Burgess, Joshua W.

ABSTRACT Eukaryotic cell proliferation and phenotype are highly regulated by contact-dependent mechanisms. We have previously shown that the binding and interaction of the opportunistic fungal pathogen Pneumocystis carinii to lung epithelial cells and extracellular matrix proteins induces mRNA expression of both the mitogen-activated protein (MAP) kinase P. carinii Ste20 (PcSte20) and the cell wall-remodeling enzyme PcCbk1 (16). Herein, we report that in addition to PcSte20 mRNA expression being upregulated, Pneumocystis PcSte20 kinase activity is increased upon interacting with these same lung targets. This activity is also significantly suppressed by Clostridium difficile toxin B, a pan-specific inhibitor of small GTPases, demonstrating the potential role of a Cdc42-like molecule in this signaling cascade. We further observed that the PcSte20 kinase physically interacts with a specific region of the P. carinii cell wall biosynthesis kinase, PcCbk1, a downstream kinase important for mating projection formation and cell wall remodeling. This direct binding was mapped to a specific region of the PcCbk1 protein. We also demonstrated that PcSte20 obtained from whole P. carinii lysates has the ability to phosphorylate PcCbk1 after the organism interacts with lung epithelial cells and extracellular matrix components. These observations provide new insights into P. carinii signaling induced by interactions of this important opportunistic fungal pathogen with lung epithelial cells and matrix.

Abstract Pneumocystis organisms are opportunistic fungal pathogens that cause significant pneumonia in immune-compromised hosts. Recent evidence has suggested that Pneumocystis carinii exists as separate mating types, and expresses and regulates proteins that govern meiosis and progression of the life cycle. This study was undertaken to investigate the activity of three life cycle–regulatory proteins in Pneumocystis, including two proteins essential in mating signaling, and a putative meiotic regulator, to determine the conditions under which they are most active. This study used V5/HIS-tagged PCRan1p, PCSte20p, and PCCbk1, purified from Saccharomyces cerevisiae strain, INVSC, as well as an in vitro Escherichia coli protein expression system to determine the optimal expression conditions of each protein in the presence of varying pH, temperature, and metal ions. These studies demonstrate an atypical enzymatic activity in PCRan1p, whereby the kinase was most active in the environmental conditions between 10 and 25°C, compared with a dramatic reduction in activity above 30°C, temperatures typically found within mammalian hosts. Circular dichroism and fluorescence spectroscopy suggest that PCRan1p becomes partially unfolded at 25°C, leading to its most active conformation, whereas continued unfolding as temperature increases results in strongly suppressed activity. These studies suggest that, in vivo, while under conditions within the mammalian lung (typically 37°C), PCRan1p kinase activity is largely suppressed, allowing better conditions for the activation of meiosis, whereas in ex vivo environments, PCRan1p kinase activity increases to arrest progression of the life cycle until conditions become more favorable.

ABSTRACT 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, PC Ran1 , a presumptive meiotic control kinase, and PC Mei2 , 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 PC Ran1 and 75% identical and 88% homologous for PC Mei2 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.