Members of the genus Pneumocystis
are opportunistic fungal pathogens that infect immunocompromised mammalian hosts causing life-threatening pneumonia. Despite available therapies, the mortality from Pneumocystis
pneumonia in humans remains high, ranging from 10 to 40% (14
). Treatment and prophylaxis failures have been demonstrated and emerging evidence indicates the development of drug resistance to the most commonly used antimicrobial agents (11
). Due to the severity of Pneumocystis
pneumonia, further investigations of this organism are warranted to develop new potential targets for therapy. Proteases represent one such potential therapeutic target. In other biological pathogens, drug targeting of proteases has proven a successful strategy (10
In addition, further evidence suggests that proteases are involved in regulation of the life cycle of P. carinii
). Although Pneumocystis
cannot be continuously cultured in vitro, the protease inhibitor leupeptin has been shown to inhibit Pneumocystis
in short-term proliferation assays, while another protease inhibitor aprotinin was demonstrated to enhance short-term organism growth (4
). These data provide intriguing indirect evidence that proteases are likely involved in regulating the organisms' life cycle. In addition, proteases are recognized as virulence factors in a number of microorganisms (10
). Unfortunately, specific knowledge of proteases in Pneumocystis
is fairly limited. Although homologues to the subtilisin clan of the serine protease family (PRT1
) have been characterized in Pneumocystis
, definitive protease function has not yet been demonstrated for these specific proteases (1
A recent report of a proteolytically active ADAM (a disintegrin and metalloprotease) homologue in A. fumigatus
has raised the possibility of the presence of ADAM homologues in other pathogenic fungi, particularly Pneumocystis
). ADAM proteins are membrane-anchored cell surface proteins that are structurally related to snake venom disintegrins (44
). ADAM proteins possess a metalloprotease domain. When active, the metalloprotease domain has a shared consensus sequence HEXXHXXGXXHD (5
). ADAM protease activities have also been demonstrated to a wide variety of target proteins (44
). Furthermore, the ADAM protein mde10 in Schizosaccharomyces pombe
has been demonstrated to play a role in the regulation of spore formation (32
). An initial search of the P. carinii
genomic sequencing project (http://pgp.cchmc.org/
) yielded a 329-bp partial sequence with homology to known ADAM proteins. Accordingly, we sought to fully clone, characterize, and assess function of this novel Pneumocystis
protein. Characterization of this molecule should provide additional important information about an important protease, with potential activity in Pneumocystis
life cycle regulation and pathogenicity.
MATERIALS AND METHODS
Strains and organisms.
All animal studies were approved by the Mayo Institutional Animal Care and Utilization Committee. P. carinii
organisms were originally derived from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in immunosuppressed rats as we previously described (19
). Female Long Evans rats (Harlan, Inc., Indianapolis, IN) were provided with dexamethasone (American Regent, Shirley, NY) at 1.2 mg/liter in their drinking water to induce immunosuppression. Antibiotics were simultaneously administered to avoid bacterial infection, specifically administering ampicillin (DAVA Pharmaceuticals, Fort Lee, NJ) at 500 mg/liter or cephalexin (Ranbaxy, India) at 500 mg/liter to the drinking water during alternate months (13
). After 5 days, rats were anesthetized and intratracheally inoculated with P. carinii
total P. carinii
pneumonia developed over 8 to 10 weeks, and the animals were sacrificed. Lungs were harvested, minced, and homogenized in a Stomacher Tissue Blender (Tekmar, Inc., Cincinnati, OH) in phosphate-buffered saline (PBS; pH 7.3). Homogenates were strained through sterile gauze and then examined by cytopreparation smears. Bacterial or fungal contaminated samples were discarded. Samples were centrifuged in Beckman J2-MC centrifuge at 4,960 × g
for 10 min at 4°C, and the supernatants were discarded. Red blood cells were lysed by using distilled deionized water, and cells were resuspended in PBS (pH 7.3) and centrifuged as described above. The cellular pellets were resuspended, and homogenates were passed through a 10-μm-pore-size filter (Millipore, Inc., Billerica, MA) (24
). When applicable, cyst and trophic forms were separated by a subsequent passage through 3-μm-pore-size filters (Millipore) as we previously reported For experiments requiring separation of the cysts and trophic forms, differential filtration through a 3-μm-pore-size filter was performed as we reported (41
). Such filtration resulted in 99.5% pure trophic forms, which pass through the filter. The cysts are retained by the filter and provide a population that is >40-fold-enriched for cysts. The P. carinii
forms were then either processed immediately for further analysis or flash frozen in a dry ice-methanol bath and stored at −80°C (41
Identification of a Pneumocystis ADAM-like gene.
In order to identify molecules with potential protease characteristics, an in silico search of the P. carinii
genome database (http://pgp.cchmc.org/
) was performed using available sequences from ADAM-like molecules present in other fungi (38
). Through this search, a 329-bp partial sequence was identified with homology to ADAM proteins known for protease and binding properties. The full-length sequence was obtained by using rapid amplification of cDNA ends (GeneRacer kit; Invitrogen) to obtain the remainder of the 5′ and 3′ coding sequences, employing gene specific antisense primers and methods we previously reported (19
). This procedure yielded a final coding sequence of 1,650-bp for the putative P. carinii ADAM
-like gene (PcADAM
MacVector 8.1.1 (Accelrys, Inc., San Diego, CA) was used to analyze the identified sequence and to predict protein size, antigenic sites, restriction enzyme sites, and protein alignments. The National Center for Biotechnology Information Basic Local Alignment Search Tool (BLAST) was used to identify protein homologues.
Molecular cloning of PcADAM.
The primers 5′-ATGCAATATCTATT-CAAATATATGA-3′ and 5′-TTACTCGCATACTGAACCTTTT-3′ were then used to create a complete 1,650-bp amplicon of the entire coding sequence using Taq
DNA polymerase (Invitrogen, Inc., Carlsbad, CA) under the following conditions over 25 cycles: 2 min at 94°C (hot start); 15 s (s) at 94°C, 30 s at 55°C, and 2 min at 72°C; with a final 15 min at 72°C. Gel electrophoresis confirmed an amplicon of ∼1,650-bp. The PCR product was then cloned into the pYES2.1/V5-His-TOPO vector (Invitrogen) into TOP10F′ OneShot Escherichia coli
cells (Invitrogen) using the TOPO cloning protocol according to the manufacturer's instructions. Sequences were confirmed, and plasmids were purified by using the QIAprep spin procedure (Qiagen, Valencia, CA). Haploid Saccharomyces cerevisiae
strain BY4741 (ATCC 201388; ATCC, Manassas, VA) and strain BY4741 (ATCC 201388) (MATahis3delta1 leu2delta0 met15delta0 ura3delta0
) were transformed with the plasmid clones as previously described (19
Southern and chromosomal hybridization of PcADAM.
To verify that the PCR product was of P. carinii origin, a radiolabeled PcADAM amplicon was hybridized both to digested P. carinii genomic DNA and separated P. carinii chromosomes. Taq DNA polymerase (Invitrogen) was used with 5′-CGAATCATGTAAAAATGAATCA-3′ and 5′-CAACCTTTTTCTCCTCCACA-3′ primers to create a 276-bp probe by PCR. Probes were radioactively labeled with [α-32P]dATP (GE Healthcare, Inc., Piscataway, NJ) by using the RadPrime DNA labeling system (Invitrogen). Radioactively labeled probes were purified by using QuickSpin columns (Roche, Basel, Switzerland). The ExpressHyb protocol (Clontech Laboratories, Inc., Mountain View, CA) was then used for PcADAM probe hybridization to Pneumocystis chromosomal contour-clamped homogenous electronic field (CHEF) nitrocellulose membranes (a gift from Melanie Cushion). Hybridized membranes were placed on Kodak Biomax film and incubated at −70°C for development.
In a parallel fashion, Pneumocystis
DNA was isolated by using the IsoQuick nucleic acid extraction system (Orca Research, Inc., Bothell, WA) and incubated with the endonucleases EcoRI, BamHI, and XbaI (Invitrogen). For comparison, rat genomic DNA was also digested with EcoRI. DNA was separated on a 1% agarose gel and transferred to nitrocellulose membranes. Radioactively labeled probes were created, hybridized, and imaged as described above. To verify RNA loading, we reprobed the membrane with a Pneumocystis
β-actin probe (19
Assessment of PcADAM mRNA expression over the life cycle of Pneumocystis.
RNA samples from separated Pneumocystis cyst and trophic forms were isolated by using the TRIzol reagent protocol (Invitrogen). Equal quantities (10 μg) of each were separated by electrophoresis through a 1% agarose gel with 30% formaldehyde. UV visualization of rRNA components was used to confirm equal nucleic acid loading. The RNA species were transferred to nitrocellulose. Radiolabeled probes were created, hybridized, and imaged as described. All findings were confirmed on at least three occasions.
Localization of PcADAMp protein in Pneumocystis life cycle forms.
We next sought to determine the relative extent of expression of the PcADAMp protein in Pneumocystis
life cycle forms. Custom-made antibodies were generated to the synthetic peptides SNKPPKTLRKRSNLYL (Ab1) and IDRNNHRVYRGDAFVT (Ab2) (Bethyl Laboratories, Montgomery, TX) predicted from the translated PcADAM
open reading frame. Immunoelectron microscopy was performed with these antibodies to identify the subcellular localization of the PcADAMp protein in separated life cycle forms of the organism (33
cyst and trophic forms were separated by differential filtration and fixed in 4% formaldehyde and 0.2% glutaraldehyde in phosphate buffer for 16 to 24 h. After fixation, the specimens were rinsed in phosphate buffer and then dehydrated in a series of graded ethyl alcohol solutions while progressively lowering the temperature to −20°C. Briefly, the specimen was treated in 60% ethanol for 15 min at 4°C, in 70% ethanol for 60 min while lowering the temperature to −20°C, in 80% for 60 min at −20°C, 95% for 60 min, and finally in absolute alcohol for 60 min and then infiltrated in 1:1 ethanol-LR White resin overnight, exposed to fresh LR White resin for 60 min, and brought to room temperature and embedded in LR White resin. Specimens were polymerized at 55°C over 2 to 3 days. Thin sections (5 μm) were generated and mounted on nickel grids and dried overnight. Nonspecific binding sites were blocked first in aqueous 1% glycine solution then in PBS and 0.05% Tween 20 (PBST) with 1% normal serum. The antibody was diluted (1:10) in PBST with 1% normal serum, and sections were incubated in primary antibody (100 μg/ml) for 2 h at room temperature. Grids were next rinsed in PBST and incubated for 60 min with goat anti-rabbit immunoglobulin conjugated to 10-nm colloidal gold (33
). After incubation, the grids were rinsed thoroughly in PBST and water and stained with uranyl lead. The sections were washed again and examined on a transmission electron microscope (model 6400; JEOL USA, Inc., Peabody, MA). During the immunoelectron condition, in excess of 100 Pneumocystis
forms were examined by electron microsopy for deposition of gold particles and representative fields were photographed.
Expression of the PcADAMp protease.
Subsequently, heterologous expression of the putative PcADAMp protease was performed in yeast. S. cerevisiae containing PcADAM in pYES2.1/V5-His-TOPO was grown in URA− minimal medium containing 2% glucose, and the expression of PcADAMp was induced in URA− minimal medium with 2% galactose. Yeast cells were lysed by using the YPER reagent (Pierce Chemical Co., Rockford, IL) and a French press. The expressed protein was purified over a nickel affinity column (Pierce) and dialyzed against Tris-buffered saline (Pierce). Expression of the protein was confirmed with Western blotting. Protein concentrations were determined by using the Coomassie protein assay reagent protocol (Pierce), and the products were stored in 40% glycerol. In some applications, protease inhibitors were use,d including EDTA (20 mM), phenylmethylsulfonyl fluoride (2 mM), and Complete Mini-Protease cocktail (Roche Applied Science, Indianapolis, IN) dissolved in YPER lysis reagent.
Determination of Pneumocystis PcADAMp proteolytic activity.
We next performed zymography to evaluate whether PcADAMp exhibited protease activity. First, zymography was performed using PcADAMp expressed heterologously in yeast. To accomplish this, S. cerevisiae was induced to express PcADAMp as described above. The yeast cells were lysed using YPER reagent (Pierce), and the lysates were purified over a nickel affinity column. Samples were resuspended in zymogram sample buffer (Bio-Rad, Inc., Hercules, CA), and proteins were separated on 12% casein zymography gels (Bio-Rad) at 100 V. The gels were then incubated in reactivation buffer containing Zn2+, previously described to be required for ADAM metalloprotease activity (50 mM Tris, 200 mM NaCl2, 5 mM CaCl2, 0.5 mM ZnCl2), at pH 7.48, over 2 h to allow protein digestion to occur. The zymograms were rinsed, fixed, and stained with Coomassie blue R-250 (Bio-Rad). To further assess the need for the essential divalent Zn2+, additional zymography was performed in parallel in the absence of Zn2+.
In addition, we also assessed the proteolytic activity of native PcADAMp. To address this, P. carinii
organisms were harvested from infected rat lungs. The organisms were incubated with lyticase (1 mg/ml; Sigma Chemical Co., St. Louis, MO) and lysed using YPER reagent (Pierce), followed by incubation with DNase I (1 mg/ml; Sigma). Next, the lysates were incubated with custom-made antibodies to PcADAMp (100 μg/ml each; Bethyl Laboratories), and protein A (Sigma) was used to immunoprecipitate the PcADAMp protease by using previously described methods (18
). These samples were resuspended in zymogram sample buffer (Bio-Rad), and proteins were separated on 12% casein zymography gels (Bio-Rad) at 100 V. The gels were then incubated in reactivation buffer (50 mM Tris, 200 mM NaCl2
, 5 mM CaCl2
, 0.5 mM ZnCl2
) at pH 7.48, over 2 h to allow protein digestion. Again, the zymograms were rinsed, fixed, and stained with Coomassie blue R-250 (Bio-Rad).
GenBank accession number.
Sequence data are available from GenBank under accession number EF025111.
These studies strongly indicate that the opportunistic fungal pathogen P. carinii expresses PcADAM, an ADAM family molecule. Our data further indicate that PcADAM is largely expressed by cystic forms of the organism, with the related protein being localized to the surface membrane, as well as being associated with intracystic bodies within mature cysts. Our data further indicate that PcADAMp possesses protease activity, which requires the divalent cation Zn2+ for optimal function.
To date, little has been reported about the proteases of Pneumocystis
. Two specific kexin family subtilisin clan serine presumptive protease genes have been partially characterized, namely, PRT1
(protease 1) and Kex1
(kexin 1) (1
). This class of proteins has been described to function as proprotein convertases, with described roles such as the activation of peptide hormones and growth factors and the degradation of cell walls in mammals and fungi, respectively. The specific proteolytic function of the Pneumocystis PRT1
homologue has never been documented. In addition, PRT1p has been shown to possess a glycosylphosphatidylinositol derivative anchor on its carboxy terminus (34
, the other putative Pneumocystis
protease described to date, has been less well characterized, and a specific function for this molecule has not been demonstrated (20
Indirect evidence of protease activity in Pneumocystis
has also been previously reported (2
). 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
). 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
In contrast, in the present study describes the first fully sequenced Pneumocystis protein that has now been directly documented to possess protease activity. The protease activity of PcADAMp was demonstrated using both heterologously expressed PcADAMp generated in yeast, as well as native PcADAMp purified via immunoprecipitation from fresh organisms. The protease domain present within the PcADAM gene sequence predicted homology to other proteolytically active ADAM family proteins and furthermore possessed the conserved HEXXHXXGXXHD zinc-binding catalytic site. Notably, PcADAMp zymography performed in the absence of Zn2+ failed to exhibit protease activity, as expected.
The life cycle of Pneumocystis
remains poorly characterized; however, the organism is well known to interact tightly with alveolar epithelial cells, with this binding stimulating proliferation of the organism (17
). Previous studies demonstrated that when binding was inhibited by preventing direct contact of the organism with lung epithelium or by adding trimethylcolchinic acid, a binding inhibitor, the proliferation of Pneumocystis
is inhibited (27
). In addition, binding of the organism leads to upregulation of Pneumocystis
expression, molecules known to signal mating and contribute to cell cycle regulation and proliferation (17
has been widely described to bind to extracellular matrix proteins, particularly fibronectin and vitronectin, promoting attachment of the organisms to lung epithelial cells (26
). Our group has recently reported the presence of a unique integrin molecule, PcINT1p, on the surface of Pneumocystis
that mediates interactions with fibronectin (16
bound extracellular matrix proteins in turn provide ligands for binding integrins on the surface of lung epithelial cells, including integrins α5, αv, β1, and α3 (36
). A direct binding mechanism of Pneumocystis
itself to lung epithelial cell integrins has not been previously documented.
ADAM family molecules have been demonstrated to play important roles in integrin-disintegrin binding in other species (30
). However, Pneumocystis
binding to lung epithelial cells has been demonstrated largely to involve the trophic form of the organism (27
). We observed that PcADAMp is expressed to a far greater extent by the cystic form of the organism rather than trophic forms, suggesting that PcADAMp likely does not participate in organism-epithelial binding. Furthermore, PcADAMp contains only a partially conserved disintegrin domain. Careful scrutiny of the full PcADAMp sequence demonstrates an RGD peptide sequence proximal to the metalloprotease domain, raising the possibility of a novel proximal disintegrin function. However, we were unable to demonstrate alteration of Pneumocystis
binding to cultured lung cells with the addition of custom-made antibodies to this proximal domain of PcADAMp. Specifically, anti-PcADAMp antibodies did not suppress specific Pneumocystis
binding to cultured A549 lung epithelial cells (data not shown).
However, effects of PcADAMp on P. carinii
life cycle regulation may involve other activities of this molecule. For instance, prior studies indicate that the PcADAM family molecule MDE10, expressed by S. pombe
, is important for the development of the spore envelope, a function that is essential for completion of meiosis and which appears to be independent of the metalloprotease activity of this molecule (32
). In light of our recent studies that verify the presence of a meiotic control system in P. carinii
, additional investigations will be required to determine these other potential functions of PcADAMp in this organism (6
In conclusion, we report here the identification, cloning, characterization, and functional activity of a novel protein PcADAMp. This is the first study to demonstrate an ADAM family member in Pneumocystis and the first to specifically identify a Pneumocystis protein demonstrating protease activity. The protein has high homology to ADM A and B from the pathogenic fungus Aspergillus and a yet-to-be-described hypothetical protein in Cryptococcus. Such ADAM family members may represent potential novel targets to better understand the life cycles, pathogenic effects, and therapeutic control of these important opportunistic fungi.