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Infection and Immunity, August 2009, p. 3328-3336, Vol. 77, No. 8
0019-9567/09/$08.00+0     doi:10.1128/IAI.01383-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of a Novel ADAM Protease Expressed by Pneumocystis carinii{triangledown} ,{dagger}

Cassie C. Kennedy,1 Theodore J. Kottom,1 and Andrew H. Limper1,2*

Thoracic Diseases Research Unit, Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,1 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 559052

Received 12 November 2008/ Returned for modification 17 December 2008/ Accepted 9 May 2009

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.

* Corresponding author. Mailing address: 8-24 Stabile Building, Mayo Clinic, Rochester, MN 55905. Phone: (507) 284-4348. Fax: (507) 284-4521. E-mail: limper.andrew{at}mayo.edu

{triangledown} Published ahead of print on 18 May 2009.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Editor: A. Casadevall

Infection and Immunity, August 2009, p. 3328-3336, Vol. 77, No. 8
0019-9567/09/$08.00+0     doi:10.1128/IAI.01383-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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