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.

Identification of novel cell wall destabilizing antifungal compounds using a conditional Aspergillus nidulans protein kinase C mutant.

OBJECTIVES: Despite the need for novel drugs to combat fungal infections, antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. The aim of this study was to screen a library of small chemical compounds to identify cell wall inhibitors using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans. This mutant is specifically susceptible to cell wall damaging compounds under PKC-repressive growth conditions. METHODS: The inhibitory effect of a library of small chemical compounds was examined in vitro using the conditional A. nidulans PKC strain and a panel of pathogenic fungal isolates. Microscopy was used to assess alterations to fungal ultrastructure following treatment. RESULTS: Three 'hit' compounds affecting cell wall integrity were identified from a screen of 5000 small chemical compounds. The most potent compound, CW-11, was further characterized and shown to specifically affect cell wall integrity. In clinical isolates of Aspergillus fumigatus, CW-11 induces morphological changes characteristic of damage to the cell wall, including wall thickening and rupturing. Analysis of the susceptibility of A. fumigatus and A. nidulans cell wall and signalling pathway mutants to CW-11 suggests that its mode of action differs from that of the antifungals caspofungin and voriconazole. CONCLUSIONS: This work demonstrates the feasibility of using a conditional Aspergillus mutant to conduct a small-molecule library screen to identify novel 'hit' compounds affecting cell wall integrity.

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.

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.

Coevolution between nonhomologous but functionally similar proteins and their conserved partners in the Legionella pathogenesis system.

Legionella pneumophila, the causative agent of Legionnaires' disease, and other pathogenic Legionella species multiply inside protozoa and human macrophages by using the intracellular multiplication (Icm)/defect in organelle trafficking (Dot) type-IV secretion system. The IcmQ protein, which possesses pore-forming activity, and IcmR, which regulates the IcmQ activity, are two essential components of this system. Analysis of the region expected to contain these two genes from 29 Legionella species revealed the presence of a conserved icmQ gene and a large hypervariable gene family [functional homologues of icmR (fir) genes], located at the icmR genomic position. Although hypervariable in their sequence, the fir genes from all 29 Legionella species were found, together with their corresponding icmQ genes, to function similarly during infection. In addition, all FIR proteins we examined were found to interact with their corresponding IcmQ proteins. Detailed bioinformatic, biochemical, and genetic analysis of the interaction between the variable FIR proteins and conserved IcmQ proteins revealed that their interaction depends on a variable region located between two conserved domains of IcmQ. This variable region was also found to be critical for IcmQ self-interaction, and the region probably coevolved with the corresponding FIR protein. A FIR-IcmQ pair was also found in Coxiella burnetii, the only known non-Legionella bacterium that contains an Icm/Dot system, indicating the significance of this protein pair for the function of this type-IV secretion system. We hypothesize that this gene variation, which is probably mediated by positive selection, plays an important role in the evolutionary arms race between the protozoan host cell and the pathogen.