macrophages than WT and reconstituted cells. These results demonstrate that GRASP, a protein involved in unconventional protein secretion, is also required for polysaccharide secretion and virulence in C. neoformans.
PMCID: PMC3124575  PMID: 21542865
12.  Optimal Conditions for Continuous Immobilization of Pseudozyma hubeiensis (Strain HB85A) Lipase by Adsorption in a Packed-Bed Reactor by Response Surface Methodology 
Enzyme Research  2012;2012:329178.
This study aimed to develop an optimal continuous process for lipase immobilization in a bed reactor in order to investigate the possibility of large-scale production. An extracellular lipase of Pseudozyma hubeiensis (strain HB85A) was immobilized by adsorption onto a polystyrene-divinylbenzene support. Furthermore, response surface methodology (RSM) was employed to optimize enzyme immobilization and evaluate the optimum temperature and pH for free and immobilized enzyme. The optimal immobilization conditions observed were 150 min incubation time, pH 4.76, and an enzyme/support ratio of 1282 U/g support. Optimal activity temperature for free and immobilized enzyme was found to be 68°C and 52°C, respectively. Optimal activity pH for free and immobilized lipase was pH 4.6 and 6.0, respectively. Lipase immobilization resulted in improved enzyme stability in the presence of nonionic detergents, at high temperatures, at acidic and neutral pH, and at high concentrations of organic solvents such as 2-propanol, methanol, and acetone.
PMCID: PMC3270537  PMID: 22315670
13.  Mycoplasma hyopneumoniae Transcription Unit Organization: Genome Survey and Prediction 
Mycoplasma hyopneumoniae is associated with swine respiratory diseases. Although gene organization and regulation are well known in many prokaryotic organisms, knowledge on mycoplasma is limited. This study performed a comparative analysis of three strains of M. hyopneumoniae (7448, J and 232), with a focus on genome organization and gene comparison for open read frame (ORF) cluster (OC) identification. An in silico analysis of gene organization demonstrated 117 OCs and 34 single ORFs in M. hyopneumoniae 7448 and J, while 116 OCs and 36 single ORFs were identified in M. hyopneumoniae 232. Genomic comparison revealed high synteny and conservation of gene order between the OCs defined for 7448 and J strains as well as for 7448 and 232 strains. Twenty-one OCs were chosen and experimentally confirmed by reverse transcription–PCR from M. hyopneumoniae 7448 genome, validating our prediction. A subset of the ORFs within an OC could be independently transcribed due to the presence of internal promoters. Our results suggest that transcription occurs in ‘run-on’ from an upstream promoter in M. hyopneumoniae, thus forming large ORF clusters (from 2 to 29 ORFs in the same orientation) and indicating a complex transcriptional organization.
PMCID: PMC3223074  PMID: 22086999
ORF cluster; intergenic regions; cotranscription; transcriptional units
14.  The Vacuolar Ca2+ Exchanger Vcx1 Is Involved in Calcineurin-Dependent Ca2+ Tolerance and Virulence in Cryptococcus neoformans▿† 
Eukaryotic Cell  2010;9(11):1798-1805.
Cryptococcus neoformans is an encapsulated yeast that causes a life-threatening meningoencephalitis in immunocompromised individuals. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this pathogen. This trait is controlled in part by the Ca2+-calcineurin pathway, which senses and utilizes cytosolic calcium for signaling. In the present study, the identification of the C. neoformans gene VCX1, which encodes a vacuolar calcium exchanger, is reported. The VCX1 knockout results in hypersensitivity to the calcineurin inhibitor cyclosporine A at 35°C, but not at 30°C. Furthermore, high concentrations of CaCl2 lead to growth inhibition of the vcx1 mutant strain only in the presence of cyclosporine A, indicating that Vcx1 acts in parallel with calcineurin. The loss of VCX1 does not influence cell wall integrity or capsule size but decreases secretion of the major capsular polysaccharide glucuronoxylomannan (GXM) in culture supernatants.Vcx1 also influences C. neoformans phagocytosis by murine macrophages and is required for full virulence in mice. Analysis of cellular distribution by confocal microscopy confirmed the vacuolar localization of Vcx1 in C. neoformans cells.
PMCID: PMC2976299  PMID: 20889719
15.  The Homeostasis of Iron, Copper, and Zinc in Paracoccidioides Brasiliensis, Cryptococcus Neoformans Var. Grubii, and Cryptococcus Gattii: A Comparative Analysis 
Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensis Pb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways.
PMCID: PMC3153025  PMID: 21833306
micronutrient homeostasis; pathogenic fungi; infection
16.  Calcium signaling components in the human pathogen 
Calcium signaling through calmodulin and the phosphatase calcineurin are required for key events of the biology of the human pathogen Cryptococcus neoformans, including mating, morphogenesis, growth at 37°C and virulence. In a recent work we described the functional characterization of a new component of this calcium signaling network: the vacuolar calcium exchanger Vcx1. This transporter is involved in calcium tolerance and virulence in C. neoformans. Two other uncharacterized calcium transporters which are putative orthologs of Saccharomyces cerevisiae PMC1 (a vacuolar calcium ATPase) and PMR1 (a Golgi calcium ATPase) are also functional in C. neoformans. No ortholog of CRZ1, the target of calcineurin in other fungi, has been identified in C. neoformans, indicating a high complexity in cryptococcal calcium-related pathways. Future studies are necessary for the complete understanding of calcium signaling regulation in C. neoformans.
PMCID: PMC3104574  PMID: 21655435
Cryptococcus neoformans; calcium; calcineurin; virulence; calcium transport
17.  Swine and Poultry Pathogens: the Complete Genome Sequences of Two Strains of Mycoplasma hyopneumoniae and a Strain of Mycoplasma synoviae†  
Vasconcelos, Ana Tereza R. | Ferreira, Henrique B. | Bizarro, Cristiano V. | Bonatto, Sandro L. | Carvalho, Marcos O. | Pinto, Paulo M. | Almeida, Darcy F. | Almeida, Luiz G. P. | Almeida, Rosana | Alves-Filho, Leonardo | Assunção, Enedina N. | Azevedo, Vasco A. C. | Bogo, Maurício R. | Brigido, Marcelo M. | Brocchi, Marcelo | Burity, Helio A. | Camargo, Anamaria A. | Camargo, Sandro S. | Carepo, Marta S. | Carraro, Dirce M. | de Mattos Cascardo, Júlio C. | Castro, Luiza A. | Cavalcanti, Gisele | Chemale, Gustavo | Collevatti, Rosane G. | Cunha, Cristina W. | Dallagiovanna, Bruno | Dambrós, Bibiana P. | Dellagostin, Odir A. | Falcão, Clarissa | Fantinatti-Garboggini, Fabiana | Felipe, Maria S. S. | Fiorentin, Laurimar | Franco, Gloria R. | Freitas, Nara S. A. | Frías, Diego | Grangeiro, Thalles B. | Grisard, Edmundo C. | Guimarães, Claudia T. | Hungria, Mariangela | Jardim, Sílvia N. | Krieger, Marco A. | Laurino, Jomar P. | Lima, Lucymara F. A. | Lopes, Maryellen I. | Loreto, Élgion L. S. | Madeira, Humberto M. F. | Manfio, Gilson P. | Maranhão, Andrea Q. | Martinkovics, Christyanne T. | Medeiros, Sílvia R. B. | Moreira, Miguel A. M. | Neiva, Márcia | Ramalho-Neto, Cicero E. | Nicolás, Marisa F. | Oliveira, Sergio C. | Paixão, Roger F. C. | Pedrosa, Fábio O. | Pena, Sérgio D. J. | Pereira, Maristela | Pereira-Ferrari, Lilian | Piffer, Itamar | Pinto, Luciano S. | Potrich, Deise P. | Salim, Anna C. M. | Santos, Fabrício R. | Schmitt, Renata | Schneider, Maria P. C. | Schrank, Augusto | Schrank, Irene S. | Schuck, Adriana F. | Seuanez, Hector N. | Silva, Denise W. | Silva, Rosane | Silva, Sérgio C. | Soares, Célia M. A. | Souza, Kelly R. L. | Souza, Rangel C. | Staats, Charley C. | Steffens, Maria B. R. | Teixeira, Santuza M. R. | Urmenyi, Turan P. | Vainstein, Marilene H. | Zuccherato, Luciana W. | Simpson, Andrew J. G. | Zaha, Arnaldo
Journal of Bacteriology  2005;187(16):5568-5577.
This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.
PMCID: PMC1196056  PMID: 16077101

Results 1-17 (17)