Functional characterization of Pneumocystis carinii brl1 by transspecies complementation analysis

Abstract

Pneumocystis jirovecii is a fungus which causes severe opportunistic infections in immunocompromised humans. The brl1 gene of P. carinii infecting rats was identified and characterized by using bioinformatics in conjunction with functional complementation in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The ectopic expression of this gene rescues null alleles of essential nuclear membrane proteins of the Brr6/Brl1 family in both yeasts.

FIG. 1.

P. carinii Brl1p (PcBrl1p) shares homology with S. cerevisiae Brl1p (ScBrl1p) and Brr6p (ScBrr6p) and with S. pombe Brl1p (SpBrl1p) (respective National Center for Research Resources Yeast Resource Center accession numbers: NP_011901, NP_011267, and SPAC8F11.06). The multiple alignment was done by using T-Coffee software and represented by using Boxshade software. Identical residues are indicated by dark areas and asterisks at the bottom of the sequence, conserved residues by gray areas and single points. The dashes within contiguous sequences are introduced by the alignment software. The black bars indicate the two putative transmembrane domains comprised within the Brl1/Brr6 homology domain (gray bar).

FIG. 2.

P. carinii Brl1p rescues inviability of the S. cerevisiae brl1 null mutant. (A) The diploid S. cerevisiae strain heterozygous for the brl1 null allele was transformed with the indicated plasmids and sporulated, and the four spores of each tetrad were separated on rich medium (shown vertically in the picture). Two dissected tetrads for each plasmid are shown. (B) The presence of the brl1 null allele (brl1Δ) and the absence of the wild-type BRL1 gene in the haploid strain complemented with P. carinii brl1 were confirmed by PCR using locus-specific primers that are able to amplify both wild-type and brl1Δ alleles and ORF-specific primers amplifying only the wild-type allele. Lane 1, heterozygous diploid; lane 2, wild-type strain; lane 3, haploid mutant complemented by P. carinii brl1. The unrelated S. cerevisiae TAF4 genomic locus was amplified as the positive DNA control. The sizes of the PCR products of the BRL1 locus, brl1Δ, BRL1 ORF, and TAF4 were, respectively, 2,008 bp, 2,092 bp, 1,416 bp, and 2,067 bp.

FIG. 3.

P. carinii Brl1p rescues the cell cycle arrest phenotype of the S. pombe brl1 null mutant. (A) The S. pombe diploid brl1 null mutant was sporulated, the four spores of each tetrad were separated on rich medium, and the phenotypes of the haploid cells arising from the dissected tetrads were examined. Two spores from each tetrad gave rise to wild-type, kanamycin-sensitive colonies, and two spores from each tetrad gave rise to a single elongated cell. (B) The presence of the brl1 null allele (brl1Δ) in the haploid strain complemented with P. carinii brl1 was confirmed by PCR using locus-specific primers that are able to amplify both wild-type and brl1Δ alleles. Lane 1, wild-type strain; lane 2, heterozygous diploid; lane 3, null mutant complemented by S. pombe brl1; and lane 4, null mutant complemented by P. carinii brl1. As predicted, the observed sizes of the amplified fragments from the S. pombe brl1 and brl1Δ alleles were 1,220 bp and 1,731 bp, respectively. (C) The diploid S. pombe brl1 null mutant was transformed with the indicated plasmids and sporulated, and the phenotypes of the haploid cells arising from the dissected tetrads were examined. The arrows indicate cells in which the mitotically unstable plasmid has probably been lost. ON and OFF indicate medium without and with thiamine, respectively, which shuts off the nmt1 promoter and, therefore, P. carinii or S. pombe brl1 expression. Scale bars, 10 μm.

FIG. 4.

Inactivation of S. pombe brl1 causes a delay in mitotic entry and progression. Haploid S. pombe brl1Δ cells covered by pREP41.Pcbrl1 cells were grown to early exponential phase in supplemented minimal medium, thiamine was added to half the culture, and the cells were grown at 25°C for 16 h. Cells were harvested by centrifugation, fixed with ethanol, and stained with DAPI and Calcofluor. (A) Induced control. (B) Cells from cultures to which thiamine had been added. Note that these latter cells are elongated compared to the induced control, which resembles the wild type. 1, cell with a single nucleus arrested in interphase; 2, cell with condensed chromosomes; 3, cell with a septum. Scale bar, 10 μm.