Dissection of A. fumigatus alkaline adaptation and virulence (with a view to inhibiting fungal growth in vivo)

Aspergillus is a common fungus in the environment, dwelling in pot plants and air conditioning ducts. Aspergillus produces huge numbers of airborne spores which disperse on air currents. The spores are small enough to reach the extremities of the human lung when inhaled. This is not usually dangerous as white blood cells are able to kill them efficiently. However, patients having white blood cell deficiency or abnormality, cannot kill spores which germinate and grow inside the body causing a disease called invasive aspergilloisis. From the lung aspergillus can penetrate the blood vessels and spread to other organs.

Invasive aspergillosis affects up to one quarter of bone marrow transplant recipients and leukaemia patients. Around 60,000 people per year become infected worldwide, as many as 9 out of 10 infected patients die. Diagnosis is difficult and comes after significant fungal growth so aggressive treatment is required to stem infections. Only a handful of antifungal drugs are available. They act on the fungal cell wall or cell membrane and are designed to kill the fungus without harming the patient. Unfortunately they have varying effectiveness, particularly against aspergillus. They are toxic to the liver and often cross-react with other medicines. Furthermore, some aspergillus species are developing resistance to these compounds in the same way that bacteria become resistant to antibiotics. There is, therefore, a desperate need for new therapies against aspergillus infection.

My work has identified aspergillus genes which allow growth inside a mouse lung by sensing the pH (concentration of hydrogen ions) and adapting accordingly. Two of the genes, called palH and palI, mediate pH-sensing at the fungal cell membrane. If palH and palI function could be impaired experimentally, fungal growth may be preventable. This project will examine how these genes mediate pH-sensing by identifying the proteins they interact with and provide a means for future screening for chemicals which impair their function by blocking the protein-protein interactions. A third gene controlling the fungal pH response in mice is pacC, which acts after pH?sensing to regulate an appropriate fungal response. Identification of functions under pacC control or which act upstream of pacC will provide further opportunities for interfering with this essential fungal adaptation mechanism.

Aspergillus fumigatus is the commonest cause of death from fungal disease worldwide, causing life-threatening infection in chronically immunosuppressed patients. No specific A. fumigatus virulence factors are identified but the repertoire of genes having demonstrated roles in vivo is growing. The majority are involved in nutrient biosynthesis or uptake. I have recently described two exceptions: sidA, catalysing siderophore biosynthesis and pacC, mediating alkaline adaptation, which together form a new class of A. fumigatus virulence determinants controlling programmed adaptive responses to the host environment. Both employ fungal-specific proteins and therefore have potential as targets for therapeutic intervention.

Alkaline adaptation is critical for fungal virulence and is mediated, in Aspergillus nidulans, by the transcription factor PacC. PacC, its appropriate processing, and six upstream pH-sensing Pal proteins are indispensable for murine pathogenicity. PacC/Rim101 fungal proteins control cell wall biosynthesis, morphogenesis, sporulation and exported enzyme and metabolite production. Many PacC targets function at, or beyond, the cell boundary.

HYPOTHESES:

Factors under PacC regulation mediate virulence as PacC is indispensable for murine pathogenicity and its constitutive activation enhances virulence.

Inhibition of PacC activation in vivo may prevent infection.

This research explores the novel concept that a non-essential fungal adaptation mechanism (which is essential for virulence) can provide valid opportunities for, and novel means of, preventing fungal growth in vivo.

Using in vivo Aspergillus fumigatus transcriptional profiling, phenotypic screening in Saccharomyces cerevisiae, and a yeast-based A. fumigatus proteomic split-ubiquitin screen, novel virulence-essential A. fumigatus functions acting up- and downstream of the transcription factor PacC, which is a pivotal regulator of Aspergillus virulence will be identified. Moreover, a new proteomic tool for identifying A. fumigatus membrane-bound protein-protein interactions will be established using the virulence-essential pH-sensing A. fumigatus PalH and PalI proteins as prototypes. This will provide a screening platform for future identification of inhibitors of characterised A. fumigatus membrane protein-protein interactions and, ultimately, a means to screen for potential inhibitors of A. fumigatus growth in vivo.

The project has three key goals.

A) Identification of functions under PacC transcriptional control in vivo.

B) Identification of novel A. fumigatus PacC regulators.

C) Establish a split-ubiquitin screening system2 for identification of A. fumigatus membrane protein-protein interactions using the pH-sensing membrane proteins PalH and PalI (having known roles in virulence) as prototypes to identify A. fumigatus PalH and PalI protein partners.