Phosphoric Metabolites Link Phosphate Import and Polysaccharide Biosynthesis for Candida albicans Cell Wall Maintenance

ACKNOWLEDGMENTS We declare no conflicts of interest. We thank Jesús Pla for his kind gift of the anti-Mkc1 antibody and Kristin Moffitt and Richard Malley for generous advice in ELISA technology and use of the ELISA reader. We thank Tahmeena Chowdhury for scientific discussions leading up to this work. We thank the Candida Genome Database. N.-N.L., M.A.-Z., W.Q., and J.R.K. were supported by R21 AI137716 and by Boston Children’s Hospital Department of Pediatrics. M.A.-Z. was partially funded by the Alfonso Martin Escudero Foundation. J.D.-A. and O.L. were funded by the Boston Children’s Hospital Department of Pediatrics and U19 AI118608-01A1. N.A.R.G. was supported by the Wellcome Trust and the Medical Research Council Centre for Medical Mycology (MR/N006364/1).Peer reviewedPublisher PD

Pneumocystis carinii Cell Wall Biosynthesis Kinase Gene CBK1 Is an Environmentally Responsive Gene That Complements Cell Wall Defects of cbk-Deficient Yeast

Pneumocystis species remain an important cause of life-threatening pneumonia in immunocompromised hosts, including those with AIDS. Responses of the organism to environmental cues both within the lung and elsewhere have been poorly defined. Herein, we report the identification of a cell wall biosynthesis kinase gene (CBK1) homologue in Pneumocystis carinii, isolated by differential display PCR, that is expressed optimally at physiological pH (7 to 8) as opposed to more acidic environments. Expression of Pneumocystis CBK1 was also induced by contact with lung epithelial cells and extracellular matrix. Translation of this gene revealed extensive homology to other fungal CBK1 kinases. Pneumocystis CBK1 expression was equal in the cyst and trophic life forms of the organisms. We further demonstrate that Pneumocystis CBK1 expressed in cbk1Δ Saccharomyces cerevisiae cells restored defective cell wall separation during proliferation. Consistent with this, Pneumocystis CBK1 expression also stimulated transcription of the CTS1 chitinase in cbk1Δ mutant yeast cells, an event necessary for cell wall separation. In addition, Pneumocystis CBK1 cDNA supported normal mating projection formation in response to α-factor in the cbk1Δ yeast cells. Site-directed mutations of serine-303 and threonine-494, potential regulatory phosphorylation sites in Pneumocystis CBK1, abolished mating projection formation, indicating a role for these amino acid residues in CBK1 activity. These findings indicate that Pneumocystis CBK1 is an environmentally responsive gene that may function in signaling pathways necessary for cell growth and mating

Pneumocystis Melanins Confer Enhanced Organism Viability

Pneumocystis continues to represent an important opportunistic fungal pathogen of those with compromised immunity. Thus, it is crucial to identify factors that affect its viability and pathogenicity. We previously reported the first identification of melanins in Pneumocystis. In the present study, we sought to further characterize these components and define the function for these melanins. Melanins extracted from Pneumocystis and melanized Pneumocystis cells were analyzed by electron spin resonance spectroscopy, revealing spectra consistent with melanins from other fungi. Immunofluorescence assays using anti-melanin monoclonal antibodies showed that melanins are widely present across Pneumocystis host species, including mouse-, ferret-, and human-derived Pneumocystis organisms, as well as Pneumocystis carinii derived from rat. Using immunoelectron microscopy, melanins were found to localize to the cell wall and cytoplasm of P. carinii cysts, as well as to intracystic bodies within mature cysts. Next, the role of melanins on the maintenance of Pneumocystis viability was determined by using quantitative reverse transcription-PCR measurement of the heat shock protein mRNA under adverse environmental conditions. Using a new method to promote the melanization of Pneumocystis, we observed that strongly melanized Pneumocystis retained viability to a greater degree when exposed to UV irradiation or desiccation compared to less-pigmented organisms. These studies support our previous identification of Pneumocystis melanins across the genus, further characterize these Pneumocystis components, and demonstrate that melanins protect Pneumocystis from environmental stressors

Evidence for a Melanin Cell Wall Component in Pneumocystis carinii

Fluorescein isothiocyanate-labeled monoclonal antibodies specific for fungal melanin were used in this study to visualize melanin-like components of the Pneumocystis carinii cell wall. A colorimetric enzyme assay confirmed these findings. This is the first report of melanin-like pigments in Pneumocystis

Pneumocystis carinii Expresses an Active Rtt109 Histone Acetyltransferase

Species in the genus Pneumocystis can cause severe pneumonia in immune-compromised hosts. The identification of specific targets present in Pneumocystis species, but lacking in mammalian hosts, is paramount to developing new means to treat this infection. One such potential protein is Rtt109, which is a type of histone acetyltransferase (HAT) required for DNA replication in fungi, but not found in mammals. Sequence orthologues of Rtt109 are present in other fungi, but are absent in mammals, making it a potential pan-specific target against medically relevant fungi. Accordingly, we sought to identify the presence of an Rtt109 in P. carinii. A Pneumocystis carinii (Pc) Rtt109 165-bp partial sequence was initially identified from the incomplete P. carinii genome database. Subsequently, a full-length, 1,128-bp cDNA with homology to Saccharomyces cerevisiae Rtt109 (39% Basic Local Alignment Search Tool (BLASTP)) was cloned and characterized. Sequence analysis of PcRtt109 indicated that the P. carinii molecule contains the putative catalytic aspartate present in yeast. We further demonstrated that the PcRtt109 expressed in rtt109Δ S. cerevisiae cells restored H3-K56 acetylation and the sensitivity toward DNA-damaging agents of rtt109Δ mutant cells. Purified PcRtt109 had the ability to acetylate lysine-56 of histone H3, similar to the ability of Schizosaccharomyces pombe Rtt109 protein. The site-directed mutagenesis of PcRtt109 D84A, a potential regulatory site in the Rtt109 HAT family, abolished H3 acetylation, whereas a DD218/219AA mutation that compromised the activity of ScRtt109 had little effect, demonstrating similarities and differences in Pneumocystis PcRtt109 compared with yeast Saccharomyces cerevisiae Rtt109. These results indicate that P. carinii contains an Rtt109 HAT molecule, and represent the complete identification and characterization of a HAT molecule from this important opportunistic fungal pathogen

Pneumocystis carinii Exhibits a Conserved Meiotic Control Pathway▿

Pneumocystis carinii is an opportunistic fungus causing severe pneumonia in immune-compromised hosts. Recent evidence suggests that Pneumocystis exists as separate sex types, though definitive evidence is currently lacking. These studies were undertaken to determine whether Pneumocystis maintains functional meiotic control molecules, which are required for sexual life stages in eukaryotes. Using the Pneumocystis carinii Genome Project database, two partial sequences for meiotic control molecules were detected, namely, PCRan1, a presumptive meiotic control kinase, and PCMei2, a homologue to a primary activator of meiosis in Schizosaccharomyces pombe. Rapid amplification of cDNA ends was employed to obtain the full open reading frames and to further investigate the functions of these proteins. These presumptive meiotic control molecules were most homologous to molecules present in S. pombe (52% identical and 67% homologous for PCRan1 and 75% identical and 88% homologous for PCMei2 by BLAST analysis). Heterologous expression of these Pneumocystis meiotic genes in corresponding temperature-sensitive and knockout strains of S. pombe, respectively, further verified the functions of the PCRan1 and PCMei2 proteins. These proteins were further shown to control downstream components of the meiotic pathway in S. pombe. Lastly, in vitro kinase assays were used to determine that PCRan1p phosphorylates PCMei2p. These experiments represent the first characterization of any proteins in P. carinii involved in meiosis and indicate the presence of a conserved meiotic pathway in Pneumocystis. Elucidation of this pathway will be essential in gaining a greater understanding of this important opportunistic fungal pathogen

Characterization of a Novel ADAM Protease Expressed by Pneumocystis carinii▿ †

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. Recent evidence has suggested that unidentified proteases are involved in Pneumocystis life cycle regulation. Proteolytically active ADAM (named for “a disintegrin and metalloprotease”) family molecules have been identified in some fungal organisms, such as Aspergillus fumigatus and Schizosaccharomyces pombe, and some have been shown to participate in life cycle regulation. Accordingly, we sought to characterize ADAM-like molecules in the fungal opportunistic pathogen, Pneumocystis carinii (PcADAM). After an in silico search of the P. carinii genomic sequencing project identified a 329-bp partial sequence with homology to known ADAM proteins, the full-length PcADAM sequence was obtained by PCR extension cloning, yielding a final coding sequence of 1,650 bp. Sequence analysis detected the presence of a typical ADAM catalytic active site (HEXXHXXGXXHD). Expression of PcADAM over the Pneumocystis life cycle was analyzed by Northern blot. Southern and contour-clamped homogenous electronic field blot analysis demonstrated its presence in the P. carinii genome. Expression of PcADAM was observed to be increased in Pneumocystis cysts compared to trophic forms. The full-length gene was subsequently cloned and heterologously expressed in Saccharomyces cerevisiae. Purified PcADAMp protein was proteolytically active in casein zymography, requiring divalent zinc. Furthermore, native PcADAMp extracted directly from freshly isolated Pneumocystis organisms also exhibited protease activity. This is the first report of protease activity attributable to a specific, characterized protein in the clinically important opportunistic fungal pathogen Pneumocystis

Characterization of PCEng2, a β-1,3-Endoglucanase Homolog in Pneumocystis carinii with Activity in Cell Wall Regulation

Pneumocystis jirovecii pneumonia is an opportunistic fungal infection that causes severe respiratory impairment in immunocompromised patients. The viability of Pneumocystis organisms is dependent on the cyst cell wall, a structural feature that is regulated by essential cell wall–associated enzymes. The formation of the glucan-rich cystic wall has been previously characterized, but glucan degradation in the organism—specifically, degradation during trophic excystment—is not yet fully understood. Most studies of basic Pneumocystis biology have been conducted in Pneumocystis carinii or Pneumocystis murina, the varieties of this genus that infect rats and mice, respectively. Furthermore, all known treatments for P. jirovecii were initially discovered through studies of P. carinii. Accordingly, in this study, we have identified a P. carinii β-1,3-endoglucanase gene (PCEng2) that is demonstrated to play a significant role in cell wall regulation. The cDNA sequence contained a 2.2-kb open reading frame with conserved amino acid domains homologous to similar fungal glycosyl hydrolases (GH family 81). The gene transcript showed up-regulation in cystic isolates, and the expressed protein was detected within both cyst and trophic forms. Complementation assays in Eng2-deleted Saccharomyces cerevisiae strains showed restoration of the cell wall separation defect during proliferation, demonstrating the importance of PCEng2 protein. during fungal growth. These findings suggest that regulation of cyst cell wall β-glucans is a fundamental process during completion of the Pneumocystis life cycle