An evolutionary genomic approach reveals both conserved and species-specific genetic elements related to human disease in closely related Aspergillus fungi.

Aspergillosis is an important opportunistic human disease caused by filamentous fungi in the genus Aspergillus. Roughly 70% of infections are caused by Aspergillus fumigatus, with the rest stemming from approximately a dozen other Aspergillus species. Several of these pathogens are closely related to A. fumigatus and belong in the same taxonomic section, section Fumigati. Pathogenic species are frequently most closely related to nonpathogenic ones, suggesting Aspergillus pathogenicity evolved multiple times independently. To understand the repeated evolution of Aspergillus pathogenicity, we performed comparative genomic analyses on 18 strains from 13 species, including 8 species in section Fumigati, which aimed to identify genes, both ones previously connected to virulence as well as ones never before implicated, whose evolution differs between pathogens and nonpathogens. We found that most genes were present in all species, including approximately half of those previously connected to virulence, but a few genes were section- or species-specific. Evolutionary rate analyses identified over 1700 genes whose evolutionary rate differed between pathogens and nonpathogens and dozens of genes whose rates differed between specific pathogens and the rest of the taxa. Functional testing of deletion mutants of 17 transcription factor-encoding genes whose evolution differed between pathogens and nonpathogens identified eight genes that affect either fungal survival in a model of phagocytic killing, host survival in an animal model of fungal disease, or both. These results suggest that the evolution of pathogenicity in Aspergillus involved both conserved and species-specific genetic elements, illustrating how an evolutionary genomic approach informs the study of fungal disease.

The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

The putative flavin carrier family FlcA-C is important for Aspergillus fumigatus virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen and the most important species causing pulmonary fungal infections. The signaling by calcium is very important for A. fumigatus pathogenicity and it is regulated by the transcription factor CrzA. We have previously used used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) aiming to identify gene targets regulated by CrzA. We have identified among several genes regulated by calcium stress, the putative flavin transporter, flcA. This transporter belongs to a small protein family composed of FlcA, B, and C. The ΔflcA null mutant showed several phenotypes, such as morphological defects, increased sensitivity to calcium chelating-agent ethylene glycol tetraacetic acid (EGTA), cell wall or oxidative damaging agents and metals, repre-sentative of deficiencies in calcium signaling and iron homeostasis. Increasing calcium concentrations improved significantly the ΔflcA growth and conidiation, indicating that ΔflcA mutant has calcium insufficiency. Finally, ΔflcA-C mutants showed reduced flavin adenine dinucleotide (FAD) and were avirulent in a low dose murine infection model.

ChIP-seq reveals a role for CrzA in the Aspergillus fumigatus high-osmolarity glycerol response (HOG) signalling pathway.

Aspergillus fumigatus is an opportunistic pathogen and allergen of mammals. Calcium signalling is essential for A. fumigatus pathogenicity and is regulated by the CrzA transcription factor. We used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) to explore CrzA gene targets in A. fumigatus. In total, 165 potential binding peaks including 102 directly regulated genes were identified, resulting in the prediction of the A[GT][CG]CA[AC][AG] CrzA-binding motif. The 102 CrzA putatively regulated genes exhibited a diverse array of functions. The phkB (Afu3g12530) histidine kinase and the sskB (Afu1g10940) MAP kinase kinase kinase of the HOG (high-osmolarity glycerol response) pathway were regulated by CrzA. Several members of the two-component system (TCS) and the HOG pathway were more sensitive to calcium. CrzA::GFP was translocated to the nucleus upon osmotic stress. CrzA is important for the phosphorylation of the SakA MAPK in response to osmotic shock. The ΔsskB was more sensitive to CaCl2 , NaCl, and paraquat stress, while being avirulent in a murine model of invasive pulmonary aspergillosis. The presence of CaCl2 and osmotic stresses resulted in synergistic inhibition of ΔcrzA and ΔsskB growth. These results suggest there is a genetic interaction between the A. fumigatus calcineurin-CrzA and HOG pathway that is essential for full virulence.

Transcriptional profiling of Brazilian Saccharomyces cerevisiae strains selected for semi-continuous fermentation of sugarcane must.

Brazil played a pioneering role in the global establishment of the sugarcane bioethanol industry. The bioethanol fermentation process currently used in Brazil is unique due to the acid wash and recycling of yeast cells. Two, industrially adopted, wild yeast strains, CAT-1 and PE-2, have become the most widely used in Brazil. How these strains respond to the unique fermentation process is poorly understood. The improved performance of CAT-1 and PE-2 is hypothesised to be related to enhanced stress tolerance. This study presents a genome-wide analysis of the CAT-1 and PE-2 transcriptomes during a small-scale fermentation process that mimicked the industrial conditions. The common and unique transcriptional responses of the two strains to the Brazilian fermentation process were identified. Environmental stress response genes were up-regulated postfermenter feeding, demonstrating the impact of the prior acid wash and high glucose environment. Cell wall and oxidative stress tolerance were subsequently demonstrated to be enhanced for the industrial strains. Conversely, numerous genes involved in protein synthesis were down-regulated at the end of fermentation revealing the later impact of ethanol-induced stress. Subsequently, the industrial strains demonstrated a greater tolerance of ethanol and the disruption of endoplasmic reticulum homoeostasis. This increased ethanol tolerance was finally correlated with an increased unfolded protein response and increased HAC1 splicing.

Sugarwin: a sugarcane insect-induced gene with antipathogenic activity.

In sugarcane fields, colonization of the stalk by opportunistic fungi usually occurs after the caterpillar Diatraea saccharalis attacks the sugarcane plant. Plants respond to insect attack by inducing and accumulating a large set of defense proteins. Two homologues of a barley wound-inducible protein (BARWIN), sugarcane wound-inducible proteins SUGARWIN1 and SUGARWIN2, have been identified in sugarcane by an in silico analysis. Antifungal properties have been described for a number of BARWIN homologues. We report that a SUGARWIN::green fluorescent protein fusion protein is located in the endoplasmic reticulum and in the extracellular space of sugarcane plants. The induction of sugarwin transcripts occurs in response to mechanical wounding, D. saccharalis damage, and methyl jasmonate treatment. The accumulation of transcripts is late induced and is restricted to the site of the wound. Although the transcripts of sugarwin genes were strongly increased following insect attack, the protein itself did not show any effect on insect development; rather, it altered fungal morphology, leading to the apoptosis of the germlings. These results suggest that, in the course of evolution, sugarwin-encoding genes were recruited by sugarcane due to their antipathogenic activity. We rationalize that sugarcane is able to induce sugarwin gene expression in response to D. saccharalis feeding as a concerted plant response to the anticipated invasion by the fungi that typically penetrate the plant stalk after insect damage.