Purification of a 68-kDa cysteine proteinase from crude extract of Pneumocystis carinii

Choi, Mihye; Chung, Byung-Suk; Chung, Yong‐An; Yu, Jae-Ran; Cho, Sang Rock; Hong, Sung Tae

doi:10.3347/kjp.2000.38.3.159

Cited by 2 publications

(4 citation statements)

References 13 publications

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“…Indirect evidence of protease activity in Pneumocystis has also been previously reported (2,4,9,37). However, prior to the present study, protease activity has not been attributed to a specific Pneumocystis protein.…”

Section: Discussioncontrasting

confidence: 40%

“…Finally, a 68-kDa protease active against collagen, hemoglobin, and fibronectin also has been extracted from Pneumocystis and found to maximally exhibit proteolytic activity at pH 5.5. Further information suggested that the molecule may function as a cysteine protease (9).…”

Section: Discussionmentioning

confidence: 99%

“…However, prior to the present study, protease activity has not been attributed to a specific Pneumocystis protein. In previous studies, total Pneumocystis lysates have been demonstrated to exhibit proteolytic activity against gelatin and elastin by zymography (9). In addition, various protease inhibitors have been shown to either prevent short-term growth (leupeptin) or enhance short-term proliferation (aprotinin) of Pneumocystis organisms (3).…”

Section: Discussionmentioning

confidence: 99%

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Characterization of a Novel ADAM Protease Expressed byPneumocystis carinii

Kennedy¹,

Kottom²,

Limper

2009

Infect Immun

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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.

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Section: Discussioncontrasting

confidence: 40%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Characterization of a Novel ADAM Protease Expressed byPneumocystis carinii

Kennedy¹,

Kottom²,

Limper

2009

Infect Immun

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“…Obligate parasitism would have important implications for the management of patients susceptible to P. jirovecii infection because the only source of infection of this pathogen to be protected from would be humans. The proteolytic activity of Pneumocystis species [27] , their surface proteases [28] , their amino acid [29] and oligopeptide (our unpublished observation) permeases, may be involved in scavenging amino acids, as described in other Ascomycetes [30] . These processes would constitute new virulence factors contributing to pathogenicity and which may be used as targets for pharmaceutical intervention.…”

Section: Resultsmentioning

confidence: 95%

Comparative Genomics Suggests that the Fungal Pathogen Pneumocystis Is an Obligate Parasite Scavenging Amino Acids from Its Host's Lungs

et al. 2010

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Pneumocystis jirovecii is a fungus causing severe pneumonia in immuno-compromised patients. Progress in understanding its pathogenicity and epidemiology has been hampered by the lack of a long-term in vitro culture method. Obligate parasitism of this pathogen has been suggested on the basis of various features but remains controversial. We analysed the 7.0 Mb draft genome sequence of the closely related species Pneumocystis carinii infecting rats, which is a well established experimental model of the disease. We predicted 8’085 (redundant) peptides and 14.9% of them were mapped onto the KEGG biochemical pathways. The proteome of the closely related yeast Schizosaccharomyces pombe was used as a control for the annotation procedure (4’974 genes, 14.1% mapped). About two thirds of the mapped peptides of each organism (65.7% and 73.2%, respectively) corresponded to crucial enzymes for the basal metabolism and standard cellular processes. However, the proportion of P. carinii genes relative to those of S. pombe was significantly smaller for the “amino acid metabolism” category of pathways than for all other categories taken together (40 versus 114 against 278 versus 427, P<0.002). Importantly, we identified in P. carinii only 2 enzymes specifically dedicated to the synthesis of the 20 standard amino acids. By contrast all the 54 enzymes dedicated to this synthesis reported in the KEGG atlas for S. pombe were detected upon reannotation of S. pombe proteome (2 versus 54 against 278 versus 427, P<0.0001). This finding strongly suggests that species of the genus Pneumocystis are scavenging amino acids from their host's lung environment. Consequently, they would have no form able to live independently from another organism, and these parasites would be obligate in addition to being opportunistic. These findings have implications for the management of patients susceptible to P. jirovecii infection given that the only source of infection would be other humans.

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Purification of a 68-kDa cysteine proteinase from crude extract of Pneumocystis carinii

Cited by 2 publications

References 13 publications

Characterization of a Novel ADAM Protease Expressed byPneumocystis carinii

Characterization of a Novel ADAM Protease Expressed byPneumocystis carinii

Comparative Genomics Suggests that the Fungal Pathogen Pneumocystis Is an Obligate Parasite Scavenging Amino Acids from Its Host's Lungs

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