SummaryThe 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.

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-Cyscontaining 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.

We have cloned the palA gene of Aspergillus nidulans, one of six genes participating in ambient pH signal transduction in a regulatory circuit mediating pH regulation of gene expression. The derived 798-residue PalA protein is 29.4% identical over its entire length to a hypothetical protein from the nematode Caenorhabditis elegans and also has possible yeast homologs.In the filamentous fungus Aspergillus nidulans, as in many other microorganisms, the syntheses of a number of permeases, secreted enzymes, and exported metabolites are appropriately regulated by ambient pH (3). For example, acid phosphatase and the acid-pH-optimum ␥-aminobutyrate permease are synthesized preferentially in acidic media, whereas alkaline phosphatase and penicillin are synthesized preferentially in alkaline media (3,7,18). pH regulation of gene expression is mediated by the zinc finger transcription factor PacC, whose 73-kDa full-length translation product is proteolyzed to yield a 29-kDa N-terminal fragment able to activate transcription of genes expressed at alkaline pH and prevent transcription of genes expressed at acid pH (15,17,19). The products of six genes, palA, -B, -C, -F, -H, and -I, form a signal transduction pathway through which alkaline ambient pH is able to elicit the conversion of the full-length form of PacC to the functional proteolyzed form (1,3,5,15,19). Loss-of-function mutations in these pal genes prevent this proteolytic conversion and mimic the effects of growth at acidic pH (1,3,5,7,15,18).The mechanism of ambient pH signal transduction is an intriguing subject of scientific and biotechnological importance. We have previously shown that the palB gene product is likely to be a cysteine protease of the calpain family, albeit not the protease responsible for the final conversion of PacC to its functional form (5). Here, we report the cloning and sequence of palA. Amino acid sequence similarity strongly suggests that the palA gene product has a homolog in the nematode Caenorhabditis elegans. It also has possible homologs in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae.The palA gene was cloned with linkage group III (containing palA) clones from a chromosome-allocated wild-type A. nidulans cosmid library (2) and pILJ16 (11) and by identification of palA ϩ cotransformants by their ability to grow on pH 8 medium (3-5) among argB ϩ transformants of a strain of genotype biA1 yA2 wA3 argB2 areA49 palA1. palA1-rescuing activity was found in cosmid W28H12 and further localized to a 5-kb XbaIXhoI fragment (Fig. 1A). To confirm that this fragment contains the palA gene itself rather than a gene associated with a fortuitous suppressor activity, strains carrying either of two