Refining the pH response in Aspergillus nidulans: a modulatory triad involving PacX, a novel zinc binuclear cluster protein.

The Aspergillus nidulans PacC transcription factor mediates gene regulation in response to alkaline ambient pH which, signalled by the Pal pathway, results in the processing of PacC(72) to PacC(27) via PacC(53). Here we investigate two levels at which the pH regulatory system is transcriptionally moderated by pH and identify and characterise a new component of the pH regulatory machinery, PacX. Transcript level analysis and overexpression studies demonstrate that repression of acid-expressed palF, specifying the Pal pathway arrestin, probably by PacC(27) and/or PacC(53), prevents an escalating alkaline pH response. Transcript analyses using a reporter and constitutively expressed pacC trans-alleles show that pacC preferential alkaline-expression results from derepression by depletion of the acid-prevalent PacC(72) form. We additionally show that pacC repression requires PacX. pacX mutations suppress PacC processing recalcitrant mutations, in part, through derepressed PacC levels resulting in traces of PacC(27) formed by pH-independent proteolysis. pacX was cloned by impala transposon mutagenesis. PacX, with homologues within the Leotiomyceta, has an unusual structure with an amino-terminal coiled-coil and a carboxy-terminal zinc binuclear cluster. pacX mutations indicate the importance of these regions. One mutation, an unprecedented finding in A. nidulans genetics, resulted from an insertion of an endogenous Fot1-like transposon.

Establishment of the ambient pH signaling complex in Aspergillus nidulans: PalI assists plasma membrane localization of PalH.

The Aspergillus nidulans ambient pH signaling pathway involves two transmembrane domain (TMD)-containing proteins, PalH and PalI. We provide in silico and mutational evidence suggesting that PalI is a three TMD (3-TMD) protein with an N-terminal signal peptide, and we show that PalI localizes to the plasma membrane. PalI is not essential for the proteolytic conversion of the PacC translation product into the processed 27-kDa form, but its absence markedly reduces the accumulation of the 53-kDa intermediate after cells are shifted to an alkaline pH. PalI and its homologues contain a predicted luminal, conserved Gly-Cys-containing motif that distantly resembles a Gly-rich dimerization domain. The Gly44Arg and Gly47Asp substitutions within this motif lead to loss of function. The Gly47Asp substitution prevents plasma membrane localization of PalI-green fluorescent protein (GFP) and leads to its missorting into the multivesicular body pathway. Overexpression of the likely ambient alkaline pH receptor, the 7-TMD protein PalH, partially suppresses the null palI32 mutation. Although some PalH-GFP localizes to the plasma membrane, it predominates in internal membranes. However, the coexpression of PalI to stoichiometrically similar levels results in the strong predominance of PalH-GFP in the plasma membrane. Thus, one role for PalI, but possibly not the only role, is to assist with plasma membrane localization of PalH. These data, considered along with previous reports for both Saccharomyces cerevisiae and A. nidulans, strongly support the prevailing model of pH signaling involving two spatially segregated complexes: a plasma membrane complex containing PalH, PalI, and the arrestin-like protein PalF and an endosomal membrane complex containing PalA and PalB, to which PacC is recruited for its proteolytic activation.

Nonsense-mediated mRNA decay mutation in Aspergillus nidulans.

An Aspergillus nidulans mutation, designated nmdA1, has been selected as a partial suppressor of a frameshift mutation and shown to truncate the homologue of the Saccharomyces cerevisiae nonsense-mediated mRNA decay (NMD) surveillance component Nmd2p/Upf2p. nmdA1 elevates steady-state levels of premature termination codon-containing transcripts, as demonstrated using mutations in genes encoding xanthine dehydrogenase (hxA), urate oxidase (uaZ), the transcription factor mediating regulation of gene expression by ambient pH (pacC), and a protease involved in pH signal transduction (palB). nmdA1 can also stabilize pre-mRNA (unspliced) and wild-type transcripts of certain genes. Certain premature termination codon-containing transcripts which escape NMD are relatively stable, a feature more in common with certain nonsense codon-containing mammalian transcripts than with those in S. cerevisiae. As in S. cerevisiae, 5' nonsense codons are more effective at triggering NMD than 3' nonsense codons. Unlike the mammalian situation but in common with S. cerevisiae and other lower eukaryotes, A. nidulans is apparently impervious to the position of premature termination codons with respect to the 3' exon-exon junction.

Virulence comparisons of Aspergillus nidulans mutants are confounded by the inflammatory response of p47phox-/- mice.

While investigating the requirement for phagosomal alkalinization in the host defense against pulmonary aspergillosis, we observed high morbidity of p47(phox)(-/-) mice infected with pH-insensitive Aspergillus nidulans mutants despite a paucity of fungal growth. Fatal infection also resulted from a normally avirulent p-aminobenzoate auxotroph. This demonstrates that p47(phox)(-/-) murine immunity contributes significantly to A. nidulans lethality. These data have wider implications for microbial virulence studies with p47(phox)(-/-) mice.

The Aspergillus pH-responsive transcription factor PacC regulates virulence.

The ability of a pathogen to adapt to the host environment is usually required for the initiation of disease. Here we have investigated the importance of the Aspergillus nidulans PacC-mediated pH response in the pathogenesis of pulmonary aspergillosis. Using mutational analysis, we demonstrate that, in neutropenic mice, elimination of the A. nidulans pH-responsive transcription factor PacC, blocking the ambient pH signal transduction pathway or prevention of PacC proteolytic processing acutely attenuates virulence. Infections caused by these alkali-sensitive mutants are characterized by limited growth in vivo and a reduction of inflammatory cell infiltration. In stark contrast, constitutive activation of PacC causes increased mortality marked by extensive fungal invasive growth. PacC action is therefore required for, and able to enhance virulence, demonstrating that the A. nidulans pH-responsive transcription factor PacC plays a pivotal role in pulmonary pathogenesis.