A Gene Complex Mediating Cyst Wall Assembly and Integrity in Pneumocystis carinii

Pneumocystis carinii remains an important fungal agent causing life-threatening pneumonia in patients with impaired immunity [1]. Elucidation of the mechanisms by which P. carinii assembles its cell wall has only recently been initiated. Our group has focused on generation of β-glucan cell wall components, which represent major structural constituents of the cyst form. The cystic form has been postulated to represent a transmissible agent capable of surviving the harsh environmental conditions outside of the mammalian host. To this end, we recently characterized P. carinii GSC-1, a gene responsible for assembly of the 1,3-β-D-glucan core carbohydrate of the cyst cell wall [2]. During screening of the P. carinii genomic library for GSC-1, we isolated an open reading frame downstream of P. carinii gsc-1 with homology to C. albicans PHR1/PHR2and S. cerevisiae GAS1.

Other opportunistic fungi such as Aspergillus nidulans and Candida albicans alter gene expression as an adaptive response to environmental pH changes in order to maintain cell wall integrity and exert virulent advantage under varying conditions [3,4]. Specifically, it has been demonstrated that C. albicans expresses a unique family of pH-regulated genes required for virulence [3]. These genes nominally include PHR1, a gene expressed maximally at pH 5.5–8.0, which encodes a protein promoting systemic infection of mice. Alternatively, C. albicans also expresses PHR2 whose transcription is greatest at acidic pH (4–5). Studies indicate that Phr1p and Phr2p act upon β-1,3 glucans of the C. albicans cell wall, mediating the attachment of β-l,6 glucosyl side chains and the attachment of β-l,6-glycosylated mannoproteins [5]. The role of glucan cross-linking in P. cariniipathogenesis is not well understood, but has been postulated to participate in maintaining cell wall integrity during environmental stress.

MATERIAL AND METHODS

In the course of screening a P. cariniiλXgt11 genomic library for the P. carinii 1,3-β-glucan synthetase GSC-1, an open reading frame was identified ∼600-bp downstream of the GSC-1 clone that contained 465-bp of nucleotide coding sequence. Translation of the ORF (BLASTX, EMBL) revealed a partial sequence clone with roughly 50% homology to the C. albicans pH-responsive protein PHR1 and containing a start site for the putative P. carinii PHR gene. To obtain the remaining 3′end of P. carinii PHR1 a modified 3′RAGE (rapid amplification of genomic ends) procedure was used. To confirm that the PHR1 gene was truly represented within the P. carinii genome, southern hybridization was performed using the PHR as a probe against freshly isolated P. carinii genomic DNA. We next evaluated the expression of P. carinii PHR1 mRNA in response to environmental pH. P. cariniiwas placed in 1.0 ml Hams F-12 tissue culture medium supplemented with 10% fetal bovine serum at the indicated pH ranges for 1.0 h at 37°C. Total RNA was hybridized to the radiolabeled PHR1 probe.

We next analyzed the ability of P. carinii Phrlp to restore cell wall integrity by transforming GAS1-deficient S. cerevisiae mutants with P. carinii PHR1 DNA. S. cerevisiae GAS1 deficient mutants have increased cellular fragility to osmotic destabilizing agents such as SDS, a stringent assessment of cell wall stability. Previous studies have shown that GAS1 mutants are unable to grow in the presence of SDS [6]. P. carinii PHR1 DNA was excised from pGEM®-T Easy by digestion with HindIII and Ndel and directionally cloned into the yeast expression vector p425GAL. The p425GAL plasmid has a leu2 gene that permits growth on medium lacking leucine. S. cerevisiae GAS1 mutants were grown to mid-log phase in YPD broth at 30°C and transformed using 10 ng of p425GAL/P. carinii PHR1 DNA or 10 ng p425GAL vector alone without insert. Transformed S. cerevisiae were plated onto minimal medium plates lacking leucine at 30°C. After 48 h, transformants were streaked onto minimal medium leucine-deficient plates containing 0.005% SDS and placed at 30°C and assessed for growth. Transformed colonies proliferating at these conditions were cultured in leucine deficient broth, and plasmid DNA extracted and sequenced to confirm the presence of P. carinii PHR1 DNA.

RESULTS AND DISCUSSION

A 2.3-kb genomic DNA fragment containing the complete PHR1gene was characterized. This single open reading frame of 1632-bp encodes a 544 amino acid peptide. Computer analysis revealed that the PHR1 sequence to be unique, yet most homologous to a protein encoded by S. cerevisiae GGP1/GAS1 (38%; BLAST X) and to the C. albicans pH responsive gene products PHR1/PHR2 (37%, each). All three proteins are thought to encode major glycoproteins localized to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor and important for the maintenance of cell wall integrity. The full length P. carinii PHR1 probe strongly hybridized to a single band on both the EcoRI and HindIII digestions of P. carinii genomic DNA. We further evaluated whether expression of this P. carinii gene was responsive to changes in the ambient pH conditions in a fashion similar to C. albicans PHR1. P. carinii PHR1 mRNA was virtually absent at pH ranging between 4.5 and 5.0. Over the pH range of 5.5 to 7.5, incremental increases of PHR1 mRNA were seen. PHR1 expression was optimal at physiological pH (7.0 to 7.5), which would appear to be the expected pH of the alveolar space.

PHR genes have been implicated in cell wall cross-linking and cell stability under adverse conditions. Accordingly, we evaluated the ability of P. carinii PHR1 to growth complement a S. cerevisiae mutant strain deficient in the PHR1 analogue GAS1, in the presence of the potent osmotic-destabilizing agent SDS. P. carinii PHR1 sequences were subcloned into the S. cerevisiae expression vector p425GAL. S. cerevisiae GAS1 mutants susceptible to growth inhibition by SDS were transformed with P. carinii PHR1 DNA in pGAL425 and selected by proliferation at 30°C on medium containing 0.005% SDS. Colonies were isolated in which the P. carinii PHR1 DNA strongly restored proliferation of the S. cerevisiae mutant parent. Complemented colonies were propagated at 30°C under the selective conditions and plasmid DNA from P. carinii PHR1 complemented colonies isolated to verify the presence of P. carinii PHR1 gene in the selected transformants. S. cerevisiae GAS1 mutant strains transformed with the pGAL425 vector alone failed to grow on selective media containing 0.005% SDS. In contrast, both the S. cerevisiae strains transformed with either the GAS1 gene or the P. carinii PHR1 gene displayed normal growth in the presence of SDS. Thus, P. carinii PHR1 is fully capable of functioning to maintain fungal cell wall integrity when expressed heterologously in this tractable fungal species [Supported by NIH grants RO1-HL-62150, RO1-HL-55934, and RO1-HL-57125].