Transcriptional and Proteomic Analysis of the Aspergillus fumigatus ΔprtT Protease-Deficient Mutant

Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, infecting immunocompromised patients. The fungus invades the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the host structural barriers. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. Unexpectedly, microarray analysis revealed that in addition to the expected decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of prtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of toxic pseurotin A. Proteomic analysis identified reduced levels of three secreted proteases (ALP1 protease, TppA, AFUA_2G01250) and increased levels of three secreted polysaccharide-degrading enzymes in the ΔprtT mutant possibly in response to its inability to derive sufficient nourishment from protein breakdown. This report highlights the complexity of gene regulation by PrtT, and suggests a potential novel link between the regulation of protease secretion and the control of iron uptake, ergosterol biosynthesis and secondary metabolite production in A. fumigatus

Transcription Factor PrtT Controls Expression of Multiple Secreted Proteases in the Human Pathogenic Mold Aspergillus fumigatus▿ †

The role of secreted proteases in the virulence of the pathogenic fungus Aspergillus fumigatus remains controversial. Recently, the Aspergillus niger transcription factor PrtT was shown to control the expression of multiple secreted proteases. In this work, the gene which encodes the PrtT homolog in A. fumigatus was cloned and its function analyzed using a deletion mutant strain. Deletion of A. fumigatus prtT resulted in the loss of secreted protease activity. The expression of six secreted proteases (ALP, MEP, Dpp4, CpdS, AFUA_2G17330, and AFUA_7G06220) was markedly reduced. Culture filtrates from the prtT deletion strain exhibited reduced killing of lung epithelial cells and lysis of erythrocytes. However, the prtT deletion strain did not exhibit altered virulence in lung-infected mice. These results suggest that PrtT is not a significant virulence factor in A. fumigatus

Phenotypic and Proteomic Analysis of the Aspergillus fumigatus ΔPrtT, ΔXprG and ΔXprG/ΔPrtT Protease-Deficient Mutants

Aspergillus fumigatus is the most common mold species to cause disease in immunocompromised patients. Infection usually begins when its spores (conidia) are inhaled into the airways, where they germinate, forming hyphae that penetrate and destroy the lungs and disseminate to other organs, leading to high mortality. The ability of hyphae to penetrate the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases that are thought to enhance penetration by degrading host structural barriers. This study explores the role of the A. fumigatus transcription factor XprG in controlling secreted proteolytic activity and fungal virulence. We deleted xprG, alone and in combination with prtT, a transcription factor previously shown to regulate extracellular proteolysis. xprG deletion resulted in abnormal conidiogenesis and formation of lighter colored, more fragile conidia and a moderate reduction in the ability of culture filtrates (CFs) to degrade substrate proteins. Deletion of both xprG and prtT resulted in an additive reduction, generating a mutant strain producing CF with almost no ability to degrade substrate proteins. Detailed proteomic analysis identified numerous secreted proteases regulated by XprG and PrtT, alone and in combination. Interestingly, proteomics also identified reduced levels of secreted cell wall modifying enzymes (glucanases, chitinases) and allergens following deletion of these genes, suggesting they target additional cellular processes. Surprisingly, despite the major alteration in the secretome of the xprG/prtT null mutant, including two to fivefold reductions in the level of 24 proteases, 18 glucanases, 6 chitinases, and 19 allergens, it retained wild-type virulence in murine systemic and pulmonary models of infection. This study highlights the extreme adaptability of A. fumigatus during infection based on extensive gene redundancy

Top 50 downregulated genes plotted in ascending order by fold change.

<p>Black bars are the iron uptake genes, white bars are genes with various other annotations.</p

qRT-PCR evaluation of transcripts levels from genes that showed a differential expression in the microarray (A) representative genes (<i>MirB, SidA, Erg3, Erg11</i> and <i>Alp1</i>) and (B) the genes <i>HapX</i>, <i>SreA, AcuM</i> and <i>SrbA</i> encoding transcription factors that regulate iron uptake.

<p>Expression rates were normalized relative to mRNA levels of the β-tubulin-encoding gene (AFUA_1G10910) and set arbitrarily to 1 for the WT strain grown in 1% SM medium. Values are given in relative quantity of template compared to the original sample (RQ). RQ values were calculated by use of the equation: RQ  =  2^–ΔΔCT, with ΔΔCT ± SD and ΔCT ± SDs. −Fe  =  growth medium lacking iron and containing 1 mM ferrozine. +Vori  =  growth medium supplemented with a sub-inhibitory concentration (0.125 µg/ml) of voriconazole. The experiment was repeated three times with similar results. Graphs show a representative experiment.</p

Selected enriched downregulated gene classes in the <i>ΔprtT</i> mutant vs. WT¹.

<p>1Top five genes with the highest fold change are shown in each category.</p>2<p>Ergosterol biosynthesis pathway.</p>3<p>Involved in amino acid catabolism.</p

Secretome proteins identified by 2D−DIGE.

1<p>genes also identified by Wartenberg ,et al. (43)</p

Significantly enriched physical clusters

<p>Significantly enriched physical clusters</p