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The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity

¹ Thoracic Disease Research Unit, Division of Pulmonary and Critical Care, Departments of Internal Medicine, and ² Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, Minnesota

Correspondence and requests for reprints should be addressed to Andrew Limper, M.D., Thoracic Disease Research Unit, 8-24 Stabile Building, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905. E-mail: limper.andrew{at}mayo.edu

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.

Key Words: Pneumocystis • meiosis • kinase • temperature

CLINICAL RELEVANCE In the current investigation, we delineate key life cycle regulatory mechanisms in Pneumocystis (meiotic control), an important cause of severe pneumonia in immunocompromised patients.

 

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