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Molecular evaluation of the plasma membrane proton pump from Aspergillus fumigatus
Henriette P Burghoorn1, Patricia Soteropoulos, Padmaja Paderu
1Public Health Research Institute, New York, New York 10016, USA.
Abstract:
The gene encoding the plasma membrane proton pump (H+ -ATPase) of Aspergillus fumigatus, PMA1, was characterized from A. fumigatus strain NIH 5233 and clinical isolate H11-20. An open reading frame of 3,109 nucleotides with two introns near the N terminus predicts a protein consisting of 989 amino acids with a molecular mass of approximately 108 kDa. The predicted A. fumigatus enzyme is 89 and 51% identical to H+ - ATPases of Aspergillus nidulans and Saccharomyces cerevisiae, respectively. The A. fumigatus PMA1 is a typical member of the P-type ATPase family that contains 10 predicted transmembrane segments and conserved sequence motifs TGES, CSDKTGT, MLTGD, and GDGVN within the catalytic region. The enzyme represents 2% of the total plasma membrane protein, and it is characteristically inhibited by orthovanadate, with a 50% inhibitory concentration of approximately 1.8 microM. H+ -ATPases from Aspergillus spp. contain a highly acidic insertion region of 60 amino acids between transmembrane segments 2 and 3, which was confirmed for the membrane-assembled enzyme with a peptide-derived antibody. An increasing A. fumigatus PMA1 copy number confers enhanced growth in low-pH medium, consistent with its role as a proton pump. These results provide support for the development of the A. fumigatus H+ -ATPase as a potential drug discovery target.
Insights
The Aspergillus fumigatus PMA1 gene encodes a plasma membrane proton pump (H+-ATPase) crucial for fungal growth. Targeting this H+-ATPase presents a potential drug discovery avenue.
Area of Science:
- Molecular Biology
- Mycology
- Biochemistry
Background:
- The plasma membrane proton pump (H+-ATPase) plays a vital role in cellular homeostasis.
- Aspergillus fumigatus is an opportunistic fungal pathogen where H+-ATPase function is critical for survival and virulence.
Purpose of the Study:
- To characterize the PMA1 gene encoding the H+-ATPase in Aspergillus fumigatus.
- To investigate the structural and functional properties of the A. fumigatus H+-ATPase.
- To evaluate the potential of A. fumigatus H+-ATPase as a drug target.
Main Methods:
- Gene sequencing and analysis of the PMA1 open reading frame.
- Bioinformatic prediction of protein structure and conserved motifs.
- Enzyme inhibition assays using orthovanadate.
- Confirmation of protein structure using peptide-derived antibodies.
Main Results:
- The A. fumigatus PMA1 gene predicts a 989-amino acid protein, a P-type ATPase with conserved motifs and 10 transmembrane segments.
- The enzyme is inhibited by orthovanadate (IC50 ≈ 1.8 μM) and shares structural similarities with other fungal H+-ATPases.
- Increased PMA1 copy number enhanced fungal growth in low-pH environments, confirming its proton pump function.
Conclusions:
- The characterized A. fumigatus H+-ATPase (PMA1) is a functional proton pump with conserved P-type ATPase features.
- The enzyme's essential role in fungal growth, particularly under acidic conditions, highlights its potential as a drug target.
- Further research into A. fumigatus H+-ATPase inhibitors could lead to novel antifungal therapies.