Eukaryotic Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
EC.00345-07v1
7/2/258    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kutty, G.
Right arrow Articles by Kovacs, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kutty, G.
Right arrow Articles by Kovacs, J. A.

 Previous Article  |  Next Article 

Eukaryotic Cell, February 2008, p. 258-267, Vol. 7, No. 2
1535-9778/08/$08.00+0     doi:10.1128/EC.00345-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Pneumocystis Encodes a Functional S-Adenosylmethionine Synthetase Gene{triangledown}

Geetha Kutty,1 Beatriz Hernandez-Novoa,1,{dagger} Meggan Czapiga,2 and Joseph A. Kovacs1*

Critical Care Medicine Department, NIH Clinical Center, National Institutes of Health, Bethesda, Maryland,1 Research Technologies Branch, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland2

Received 18 September 2007/ Accepted 23 November 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
S-Adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6) is the enzyme that catalyzes the synthesis of AdoMet, a molecule important for all cellular organisms. We have cloned and characterized an AdoMet synthetase gene (sam1) from Pneumocystis spp. This gene was transcribed primarily as an ~1.3-kb mRNA which encodes a protein containing 381 amino acids in P. carinii or P. murina and 382 amino acids in P. jirovecii. sam1 was also transcribed as part of an apparent polycistronic transcript of ~5.6 kb, together with a putative chromatin remodeling protein homologous to Saccharomyces cerevisiae, CHD1. Recombinant Sam1, when expressed in Escherichia coli, showed functional enzyme activity. Immunoprecipitation and confocal immunofluorescence analysis using an antipeptide antibody showed that this enzyme is expressed in P. murina. Thus, Pneumocystis, like other organisms, can synthesize its own AdoMet and may not depend on its host for the supply of this important molecule.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pneumocystis jirovecii is a pathogen that causes pneumonia in patients with AIDS and in other immunocompromised patients (22, 27). Pneumocystis organisms that infect different mammalian hosts are unique and genetically divergent (9, 31, 33, 37) and are designated as different species (6, 14, 32). Because a reliable in vitro culture system is lacking, it is difficult to study the biology of this organism directly. One group has reported that the continuous addition of S-adenosylmethionine (AdoMet) can enhance the survival of the organism in culture (25, 26). The failure to detect AdoMet synthetase (EC 2.5.1.6) activity in Pneumocystis carinii homogenates led to the conclusion that Pneumocystis is unable to express functional AdoMet synthetase and thus must rely on its hosts for its supply of AdoMet (24).

AdoMet synthetase catalyzes the formation of AdoMet from methionine and ATP (15). AdoMet is an essential molecule in cellular metabolism: it is the methyl donor for most methylation reactions, such as the methylation of proteins, nucleic acids, lipids, and polysaccharides (19). It also serves as a precursor for polyamines and glutathione synthesis. Almost all organisms have a functional AdoMet synthetase and are able to synthesize this molecule de novo.

Genes encoding AdoMet synthetase have been characterized from prokaryotes and eukaryotes and are highly conserved (15). In perusing the Pneumocystis genome project database (8), we noted a partial sequence homologous to AdoMet synthetase for other organisms, including fungi. Our aim in the current study was to determine whether the Pneumocystis genome contained an AdoMet synthetase gene capable of encoding a functional enzyme that is expressed by Pneumocystis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
RNA and DNA extraction. Total RNA was extracted, using RNAzol B (Tel-Test Inc., Friendswood, TX) from, P. carinii- or Pneumocystis murina-infected lungs and from P. carinii or P. murina organisms partially purified from the infected lungs of rats or mice by Ficoll-Hypaque density gradient centrifugation, as described previously (16). Genomic DNA was isolated using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA). For P. jirovecii, genomic DNA was extracted from autopsy lung samples.

PCR and DNA sequencing. PCR and sequencing were performed as described previously (17). PCR was performed using High Fidelity PCR master mix (Roche Diagnostics Corp., Indianapolis, IN) and genomic DNA or cDNA from P. carinii or P. murina organisms or from P. jirovecii-infected lung samples as templates. The sequences of the primers used for the amplifications are listed in Table 1. In certain experiments, AccuPrime Pfx (Invitrogen, Carlsbad, CA) or HotStar Taq (Qiagen) was used.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Sequences of oligonucleotides used for PCRs

 
Partial genomic sequences of sam1 from both P. murina and P. jirovecii were obtained by sequencing the PCR products generated by the amplification of genomic DNA, using primers designed from the P. carinii sam1 gene sequence obtained from the Pneumocystis genome project database (7).

For reverse transcription (RT)-PCR, first-strand cDNA was synthesized from total RNA preparations obtained from partially purified P. carinii organisms or from P. murina-infected lung samples, using AP primer and Superscript II reverse transcriptase (Invitrogen). PCR was performed utilizing primers designed from the known sam1 gene sequence. For 3' rapid amplification of cDNA ends (3' RACE), primer UAP (3' RACE kit), and the sam1 gene-specific primers were used. RNA isolated from P. carinii organisms or from P. murina-infected lung samples was subjected to RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), using a First Choice RLM-RACE kit (Ambion Inc., Austin, TX) according to the manufacturer's protocol. The first- and second-round PCR were performed using outer and inner adapter primers along with the sam1 gene-specific primers.

Inverse PCR was done as described previously (20). Briefly, genomic DNA extracted from lung samples infected with P. carinii, P. murina, or P. jirovecii was digested with restriction enzyme HindIII or MboI (New England Biolabs, Beverly, MA). The digested product was ligated using T4 DNA ligase (New England Biolabs, Beverly, MA) and subjected to nested PCR.

In some cases, PCR products were subcloned into TOPO TA cloning PCR 2.1 vector (Invitrogen). The clones were verified by sequencing PCR products generated using M13 forward and reverse primers.

Southern and Northern blotting analyses. Southern and Northern blotting analyses were performed as described previously (17). Southern blotting analysis was performed using genomic DNA from P. carinii-infected lung samples digested with different restriction enzymes. The blots were hybridized with a digoxigenin (DIG)-labeled PCR product spanning nucleotides 552 to 2020 of the P. carinii sam1 genomic sequence (DIG-High Prime; Roche) or a DIG-dUTP-labeled oligonucleotide (DIG oligonucleotide tailing kit; Roche). For Northern blotting analysis, total RNA from P. carinii- or P. murina-infected lung samples was subjected to agarose gel electrophoresis in the presence of formaldehyde, transferred to a Nytran membrane, and hybridized as described above for Southern blots.

Protein expression and refolding. The coding region of the P. carinii or P. murina sam1 gene (1,143 bp) was amplified, cloned into the pET 28 expression vector (EMD Biosciences, San Diego, CA), and transformed into Escherichia coli strain BL21(DE3) RIL (Stratagene, Cedar Creek, TX). Recombinant protein was induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3 h at 30°C. Recombinant protein and controls used for the AdoMet synthetase activity assay were expressed using Overnight Express Autoinduction System 1 (EMD Biosciences). Cells were harvested and resuspended in buffer containing 0.5 M NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% β-mercaptoethanol, and a cocktail of protease inhibitors. Following cell sonication, the lysate was centrifuged at 10,000 x g for 10 min, and the pellet was processed as described previously (29). Briefly, the pellet was washed three times with the buffer containing 0.1 M Tris-HCl (pH 7.5), 10 mM MgSO4, 5% Triton X-100, and 4 M urea, followed by two washes using the same buffer without Triton X-100 or urea, and subsequently solubilized at 10°C in buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgSO4, and 8 M urea. The samples were dialyzed (three times within 24 h) at 4°C, using refolding buffer (50 mM Tris-HCl [pH 8.0], 10 mM MgSO4, and 10 mM dithiothreitol). Prior to dialysis, the samples were diluted fourfold by using refolding buffer.

AdoMet synthetase assay. AdoMet synthetase activity was measured as described previously (4). The reaction mixture (250 µl) contained 100 mM Tris-HCl (pH 7.5), 200 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 5 mM ATP, 1 mM L-methionine, and 0.2 µCi of L-[methyl-14C]methionine (GE Health Care, Pittsburgh, PA). Samples (125 µl) along with the reaction buffer were incubated at 37°C, and the reaction was stopped at different time points by the addition of 10 ml of cold water. The reaction samples were loaded onto AG 50 W-X2 cation-exchanger columns (NH4+; Bio-Rad, Hercules, CA) and washed with 20 ml of water. AdoMet was eluted in two 4-ml aliquots of 3 M NH4OH. Each eluate was added to 10 ml of Optiphase-Hisafe 3 (Perkin Elmer, Wellesley, MA), and the radioactivity was measured in a scintillation counter.

Peptide antibodies. A synthetic peptide, ISTEKIREEILEKIVKKVIPS (corresponds to amino acids 199 to 219 of P. murina Sam1) was used to commercially raise antibodies in rabbits and to affinity purify the hyperimmune sera (SigmaGenosys, The Woodlands, TX).

Immunofluorescence and confocal microscopic analysis. Immunofluorescent staining was performed by Histoserv, Inc. (Germantown, MD). P. murina-infected or uninfected lung tissue sections were costained with affinity purified anti-Sam1 antibody and anti-Pneumocystis monoclonal antibody 4D7 (1, 23). Antibody 4D7 recognizes a Pneumocystis-specific antigen that, based on immunofluorescence, appears to be present on both cysts and trophozoites. Alexa Fluor 488 goat anti-rabbit immunoglobulin G (IgG) was used for the detection of Sam1, while biotin-conjugated anti-mouse IgG and streptavidin-conjugated Alexa Fluor 594 were used for staining Pneumocystis organisms. Nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI). In certain experiments, Sam1 antibody was preincubated with the purified recombinant P. murina Sam1 (insoluble form), cells were centrifuged, and the supernatant was used for immunohistochemistry.

Confocal microscopy images were collected with a Leica SP5 confocal microscope (Leica Microsystems, Exton, PA) using an x63 oil immersion objective with a numerical aperture of 1.4, and zoom 4. Fluorochromes were excited by using an argon laser (Enterprise model 651; Coherent, Inc.) at 364 nm for DAPI, an argon laser at 488 nm for Alexa Fluor 488, and an orange helium-neon laser at 594 nm for Alexa Fluor 594. To avoid possible cross-talk, the wavelengths were collected separately and were merged later. Images were processed using Leica LAS-AF software (version 1.7.0, build 1240).

Immunoprecipitation. Partially purified P. murina organisms were resuspended in 20 mM HEPES (pH 7.5), 150 mM NaCl, 1% sodium dodecyl sulfate (SDS) and a cocktail of protease inhibitors containing EDTA. The extracts were boiled for 10 min and then centrifuged for 15 min at 13,000 rpm, and the supernatant was adjusted to a final concentration of 0.12% SDS, 1% Triton X-100, 20 mM HEPES, and 150 mM NaCl. The samples were incubated overnight at 4°C with the affinity purified anti-Sam1 antibody. Simultaneously, P. murina extracts were treated with preimmune serum to be used as a negative control. The samples were incubated with protein A-Sepharose beads for 2 h at 4°C. The beads were washed twice with Tris-buffered saline containing 0.1% Tween 20, followed by a final wash with Tris-buffered saline and then boiled in SDS sample denaturing buffer before they were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting analysis.

SDS-PAGE and Western blotting analysis. Recombinant Sam1 was electrophoretically separated on 10 to 20% Tricine gels (Invitrogen) and stained with Coomassie brilliant blue R250. Western blotting analysis of the recombinant Sam1 protein was performed using peroxidase-conjugated anti-His tag antibody (Roche). Immunoprecipitated samples from partially purified P. murina preparations were used for SDS-PAGE and Western blotting analysis. The blots were then probed with Sam1 antibody and peroxidase-conjugated anti-rabbit IgG (ReliaBlot; Bethyl Laboratories, Inc. Montgomery, TX). Immunoreactive bands were visualized using BM Blue POD Substrate precipitating (Roche). In certain experiments, anti-Sam1 antibody was preincubated with excess antigen (refolded recombinant P. murina Sam1) before being used for Western blotting analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of AdoMet synthetase genes of Pneumocystis. Following the identification of a partial genomic sequence (~1,600 bp) of AdoMet synthetase in the Pneumocystis genome project database, we undertook to determine whether a full-length functional enzyme was carried by the organism. We obtained a partial cDNA sequence of AdoMet synthetase from P. carinii by sequencing an RT-PCR product generated by using RNA preparations from partially purified P. carinii organisms and primers designed from the known genomic sequence. Partial cDNA and genomic sequences from P. murina and P. jirovecii were determined by sequencing RT-PCR or PCR products obtained using RNA or genomic DNA from P. murina- or P. jirovecii-infected lung samples and primers designed from conserved areas of the P. carinii AdoMet synthetase sequence. Inverse PCR was utilized to obtain upstream and downstream genomic sequences. 3' and 5' RACE were employed to obtain the complete cDNA sequence of P. carinii (1,232 bp) or P. murina (1,309-bp) AdoMet synthetase (GenBank accession numbers EF377365 and EF377360, respectively). For P. jirovecii, the cDNA sequence (GenBank accession number EF377362) for the coding region was deduced by comparing the genomic sequence with the cDNA sequences of P. carinii and P. murina AdoMet synthetase. The AT content of all three genes was 70 to 72%, consistent with an origin from Pneumocystis rather than from a mammalian host. The accuracy of the genomic and cDNA sequences was confirmed for P. carinii and P. murina by PCR amplification and sequencing of the entire coding region, using both genomic and cDNA as a template. Figure 1 shows P. carinii AdoMet synthetase genomic sequence together with the deduced amino acid sequences. The figure also contains a partial sequence of the adjacent gene located immediately upstream of AdoMet synthetase, which has homology to the chromatin remodeling protein CHD1 of Saccharomyces cerevisiae (see below). A comparison of genomic and cDNA sequences of AdoMet synthetase identified seven introns. The GenBank accession numbers of P. carinii, P. murina, and P. jirovecii AdoMet synthetase genomic sequences are EF377364, EF377361, and EF377363, respectively.


Figure 1
View larger version (63K):
[in this window]
[in a new window]

  		FIG. 1. Nucleotide and deduced amino acid sequences of P. carinii sam1 and partial sequences of the putative chd1 gene. Initiation and termination codons are shown in bold and are underlined. The transcription start site is indicated by an arrow, the introns are shown in lowercase letters, and the XbaI site is marked by a solid line above the sequence.

 
Most organisms have at least two forms of AdoMet synthetase (15). In Saccharomyces cerevisiae, two isozymes encoded by the two genes sam1 and sam2 have been reported (35, 36), but in Schizosaccharomyces pombe, only sam1 has been identified (11). We report the Pneumocystis AdoMet synthetase gene as sam1, since it has high homology to the sam1 gene of S. pombe.

Deduced amino acid sequences of Pneumocystis Sam1. The cDNA sequence of P. carinii sam1 or P. murina sam1 contains an open reading frame encoding a protein containing 381 amino acids, while P. jirovecii Sam1 contains 382 amino acids. Figure 2 shows alignment of the deduced amino acid sequences of AdoMet synthetase from Pneumocystis, yeast, E. coli, rats, mice, and humans (11-13, 21, 30, 36). The two AdoMet synthetase signature motifs GAGDQGIMFGY and GGGAFSGKD are 100% conserved among these species (11). ATP binding sites are also highly conserved (15, 28, 34). The Sam1 protein sequence is highly conserved among Pneumocystis: P. carinii Sam1 showed 94% identity to that of P. murina, and both showed 83% identity to that of P. jirovecii. Pneumocystis Sam1 (all three species) showed 75% identity to S. pombe Sam1 and 71% to that of S. cerevisiae. Identity to human, mouse, and rat sequences (GenBank accession numbers NM_000429, NM_133653, and NM_012860, respectively) ranged from 64% to 67%, and the E. coli sequence (GenBank accession number NP_289514 [GenBank] ) showed 53% to 55% identity.


Figure 2
View larger version (124K):
[in this window]
[in a new window]

  		FIG. 2. Alignment of the deduced Pneumocystis Sam1 amino acid sequences with those of other organisms. Sam1 sequences from P. carinii, P. murina, P. jirovecii, S. pombe, and S. cerevisiae, MetK sequence from E. coli, and MAT1 sequences from mice (Mus musculus), rats (Rattas norvegicus), and humans (Homo sapiens) were aligned using Clustal W. Identical amino acid residues are boxed. AdoMet synthetase signature motifs are underlined. ATP binding motifs are boxed in bold.

 
Northern and Southern blotting analyses. To see whether the sam1 gene was being transcribed, Northern blotting analysis was performed using RNA extracted from partially purified P. carinii organisms or from P. murina-infected mouse lung (Fig. 3A). A DIG-labeled PCR product corresponding to nucleotides 552 to 2020 of the P. carinii sam1 genomic sequence was used as the probe for hybridization. In both P. carinii (Fig. 3, lane 1) and P. murina (Fig. 3, lane 2) preparations, an ~1.3-kb hybridization signal was observed, which is consistent with the size expected for the sam1 gene transcript. However, a second, less intense band of ~5.6 kb was observed consistently in the RNA preparations from both organisms.


Figure 3
View larger version (55K):
[in this window]
[in a new window]

  		FIG. 3. Northern and Southern blotting analyses of Pneumocystis sam1. (A) Northern blotting analysis of total RNA from P. carinii organisms or P. murina-infected mouse lung. The blots were hybridized with a DIG-labeled PCR product spanning 552 to 2,020 bp of P. carinii sam1 genomic sequence. A strong hybridization signal (~1.3 kb) indicated by the solid arrow is seen in P. carinii (lane 1) or P. murina (lane 2) RNA preparations, which is consistent with the size of Pneumocystis sam1 cDNA. The probe also recognized a minor band of ~5.6 kb, which is indicated by an open arrow. When the blot containing P. carinii RNA was reprobed with DIG-labeled oligonucleotides designed from the coding region of the putative chd1 gene, only the 5.6-kb band was seen (lane 3). (B) Southern blotting analysis of genomic DNA from P. carinii. Genomic DNA extracted from P. carinii-infected rat lung was digested with different restriction endonucleases, and the blots were probed with a DIG-labeled PCR product spanning 552 to 2,020 bp of P. carinii sam1 genomic sequence. DNA digested with AseI (lane 1) showed a single band; XbaI (lane 2) gave two bands due to the presence of a restriction site within the region of the probe. When the same blot was reprobed with the DIG-labeled oligonucleotide designed from the putative chd1 gene, a single band was seen following digestion with AseI (lane 3) or XbaI (lane 4). The hybridization signals obtained correspond to the same size as the band recognized by the sam1 probe, consistent with a tandem genetic arrangement of sam1 and the putative chd1 gene. Molecular size markers (kb) are shown to the right of each gel.

 
The 5.6-kb transcript could be derived from a gene which contained sequences with homology to the probe. To examine this, a Southern blotting analysis was performed using restriction endonuclease-digested genomic DNA from P. carinii-infected rat lung (Fig. 3B); the blot was hybridized with the same probe. Genomic DNA digested with AseI (Fig. 3B, lane 1) showed a single band, while that digested with XbaI (Fig. 3B, lane 2) showed two bands. This is due to the presence of one XbaI site in the probe-spanning region. Thus, the Southern blot shows that P. carinii sam1 appears to be a single-copy gene, and no second gene with homology to the probe that could account for the higher band on the Northern blot was identified.

We also excluded the possibility that the higher transcript was related to the host sam1 gene: the same probe did not show any reactivity with RNA from a normal (uninfected) mouse lung, while an oligonucleotide specific for mouse sam1 gave a band of the appropriate size (~3.5 kb; data not shown).

An alternative explanation is that the 5.6-kb band represented a polycistronic RNA. Adjacent genes are rarely cotranscribed as a polycistronic RNA in eukaryotes (2). The open reading frame of the sam1 gene is downstream of another gene that shows homology to the chromatin remodeling protein, CHD1, of S. cerevisiae (GenBank accession no. U18917; Fig. 1). These two genes are separated by an intergenic region of 238 bp (Fig. 1). RT-PCR performed using an upstream oligonucleotide designed from the coding region of the chd1 homologue gene along with a downstream oligonucleotide designed from the sam1 cDNA sequence amplified an ~2,000-bp product that contained part of the putative chd1 cDNA sequence as well as sam1 cDNA, confirming that these two genes are transcribed as a single RNA. The elimination of reverse transcriptase and pretreatment of the RNA with RNase-free DNase confirmed that it was RNA, not DNA, that was being amplified (data not shown). A comparison of genomic and cDNA sequences identified one intron in the partial sequence of the putative chd1 gene and two introns in the intergenic region (Fig. 1).

To confirm that the 5.6-kb band in the Northern blotting analysis was derived from this cotranscribed message, the same blot was reprobed with an oligonucleotide corresponding to the coding region of the putative chd1 gene. The probe hybridized to the 5.6-kb band but not to the 1.3-kb band (Fig. 3A, lane 3). In S. cerevisiae, the size of chd1 cDNA is ~4.4 kb. This suggests that the 5.6-kb band represents a bicistronic RNA containing the chd1 and sam1 transcripts. We also reprobed the Southern blots with the chd1 gene-specific oligonucleotide. A single band was observed for DNA digested with either AseI (Fig. 3B, lane 3) or XbaI (Fig. 3B, lane 4). In both digests, the bands observed correspond to bands seen when the blot was hybridized with the sam1 probe.

Expression of the P. carinii Sam1 protein in E. coli. The coding region from P. carinii sam1 cDNA (1,145 bp) was amplified and cloned into the pET 28 expression vector and expressed as a His tag fusion protein in bacteria. SDS-PAGE analysis of whole-bacterium extract expressing recombinant protein showed a prominent band of ~45-kDa protein band when stained with Coomassie blue (Fig. 4A, lane 1), which is the size expected for Sam1, but this band was not seen when bacteria transfected with a control vector with no insert were analyzed (Fig. 4A, lane 2). The expressed protein showed immunoreactivity to His tag antibody when analyzed by Western blotting (Fig. 4B, lane 1), but no immunoreactivity was seen with the control preparation (Fig. 4B, lane 2).


Figure 4
View larger version (48K):
[in this window]
[in a new window]

  		FIG. 4. Expression of P. carinii recombinant protein Sam1. (A) SDS-PAGE analysis of the bacterial cells expressing recombinant protein showed a band of the expected size (~45 kDa, indicated by the arrows), when stained with Coomassie blue (lane 1). When vector with no insert was analyzed, no band of that size was observed (lane 2). (B) Bacterial cells expressing the Sam1 protein showed immunoreactivity with the expected size band when probed in a Western blot with His tag antibody (lane 1), while there was no immunoreactivity when vector alone was analyzed (lane 2). (C) Refolded protein showed a band of ~45 kDa when stained with Coomassie blue (lane 1), while no band of that size was seen when vector alone was analyzed (lane 2). (D) A 45-kDa band showed immunoreactivity when Western blotting analysis shown in panel C was performed using His tag antibody (lane 1), but no immunoreactivity was observed when vector alone (negative control) was analyzed (lane 2). (E) Immunoreactivity to a 45-kDa band (lane 1) was lost when the anti-Sam1 antibody was preincubated with the antigen (recombinant protein) (lane 2). Molecular size markers (kDa) are shown to the right of panels B and D.

 
Most of the expressed protein was in insoluble bacterial inclusions (data not shown). To obtain soluble, potentially functional protein, expressed recombinant protein was denatured and allowed to refold as described for AdoMet synthetase in Leishmania donovani (29). Control samples were processed in parallel. SDS-PAGE followed by Coomassie blue staining (Fig. 4C) and Western blotting (Fig. 4D) demonstrated solubilization of the recombinant protein.

AdoMet synthetase assay. The refolded protein was analyzed for AdoMet synthetase activity using 14C-labeled methionine as described previously (4). Figure 5 shows the AdoMet synthetase activity of P. carinii refolded recombinant protein, demonstrating increased production of AdoMet over time. The control preparation (vector alone) showed no enzyme activity. When ATP was omitted from the reaction mixture, no product was detected (data not shown). The activity was also dependent on enzyme concentration (data not shown).


Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 5. AdoMet synthetase activity. AdoMet synthetase activity of recombinant, refolded P. carinii protein was measured at different time intervals. The figure shows the activity measured in two different experiments and performed in duplicate. Values are means ± standard deviations (error bars) (n = 4). The enzyme activity is expressed as nmol of AdoMet formed/mg protein. The protein preparation obtained by using the vector alone showed no activity.

 
Immunochemical analysis of Sam1 in Pneumocystis. Given that the sam1 mRNA was expressed by Pneumocystis and that the mRNA encoded a functional enzyme, we were interested in demonstrating the expression of Sam1 in Pneumocystis by immunochemical analysis. For that experiment, a polyclonal antibody was generated against a synthetic peptide corresponding to amino acid residues 199 to 219 of P. murina Sam1. The antibody recognized the expressed recombinant protein (Fig. 4E, lane 1), and immunoreactivity was effectively blocked when the antibody was preincubated with excess refolded recombinant P. murina Sam1 (Fig. 4E, lane 2). To concentrate native Sam1, partially purified P. murina extracts were subjected to immunoprecipitation, using this antibody; preimmune serum was used as a negative control. Western blotting analysis of immunoprecipitated samples identified an ~45-kDa band (Fig. 6, lane 1) when the antipeptide antibody was used that was not seen with preimmune serum (Fig. 6, lane 2). Preincubation of the antibody with recombinant protein led to a loss of reactivity (Fig. 6, lane 3).


Figure 6
View larger version (62K):
[in this window]
[in a new window]

  		FIG. 6. Western blotting analysis of immunoprecipitated samples from P. murina. Partially purified P. murina extracts were subjected to immunoprecipitation, using anti-Sam1 antibody (lane 1) or preimmune serum as a negative control (lane 2), followed by Western blotting analysis, using the anti-Sam1 antibody. The former (lane 1) showed a reactive band of 45 kDa (arrow), the size expected for Sam1, while the latter (lane 2) showed no immunoreactivity. The bands corresponding to IgG are marked. When anti-Sam1 antibody that was preincubated with recombinant Sam1 was used for the Western blot, immunoreactivity to the 45-kDa band was blocked (lane 3), demonstrating that the immunoreactivity is specific for Sam1.

 
To examine the expression of Sam1 protein in P. murina-infected tissue, lung sections were costained with anti-Sam1 and anti-Pneumocystis (4D7) antibodies and subjected to confocal microscopic analysis (Fig. 7). The immunoreactivity toward Sam1 antibody colocalized with the staining of Pneumocystis using 4D7 antibody, indicating the expression of Sam1 protein in Pneumocystis (Fig. 7A). It was noted that structures consistent with Pneumocystis cysts did not stain with the anti-Sam1 antibody, suggesting that Sam1 expression may be decreased or absent in cysts. When immunofluorescence analysis was done using anti-Sam1 antibody that was preabsorbed with recombinant P. murina Sam1, the immunoreactivity was lost (Fig. 7B).


Figure 7
View larger version (65K):
[in this window]
[in a new window]

  		FIG. 7. Confocal immunofluorescence microscopic detection of Sam1 in P. murina-infected mouse lung. (A) Dual immunofluorescence staining of P. murina-infected mouse lung tissue sections using anti-Sam1 and anti-Pneumocystis (4D7) antibodies. (B) Dual immunofluorescence staining of P. murina-infected mouse lung tissue sections using anti-Sam1 antibody preabsorbed with insoluble recombinant AdoMet protein and anti-Pneumocystis (4D7) antibody. Panel 1, blue indicates the cell nuclei stained with DAPI. Panel 2, staining of Pneumocystis using 4D7 antibody, biotin-conjugated anti-mouse IgG and streptavidin-conjugated Alex Fluor 594. Red indicates immunoreactivity. Panel 3, staining with rabbit anti-P. murina Sam1 antibody, using Alexa Fluor 488-conjugated goat anti-rabbit IgG as the secondary antibody. Green indicates the immunoreactivity with the anti-Sam1 antibody. Panel 4, merged images. The green anti-Sam1 fluorescence colocalizes with the anti-Pneumocystis red fluorescence staining. An arrow indicates a structure that is consistent with a cyst, within which are multiple DAPI-stained nuclei that, however, did not stain with the anti-Sam1 antibody. The anti-Sam1 immunoreactivity was blocked when the antibody was preabsorbed with recombinant protein, showing that the immunoreactivity is specific for Sam1. The thickness of the slides used for the confocal examination was 5 µm, and each confocal picture is estimated to be ~1 µm.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study we have identified genes encoding AdoMet synthetase (sam1) in Pneumocystis and have demonstrated that sam1 mRNA is transcribed, that recombinant Sam1 is enzymatically active, and that immunoreactive Sam1 can be identified in Pneumocystis preparations. These data strongly support the conclusion that Pneumocystis can synthesize AdoMet de novo and suggest that the organism does not need to rely on exogenous, host-derived AdoMet for survival.

At least two isozymes of AdoMet synthetase encoded by different but closely related genes are present in many eukaryotes. The sequences are highly conserved among species (15). With Pneumocystis, we were able to identify only one gene, sam1, for encoding AdoMet synthetase, similar to what has been reported for S. pombe (11). Pneumocystis AdoMet synthetase showed high homology to that of yeast and less so to those of E. coli and mammalian species (11-13, 21, 30, 35, 36). AdoMet synthetase signature motifs and ATP binding sites are highly conserved in all these sequences (28, 34). While the recombinant protein demonstrated AdoMet synthetase activity, the specific activity under nonoptimized conditions, ~0.64 nmol/min/mg protein, was less than that seen with recombinant rat (1.5 to 30 nmol/min/mg protein, depending on the conditions for refolding) or Leishmania enzyme (~80 nmol/min/mg protein), though it was similar to that seen with crude extracts obtained from S. pombe (~0.05 to 0.55 nmol/min/mg protein, depending on the phase of growth) (11, 18, 29).

It is of interest that Northern blotting analysis identified two transcripts, a major band (~1.3 kb) that corresponded to the expected size of the sam1 mRNA, and a minor band (~5.6 kb) that is consistent with a bicistronic mRNA containing sam1 and a putative chd1 homologue. The presence of polycistronic RNA is very rare in eukaryotes, and the genes derived from the polycistronic RNA are usually functionally related (3). Cotranscription of these genes was seen for both rat and mouse Pneumocystis, suggesting that this organization dates to the ancestor of at least these two species. Insufficient RNA was available to examine transcription in P. jirovecii.

A probe corresponding to the chd1 gene recognized only the larger size band; thus, chd1 appears to be invariably transcribed with sam1, while the majority of sam1 transcripts are monocistronic. It is unknown whether the translation of both proteins occurs from the cotranscribed message and what effect the excision of introns from the intergenic region may have on the synthesis of Sam1. Bicistronic RNA in which dmc1 and rad 24 are cotranscribed has been reported in S. pombe (10). The functional significance of bicistronic RNA in fungi is currently unknown.

Our findings do not support the conclusion of Merali and Clarkson (24) that Pneumocystis does not possess a functional AdoMet synthetase gene. This group reported that Pneumocystis cannot synthesize AdoMet and that this pathogen has to depend on its hosts for the supply of this important molecule (26). AdoMet is an important molecule, and all organisms, with the exception of the xD strain of Amoeba proteus, are known to synthesize this molecule. It has been reported that the xD strain of Amoeba proteus, which originated following the spontaneous infection of the D strain of Amoeba proteus by X-bacteria, must depend on its symbiont X-bacteria for its supply of AdoMet (5). Merali and Clarkson (24) noted the existence of a possible Pneumocystis-specific AdoMet synthetase gene in the genome project but proposed that ATP binding sites of Pneumocystis AdoMet synthetase might be mutated, resulting in a nonfunctional enzyme. However, our study demonstrates that this is not the case; Pneumocystis Sam1 retains all the consensus ATP binding sites (28, 34).

By immunochemical analysis, we were able to show the expression of the Sam1 protein in P. murina. The affinity purified antibody raised against a peptide that corresponds to amino acid residues 199 to 219 of P. murina Sam1 reacted with a 45-kDa protein in the extracts of partially purified P. murina organisms isolated from infected mouse lung, when analyzed by immunoprecipitation. The size is consistent with the expected size of Sam1. Preincubation of anti-Sam1 antibody with the refolded recombinant P. murina Sam1 completely removed the 45-kDa band, indicating that it is specific for Sam1. Confocal immunofluorescence analysis of P. murina-infected lung tissue sections using the same antibody showed that the immunostaining of Sam1 is colocalized to Pneumocystis organisms detected with a specific monoclonal antibody (1, 23). While there was some nonspecific activity seen with the anti-Sam1 antibody, preabsorption of the antibody with the antigen (recombinant P. murina Sam1) blocked the immunoreactivity, supporting its specificity for Sam1.

In summary, we have characterized the sam1 gene from Pneumocystis, which is transcribed into an ~1.3-kb mRNA. The recombinant protein expressed in E. coli showed functional enzyme activity. Our study clearly shows that Pneumocystis has a sam1 gene that can encode a functional AdoMet synthetase. We were able to detect the expression of Sam1 protein in P. murina by immunoprecipitation and confocal immunofluorescence analyses. Thus, Pneumocystis, like other organisms, could synthesize its own AdoMet and does not need to depend on its hosts for the supply of this important molecule, as reported by Merali and Clarkson (24).


   ACKNOWLEDGMENTS
 
We thank Rene Costello and Howard Mostowski for their assistance with the animal studies.

This research was supported by the Intramural Research Program of the NIH Clinical Center and the National Institute of Allergy and Infectious Diseases.


   FOOTNOTES
 
* Corresponding author. Mailing address: Building 10, Room 2C145, MSC 1662, Bethesda, MD 20892-1662. Phone: (301) 496-9907. Fax: (301) 402-1213. E-mail: jkovacs{at}nih.gov Back

{triangledown} Published ahead of print on 7 December 2007. Back

{dagger} Present address: Servicio de Enfermedades Infecciosas, 4a Planta Centro. Control A, Hospital Ramón y Cajal, Ctra. de Colmenar Km 9.100, 28034 Madrid, Spain. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Angus, C. W., A. Tu, P. Vogel, M. Qin, and J. A. Kovacs. 1996. Expression of variants of the major surface glycoprotein of Pneumocystis carinii. J. Exp. Med. 183:1229-1234.[Abstract/Free Full Text]
  2. Blumenthal, T. 1998. Gene clusters and polycistronic transcription in eukaryotes. Bioessays 20:480-487.[CrossRef][Medline]
  3. Blumenthal, T. 2004. Operons in eukaryotes. Brief. Funct. Genomic Proteomic 3:199-211.[Abstract/Free Full Text]
  4. Chiang, P. K., and G. L. Cantoni. 1977. Activation of methionine for transmethylation. Purification of the S-adenosylmethionine synthetase of bakers' yeast and its separation into two forms. J. Biol. Chem. 252:4506-4513.[Free Full Text]
  5. Choi, J. Y., T. W. Lee, K. W. Jeon, and T. I. Ahn. 1997. Evidence for symbiont-induced alteration of a host's gene expression: irreversible loss of SAM synthetase from Amoeba proteus. J. Eukaryot. Microbiol. 44:412-419.[Medline]
  6. Cushion, M. T. 2004. Molecular and phenotypic description of Pneumocystis wakefieldiae sp. nov., a new species in rats. Mycologia 96:429-438.[Abstract/Free Full Text]
  7. Cushion, M. T., and J. Arnold. 1997. Proposal for a Pneumocystis genome project. J. Eukaryot. Microbiol. 44:7S.[Medline]
  8. Cushion, M. T., A. G. Smulian, B. E. Slaven, T. Sesterhenn, J. Arnold, C. Staben, A. Porollo, R. Adamczak, and J. Meller. 2007. Transcriptome of Pneumocystis carinii during fulminate infection: carbohydrate metabolism and the concept of a compatible parasite. PLoS ONE 2:e423.[CrossRef]
  9. Dei-Cas, E., E. Mazars, C. O. Ferragut, I. Durand, E. M. Aliouat, M. Dridba, F. Palluault, J. C. Cailliez, N. Seguy, M. Tibayrenc, et al. 1994. Ultrastructural, genomic, isoenzymatic and biological features make it possible to distinguish rabbit Pnenumocystis from other mammal Pneumocystis strains. J. Eukaryot. Microbiol. 41:84S.[Medline]
  10. Fukushima, K., Y. Tanaka, K. Nabeshima, T. Yoneki, T. Tougan, S. Tanaka, and H. Nojima. 2000. Dmc1 of Schizosaccharomyces pombe plays a role in meiotic recombination. Nucleic Acids Res. 28:2709-2716.[Abstract/Free Full Text]
  11. Hilti, N., R. Graub, M. Jorg, P. Arnold, A. M. Schweingruber, and M. E. Schweingruber. 2000. Gene sam1 encoding adenosylmethionine synthetase: effects of its expression in the fission yeast Schizosaccharomyces pombe. Yeast 16:1-10.[CrossRef][Medline]
  12. Horikawa, S., M. Ishikawa, H. Ozasa, and K. Tsukada. 1989. Isolation of a cDNA encoding the rat liver S-adenosylmethionine synthetase. Eur. J. Biochem. 184:497-501.[Medline]
  13. Horikawa, S., and K. Tsukada. 1991. Molecular cloning and nucleotide sequence of cDNA encoding the human liver S-adenosylmethionine synthetase. Biochem. Int. 25:81-90.[Medline]
  14. Keely, S. P., J. M. Fischer, M. T. Cushion, and J. R. Stringer. 2004. Phylogenetic identification of Pneumocystis murina sp. nov., a new species in laboratory mice. Microbiology 150:1153-1165.[Abstract/Free Full Text]
  15. Kotb, M., and A. M. Geller. 1993. Methionine adenosyltransferase: structure and function. Pharmacol. Ther. 59:125-143.[CrossRef][Medline]
  16. Kovacs, J. A., J. L. Halpern, J. C. Swan, J. Moss, J. E. Parrillo, and H. Masur. 1988. Identification of antigens and antibodies specific for Pneumocystis carinii. J. Immunol. 140:2023-2031.[Abstract/Free Full Text]
  17. Kutty, G., and J. A. Kovacs. 2007. Identification and characterization of rad51 of Pneumocystis. Gene 389:204-211.[CrossRef][Medline]
  18. Lopez-Vara, M. C., M. Gasset, and M. A. Pajares. 2000. Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies. Protein Expr. Purif. 19:219-226.[CrossRef][Medline]
  19. Lu, S. C. 2000. S-adenosylmethionine. Int. J. Biochem. Cell. Biol. 32:391-395.[CrossRef][Medline]
  20. Ma, L., L. Borio, H. Masur, and J. A. Kovacs. 1999. Pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim-sulfamethoxazole or dapsone use. J. Infect. Dis. 180:1969-1978.[CrossRef][Medline]
  21. Markham, G. D., J. DeParasis, and J. Gatmaitan. 1984. The sequence of metK, the structural gene for S-adenosylmethionine synthetase in Escherichia coli. J. Biol. Chem. 259:14505-14507.[Abstract/Free Full Text]
  22. Masur, H., M. A. Michelis, J. B. Greene, I. Onorato, R. A. Stouwe, R. S. Holzman, G. Wormser, L. Brettman, M. Lange, H. W. Murray, and S. Cunningham-Rundles. 1981. An outbreak of community-acquired Pneumocystis carinii pneumonia: initial manifestation of cellular immune dysfunction. N. Engl. J. Med. 305:1431-1438.[Abstract]
  23. Mei, Q., Q. Wang, W. Fan, B. Li, Y. Chen, and Y. Liu. 1994. Experimental and clinical study on pneumocystosis. III. Development and characterization of monoclonal antibody against Pneumocystis carinii. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Zuzhi. 12:188-191. (In Chinese.)
  24. Merali, S., and A. B. Clarkson, Jr. 2004. S-adenosylmethionine and Pneumocystis. FEMS Microbiol. Lett. 237:179-186.[Medline]
  25. Merali, S., U. Frevert, J. H. Williams, K. Chin, R. Bryan, and A. B. Clarkson, Jr. 1999. Continuous axenic cultivation of Pneumocystis carinii. Proc. Natl. Acad. Sci. USA 96:2402-2407.[Abstract/Free Full Text]
  26. Merali, S., D. Vargas, M. Franklin, and A. B. Clarkson, Jr. 2000. S-adenosylmethionine and Pneumocystis carinii. J. Biol. Chem. 275:14958-14963.[Abstract/Free Full Text]
  27. Munoz, A., L. K. Schrager, H. Bacellar, I. Speizer, S. H. Vermund, R. Detels, A. J. Saah, L. A. Kingsley, D. Seminara, and J. P. Phair. 1993. Trends in the incidence of outcomes defining acquired immunodeficiency syndrome (AIDS) in the Multicenter AIDS Cohort Study: 1985-1991. Am. J. Epidemiol. 137:423-438.[Abstract/Free Full Text]
  28. Perez-Pertejo, Y., R. M. Reguera, C. Garcia-Estrada, R. Balana-Fouce, and D. Ordonez. 2004. Mutational analysis of methionine adenosyltransferase from Leishmania donovani. Eur. J. Biochem. 271:2791-2798.[Medline]
  29. Perez-Pertejo, Y., R. M. Reguera, H. Villa, C. Garcia-Estrada, R. Balana-Fouce, M. A. Pajares, and D. Ordonez. 2003. Leishmania donovani methionine adenosyltransferase. Role of cysteine residues in the recombinant enzyme. Eur. J. Biochem. 270:28-35.[Medline]
  30. Sakata, S. F., L. L. Shelly, S. Ruppert, G. Schutz, and J. Y. Chou. 1993. Cloning and expression of murine S-adenosylmethionine synthetase. J. Biol. Chem. 268:13978-13986.[Abstract/Free Full Text]
  31. Sinclair, K., A. E. Wakefield, S. Banerji, and J. M. Hopkin. 1991. Pneumocystis carinii organisms derived from rat and human hosts are genetically distinct. Mol. Biochem. Parasitol. 45:183-184.[CrossRef][Medline]
  32. Stringer, J. R., C. B. Beard, R. F. Miller, and A. E. Wakefield. 2002. A new name (Pneumocystis jirovecii) for Pneumocystis from humans. Emerg. Infect. Dis. 8:891-896.[Medline]
  33. Stringer, J. R., S. L. Stringer, J. Zhang, R. Baughman, A. G. Smulian, and M. T. Cushion. 1993. Molecular genetic distinction of Pneumocystis carinii from rats and humans. J. Eukaryot. Microbiol. 40:733-741.[Medline]
  34. Taylor, J. C., and G. D. Markham. 2000. The bifunctional active site of S-adenosylmethionine synthetase. Roles of the basic residues. J. Biol. Chem. 275:4060-4065.[Abstract/Free Full Text]
  35. Thomas, D., R. Rothstein, N. Rosenberg, and Y. Surdin-Kerjan. 1988. SAM2 encodes the second methionine S-adenosyl transferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes. Mol. Cell. Biol. 8:5132-5139.[Abstract/Free Full Text]
  36. Thomas, D., and Y. Surdin-Kerjan. 1987. SAM1, the structural gene for one of the S-adenosylmethionine synthetases in Saccharomyces cerevisiae. Sequence and expression. J. Biol. Chem. 262:16704-16709.[Abstract/Free Full Text]
  37. Wright, T. W., T. Y. Bissoondial, C. G. Haidaris, F. Gigliotti, and P. J. Haidaris. 1995. Isoform diversity and tandem duplication of the glycoprotein A gene in ferret Pneumocystis carinii. DNA Res. 2:77-88.[Abstract]

Eukaryotic Cell, February 2008, p. 258-267, Vol. 7, No. 2
1535-9778/08/$08.00+0     doi:10.1128/EC.00345-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
EC.00345-07v1
7/2/258    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kutty, G.
Right arrow Articles by Kovacs, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kutty, G.
Right arrow Articles by Kovacs, J. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Appl. Environ. Microbiol. Infect. Immun. J. Bacteriol.
Mol. Cell Biol. Microbiol. Mol. Biol. Rev. ALL ASM JOURNALS