Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis.

BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.

Complement component 3C3 and C3a receptor are required in chitin-dependent allergic sensitization to Aspergillus fumigatus but dispensable in chitin-induced innate allergic inflammation.

Levels of the anaphylatoxin C3a are increased in patients with asthma compared with those in nonasthmatics and increase further still during asthma exacerbations. However, the role of C3a during sensitization to allergen is poorly understood. Sensitization to fungal allergens, such as Aspergillus fumigatus, is a strong risk factor for the development of asthma. Exposure to chitin, a structural polysaccharide of the fungal cell wall, induces innate allergic inflammation and may promote sensitization to fungal allergens. Here, we found that coincubation of chitin with serum or intratracheal administration of chitin in mice resulted in the generation of C3a. We established a model of chitin-dependent sensitization to soluble Aspergillus antigens to test the contribution of complement to these events. C3(-/-) and C3aR(-/-) mice were protected from chitin-dependent sensitization to Aspergillus and had reduced lung eosinophilia and type 2 cytokines and serum IgE. In contrast, complement-deficient mice were not protected against chitin-induced innate allergic inflammation. In sensitized mice, plasmacytoid dendritic cells from complement-deficient animals acquired a tolerogenic profile associated with enhanced regulatory T cell responses and suppressed Th2 and Th17 responses specific for Aspergillus. Thus, chitin induces the generation of C3a in the lung, and chitin-dependent allergic sensitization to Aspergillus requires C3aR signaling, which suppresses regulatory dendritic cells and T cells and induces allergy-promoting T cells.

Phylogenetic analysis reveals a high level of speciation in the Paracoccidioides genus.

Paracoccidioidomycosis (PCM) is a systemic disease endemic to most of Latin America, with greatest impact in rural areas. The taxonomic status of one of the best studied Paracoccidioides isolates (Pb01) as P. brasiliensis remains unresolved due to its genomic differences from the other three previously described phylogenetic species (S1, PS2 and PS3; Carrero et al., 2008. Fungal Genet. Biol. 45, 605). Using the genealogic concordance method of phylogenetic species recognition (GCPSR) via maximum parsimony and Bayesian analysis, we identified a clade of 17 genotypically similar isolates, including Pb01, which are distinct from the S1/PS2/P3 clade. Consistent with GCPSR, this "Pb01-like" group can be considered a new phylogenetic species, since it is strongly supported by all independent and concatenated genealogies. "Pb01-like" species exhibit great sequence and morphological divergence from the S1/PS2/PS3 species clade, and we estimate that these groups last shared a common ancestor approximately 32 million years ago. In addition, recombination analysis revealed independent events inside both main groups suggesting reproductive isolation. Consequently, we recommend the formal description of the "Pb01-like" cluster as the new species Paracoccidioides lutzii, a tribute to Adolpho Lutz, discoverer of P. brasiliensis in 1908.

Insights into the pathobiology of Paracoccidioides brasiliensis from transcriptome analysis--advances and perspectives.

Paracoccidioiddes brasiliensis is a thermodimorphic fungus endemic to Latin America, where it causes the most prevalent systemic mycosis, paracoccidioidomycosis (PCM). DNA microarray technology has been used to identify patterns of gene expression when a microbe is confronted with conditions of interest, such as in vitro and/or ex vivo interaction with specific cells. P. brasiliensis is one organism that has benefited from this approach. Even though its genome has not been sequenced yet, much has been discovered from its transcriptome and DNA array analyses. In this review, we will outline the current knowledge in P. brasiliensis transcriptome, with focus on differential expression analysis in vitro and on the discussion of the genes that are controlled during the host-pathogen interaction ex vivo in order to give insights into the pathobiology of this fungus. In vitro experiments enabled the delineation of whole metabolic pathways; the description of differential metabolism between mycelium and yeast cells and of the mainly signaling pathways controlling dimorphism, high temperature growth, thermal and oxidative stress, and virulence/ pathogenicity. Recent ex vivo experiments provided advances on the comprehension of the plasticity of response and indicate that P. brasiliensis is not only able to undergo fast and dramatic expression profile changes but can also discern subtle differences, such as whether it is being attacked by a macrophage or submitted to the bloodstream route conditions.

Structural and ultrastructural alterations of Malpighian tubules of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae infected with different Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) recombinant viruses.

Malpighian tubules constitute the main excretion organ of insects. Infection by egt(-) recombinant AcMNPV baculovirus in lepidopteran larvae promotes early degeneration of these structures, which has been correlated with earlier death of the host. However, no trace of viral infection has been detected in that tissue. We constructed two AgMNPV recombinants with the egfp gene under control of the hsp70 promoter, one being egt(-), and used another two recombinants (one egt(-)) containing the lacZ gene. Morphological alterations in the tubules were analyzed by light and electron microscopies. Bioassays were conducted to compare the pathogenicity of recombinants. Results showed progressive presence of marker proteins and tissue degeneration without signals of infection in the tissue. Morphological and bioassay results showed increased pathogenicity for lacZ-containing recombinants compared to the egfp ones; as for egt(-) viruses, we noted higher intensity and earlier onset of alterations. The absence of infection led us to believe that Malpighian tubules degeneration is provoked initially by the death of tracheal cells attached to the tubules and later, by the death of Malpighian tubule cells themselves. Tubule cell death might be due to oncosis and apoptosis, which may be activated by depletion of energy reserves and by accumulation of marker proteins, respectively. Absence of the egt gene may be leading to a higher energetic expense due to molting, thus aggravating tubule cell death, resulting in faster death of host.

Transcriptional profile of ras1 and ras2 and the potential role of farnesylation in the dimorphism of the human pathogen Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes a human systemic mycosis with high incidence in Latin America. Owing to their participation in the control of pathogen morphogenesis, differentiation and virulence, it was decided to characterize ras genes in P. brasiliensis. ras1 and ras2 were identified to be coding for two different proteins with high identity. The ras transcriptional pattern was investigated by reverse transcription PCR (RT-PCR) during mycelium-to-yeast (M-->Y) transition, heat shock at 42 degrees C and after internalization of yeast cells by murine macrophages. Both genes were downregulated inside macrophages and ras1, at 42 degrees C. In contrast, ras genes did not show any transcriptional variation during the M-->Y transition. The fact that Ras proteins are attached to the membrane via farnesylation prompted the use of a farnesyltransferase inhibitor to investigate the importance of this process for vegetative growth and dimorphic transition. Farnesylation blockage interfered with the vegetative growth of yeast cells and stimulated germinative tube production even at 37 degrees C. During Y-->M transition, the inhibitor increased filamentation in a dose-dependent manner, indicating that impaired farnesylation favours the mycelium form of P. brasiliensis. The results suggest that ras genes might have a role in dimorphism, heat shock response and in host-pathogen interaction.

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells.

BACKGROUND: Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. RESULTS: In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation - cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-beta-glucosidase) in mycelium cells; and ags (an alpha-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport - two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells - isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. CONCLUSION: Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.