Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
Correspondence
James R. Stringer
stringjr{at}ucmail.uc.edu
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ABSTRACT |
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The GenBank accession numbers for the MSG gene sequences determined in this work are: S5, AF164561; 60A9, AF169405; J3, AF169406.
Present address: House Staff Office, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45227, USA.
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INTRODUCTION |
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P. carinii organisms express a surface antigen known as the major surface glycoprotein (MSG) (Walzer & Linke, 1987; Kovacs et al., 1988
; Linke et al., 1989
; Tanabe et al., 1989
; Linke & Walzer, 1989
, 1991
; Nakamura, 1998
; Stringer & Keely, 2001
). Multiple forms of MSG are encoded by a family of approximately 100 genes, members of which are located at 34 telomeric sites (Stringer et al., 1991
; Kovacs et al., 1993
; Sunkin et al., 1994
; Sunkin & Stringer, 1996
, 1997
; Stringer & Keely, 2001
; Cornillot et al., 2002
; Keely et al., 2003
). Other species of Pneumocystis, including the species that infects humans, also have families of MSG genes (Haidaris et al., 1991
; Stringer et al., 1993
; Garbe & Stringer, 1994
; Wright et al., 1995
; Mei et al., 1998
; Haidaris et al., 1998
; Schaffzin et al., 1999
; Kutty et al., 2001
).
We hypothesize that only one P. carinii MSG gene is expressed at a time in a given organism and that this control is exerted in cis by a unique locus named the upstream conserved sequence (UCS). The UCS encodes a tract of 365 ribonucleotides that is found at the 5' ends of mRNAs encoding different MSG forms (Wada et al., 1995; Edman et al., 1996
). The UCS RNA leader sequence encodes a peptide that served as a signal sequence for transport of nascent protein into the secretory apparatus when expressed in insect cells, suggesting that one role of the UCS is to direct MSG precursors into the secretory pathway (Sunkin et al., 1998
). Epitopes on the UCS-encoded peptide are found on MSG in whole P. carinii extracts, but not on MSG extracted from the P. carinii cell wall, suggesting that MSG is synthesized as a preprotein beginning with the UCS and that the UCS is removed proteolytically to produce the mature form of MSG found on the cell surface (Sunkin et al., 1998
). Thus, the UCS appears to be necessary for MSG translation and surface expression, but not for function of mature MSG proteins.
Previous studies on populations of P. carinii obtained from different rats showed that each population contained transcripts representing different subsets of the MSG gene family. These differences allowed experiments showing that MSG genes that are linked to the UCS locus are represented as UCS-containing mRNAs, suggesting that linkage to the UCS locus is necessary and sufficient for transcription of an MSG gene (Sunkin & Stringer, 1996, 1997
).
If linkage of an MSG gene to the UCS locus indeed controls surface expression of the protein encoded by that gene, then P. carinii populations rich in a particular MSG protein should contain many organisms that have the DNA sequence encoding that protein at the UCS locus. To test this hypothesis, we developed probes that identify a specific MSG sequence at both the protein and DNA levels. To detect specific MSG proteins, a monoclonal antibody (mAb), RA-C11, was used (Linke et al., 1998). RA-C11 has been shown to recognize very few of the peptides encoded by the MSG gene family, suggesting that it recognizes an epitope that is not present in most forms of MSG. The evidence suggesting that the RA-C11 epitope is rare came from two sets of experiments. The first experiments used Western blotting to show that mAb RA-C11 reacted with MSG from only 25 % of populations examined (Vasquez et al., 1996
). The second set of experiments used an expression library to show that mAb RA-C11 reacted with only 1 % of the
gt11 plaques bearing MSG epitopes (Linke et al., 1998
).
The work described herein began by determining the sequence of the DNA that encodes the RA-C11 epitope (i.e. the epitope-encoding sequence, EES). Next, six populations of P. carinii that differed with respect to expression of RA-C11-reactive proteins were identified. Analysis of the fractions of organisms with the RA-C11 EES at the UCS showed that this parameter correlated directly with the abundance of RA-C11-reactive protein. These results support the hypothesis that residence of a particular MSG gene at the UCS locus is necessary and sufficient for expression the MSG protein encoded by that gene.
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METHODS |
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To map the RA-C11 epitope, overlapping fragments of the clone C11-S5 MSG gene were generated using PCR and subcloned into the pET30 expression vector (Novagen). A detailed description of the primers and conditions used in the PCR experiments can be found elsewhere (Schaffzin et al., 1999). Inserts in recombinant plasmids were sequenced to confirm correct amplification and reading frame. Recombinant plasmids were established in strain BL21(DE3) (Novagen). Production of recombinant proteins was induced by adding IPTG (final concentration, 1 mM) to bacterial cultures. After 2 h at 37 °C, cells were collected by centrifugation, resuspended in SDS-PAGE loading buffer, boiled at 100 °C for 2 min and immediately loaded onto a 1020 % Tricine SDS-PAGE gel (Novex). Immunoblots were prepared by standard methods (Sambrook et al., 1989
). Proteins produced carried an S protein tag encoded by the Pet30 vector (Novagen), so proteins of interest could be located on immunoblots using an S-tag reagent purchased from Novagen. This reagent carries an alkaline phosphatase conjugated to S protein (AP-S), which binds the S-tag. The AP-S reagent was used according to the instructions provided by the vendor. Antibodies bound to immunoblots were visualized using secondary antibodies conjugated to either AP or horseradish peroxidase. Standard methods were employed in these experiments (Sambrook et al., 1989
).
Measurement of the fraction of UCS-MSG junctions that contain the C11 EES.
UCS-MSG junctions were obtained by amplifying P. carinii genomic DNA. DNA was subjected to PCR using the UCS1 primer (5'-GTTGTCCGAAGAGGTGGCA-3'), paired with MSG primer C7 (5'-GTCTTGTCCCTTTTTATAGCA-3') under the following conditions: 95 °C for 5 min, 35 cycles of incubation at 95 °C for 60 s, 48 °C for 2 min and 72 °C for 90 s, followed by 72 °C for 10 min.
The fraction of amplified UCS-MSG junctions that contain the C11 EES was measured using an allele-specific hybridization method adapted from that described by Kren et al. (1997). PCR amplicons were attached to nylon membranes, and incubated with two radiolabelled oligonucleotides in succession. First, the filter was incubated with the C11 EES oligonucleotide (5'-TTGAGGGAGAAATGTTACGAATTGAAGCG-3') for 18 h at 50 °C in 2x SSC, 1 % SDS, 5x Denhardt's solution, and 0·2 mg ml-1 sheared salmon sperm DNA (Sambrook et al., 1989
). Filters were washed in 1x SSPE, 0·5 % SDS at 50 °C for 30 min and immediately washed in tetramethylammonium chloride (TMAC) buffer (50 mM Tris/HCl pH 8·0, 2 mM EDTA pH 8·0, 0·1 % SDS, 3 M TMAC) at 70 °C for 1 h (Kren et al., 1997
). Radioactive emissions were detected by exposing the membrane to phosphor-imaging plates and reading the signal in a PhosphorImager followed by analysis with ImageQuant software (Molecular Dynamics, Amersham Biosciences). Signals obtained using the C11 EES probe were corrected for any differences in the amount of target DNA on the filter by comparing them to signals obtained with the CRJE oligonucleotide (5'-ATGGCACGGCCGGTTAAGAG-3'). This oligonucleotide corresponds to 20 of the nucleotides in the 28 bp sequence known as the conserved recombination junction element (CRJE) (Wada et al., 1995
). The CRJE is present between the UCS and the MSG-encoding regions of all UCS-linked MSG genes (Keely et al., 2003
). To perform CRJE-hybridization, the filter was stripped of the C11 EES probe by boiling in 1 % SDS for 15 min. Absence of the C11 EES probe was confirmed by exposing the membrane to phosphor-imaging plates. The stripped filter was then incubated with the CRJE probe at 42 °C in 6x SSC, 0·1 % SDS, 1x Denhardt's solution, 0·08 mg ml-1 sheared salmon sperm DNA. Filters were washed at 65 °C in 0·5x SSC, 0·1 % SDS twice for 10 min. Hybridization experiments were performed in triplicate.
The C11 EES probe was shown to be specific for the target sequence by the following experiments. First, DNA molecules with a sequence known to match 29 of the 30 nucleotides in the reverse complement of the C11 EES oligonucleotide were attached to nylon membranes and tested for hybridization to the C11 EES oligonucleotide. Prior to washing in TMAC, the mismatched hybrids were detected, but the TMAC washing procedure eliminated these. By contrast, TMAC washing did not remove the signal from a control filter carrying DNA that matched the C11 EES exactly. Second, a library of plasmids carrying hundreds of different MSG genes was screened by hybridization to the C11 EES oligonucleotide. Plasmids from 10 positive colonies were subjected to DNA sequence analysis, which showed that all of them carried an exact copy of the C11 EES.
Immunoblot detection of P. carinii antigens.
P. carinii organisms were extracted from the lungs of infected rats and enumerated by light microscopy as previously described (Cushion et al., 1988, 1998a
). For protein separation, samples containing the same number of organisms were boiled in SDS-PAGE loading buffer for 2 min and insoluble material was removed by centrifugation. SDS-soluble material was diluted by a series of six twofold dilutions in SDS-PAGE loading buffer, boiled for 1 min, and loaded onto an 816 % Tricine SDS-PAGE gel (Novex). Duplicate gels and Western blots were prepared and tested for reactivity with either mAb RA-C11 or mAb RA-F1 (Linke et al., 1998
). Signals obtained with mAb RA-F1 were used to normalize the RA-C11 signals. Developed blots were scanned digitally and the relative intensity of the 116 kDa MSG band in each lane was evaluated using ImageQuant software. The relationship between sample dilution factor and intensity of staining with mAbs was examined and this relationship generally appeared to be linear. However, linearity could not be firmly established in samples with relatively little mAb RA-C11 reactivity because it was not possible to detect bands at more than two dilutions.
Immunofluorescence.
P. carinii organisms that had been cryogenically preserved in DMSO freezing medium (7·5 %, DMSO) were diluted in 1x PBS and passed thrice through a 10 µm filter (Millipore) (Cushion et al., 2001). Organisms were prepared for immunofluorescence assay by methods described by Broomall et al. (1998)
. Briefly, organisms were applied to glass slides and fixed by air-drying followed by immersion in ice-cold acetone for 5 min. Slides were stored at -80 °C. Slides were thawed in room-temperature acetone and rehydrated in 1x PBS, then incubated in a moist chamber at 37 °C for 30 min with mAb diluted 1 : 40 in PBS (Sambrook et al., 1989
), after which they were washed in PBS containing 0·05 % Tween 20 twice for 10 min at room temperature. Slides were next incubated with FITC-conjugated goat-anti-mouse IgG (1 : 80 dilution, Biomeda), and washed as above. Organisms were counterstained by incubation in 0·1 mg ml-1 rhodamine-conjugated concanavalin A (ConA, Sigma). Control slides were incubated with either FITC goat-anti-mouse IgG plus ConA, or goat IgG alone. Organisms were visualized using a fluorescence microscope under oil immersion (1000x magnification).
Statistical analysis.
Analysis of variance (ANOVA) was performed on the data from hybridization, Western blotting and immunofluorescence experiments. The Tukey procedure was used to test for significance between pairs of populations. Correlation coefficients were calculated and evaluated for significance using a one-tailed t-test (Stevens, 1990).
GenBank accession numbers.
Sequences from previous studies were obtained from GenBank using the following accession numbers: MSGB, L29497; MSG 100, D31909; MSG 101, D31910; MSG 102 and MSG 104, D31911; MSG 103, D31912; MSG 105, D82031; GP3, L05906; PC3, L05904; PC5, L05907; C10, U83323; MSG 1, D17438; MSG 2, D17439; MSG 3, D17440; 3G5, AL592382; 22C8, AL592263.
Sequences obtained in the current study were submitted to GenBank under the following accession numbers: S5, AF164561; 60A9, AF169405; J3, AF169406.
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RESULTS |
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To identify the RA-C11 epitope-encoding sequence (EES) in the C11-S5 insert, PCR was used to generate five overlapping segments of the insert, which were each inserted into a plasmid expression vector. Recombinant proteins in lysates from bacteria carrying these plasmids were tested by Western blotting for mAb RA-C11 reactivity (Fig. 1A). As expected, only some of the plasmids produced a band of RA-C11 reactivity (see lanes 11 and 12). Positive control experiments using alkaline phosphatase-conjugated S-protein (AP-S) showed that lack of mAb RA-C11 reactivity was not due to failure of some plasmids to produce a fusion protein (lanes 15). The fusion proteins were also tested for reactivity with mAb RA-F1, which is an antibody to a defined epitope that is known to be present on a broad spectrum of MSGs (Linke et al., 1998
). Two fusion proteins reacted with mAb RA-F1 (Fig. 1A
, lanes 7 and 8). The reactive proteins covered the region where the mAb RA-F1 epitope was previously mapped (Linke et al., 1998
).
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Abundance of the RA-C11 epitope varies among populations of P. carinii
Previous studies on mRNA had shown that populations of P. carinii from individual rats tended to have transcripts encoding many different MSG isoforms, and that populations from different rats tended to differ with respect to the MSG sequences represented by mRNAs (Sunkin & Stringer, 1997). This variation suggested that some populations might express very little of the RA-C11 epitope, while others might express much more. To determine if this were true, populations of P. carinii were screened for mAb-reactive proteins by immunoblotting of total proteins that had been separated by denaturing gel electrophoresis. Duplicate blots were reacted with either mAb RA-C11, to measure RA-C11 epitope abundance, or mAb RA-F1, to assess the amount of MSG. The choice of mAb RA-F1 for this purpose was based on previous work which showed that this antibody recognized 100 times as many plaques in the
gt11 expression library as mAb RA-C11 did, indicating that the RA-F1 epitope is very common among MSG forms (Linke et al., 1998
). In addition, the DNA sequence encoding the RA-F1 epitope is present in all known MSG genes (not shown).
The immunoblotting experiments identified three populations of P. carinii that differed with respect to reactivity with mAb RA-C11. Population 1160 had the highest reactivity, which was estimated to be eightfold greater than population 1153 and 44-fold greater than the population of lowest reactivity, 1140 (Fig. 4, black bars).
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Fig. 4 shows that the relative hybridization intensity (white bars) correlated with mAb RA-C11 reactivity (black bars) (r=0·986). This correlation was strong enough to support the expression-site hypothesis with 90 % confidence (P<0·1). Analysis of variance (ANOVA) performed on the hybridization values demonstrated a significant difference between the three populations, particularly between population 1160 and the other two (P<0·01).
Residence of the C11 EES at the UCS locus correlates with surface reactivity
The expression-site hypothesis predicts that populations of P. carinii that exhibit high abundance of the RA-C11 epitope by immunoblotting will have more organisms with this epitope on their surface. To test this prediction, indirect immunofluoresence (IFA) experiments were attempted on organisms from populations 1140, 1153 and 1160. No staining was observed with either mAb RA-C11 or mAb RA-F1. Poor cryopreservation of the samples may have been at fault (A. G. Smulian, personal communication). In any case, it became necessary to use different populations of P. carinii for IFA measurements.
Samples from properly cryopreserved populations of P. carinii were screened by DNA hybridization (as described above) to identify those that varied with respect to representation of the C11 EES at the UCS locus. Three populations (M25-14, M54-12 and M54-27) that differed over a 10-fold range were identified. Specifically, population M25-14 exhibited the highest hybridization signal, which was approximately fourfold greater than that of population M54-12 and 10-fold greater than that of population M54-27 (Fig. 5).
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DISCUSSION |
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Previous work had suggested that the RA-C11 epitope is rare, and therefore would serve to allow this prediction to be tested. Identification of the C11 epitope showed that 24 of the 26 known MSG isoforms do not contain it. Furthermore, preliminary experiments (not presented here), in which the C11 EES was used as a hybridization probe to screen P. carinii genomic libraries, suggested that only 3 of the 100 MSG genes in the P. carinii genome contain the DNA sequence that encodes the S5 RA-C11 epitope.
The rarity of the C11 epitope suggested that its abundance might vary among populations of P. carinii. This was the case, and three populations that differed in this regard were readily identified by immunoblotting experiments. Subsequent analysis of the UCS locus in these populations showed that the C11 EES was linked to it in proportion to the level of RA-C11 immunoblot reactivity. To confirm the correlation, and to focus the analysis on surface antigens, DNA hybridization was used to identify a second set of P. carinii populations that differed with respect to the fraction of UCS-linked DNA that had the C11 EES. Subsequent analysis of these populations by IFA showed that the fraction of organisms labelled by the RA-C11 antibody was in proportion to UCS-linked C11 EES. These findings support the expression-site model of MSG regulation.
Details regarding the mechanism of regulation of expression aside, these studies show that P. carinii can vary with respect to expression of the RA-C11 epitope. In each of the three populations studied with IFA, some organisms did not bind the RA-C11 mAb. Hence, it appears that not all MSG isoforms are expressed in a given organism at the same time. The heterogeneity observed was not surprising for two reasons. First, heterogeneous staining of P. carinii populations by antibodies that recognize MSG epitopes has been reported before (Angus et al., 1996). Second, heterogeneity is consistent with expectation based on previous studies of the UCS locus, which showed that heterogeneity in P. carinii populations is high when rats are kept in an open-air colony, where they are exposed for extended periods of time to high numbers of airborne P. carinii produced by other heavily infected rats in the colony (Keely et al., 2003
). The rats used in the study described above were from such a colony. By contrast, P. carinii populations that originated via proliferation of one or a few progenitors were much less heterogeneous at the UCS locus (Keely et al., 2003
). Such populations might also be less heterogeneous with respect to surface MSG epitopes. Studies to test this idea are under way.
Expression of one MSG on the surface at a time would mimic the strategy employed by other pathogens that establish themselves in immunocompetent mammalian hosts. Two well-studied examples of such pathogens are African trypanosomes and Borrelia spirochaetes (Donelson, 1995; Zhang et al., 1997
; Rudenko et al., 1998
). In each microbe, only one version of a major surface antigen is expressed per organism, and the version expressed can be changed by suppressing expression of the active gene while activating expression of an inactive gene. P. carinii can accomplish such a concerted change by moving a silent gene into the expression site.
The purpose of MSG expression changes might be to allow P. carinii to colonize immunocompetent rats. While well known as a pathogen of rats that lack a normal immune system, P. carinii can remain in immunocompetent rats for prolonged periods of time (Vargas et al., 1995). Colonization may be the primary means by which P. carinii survives. Three observations support this conjecture. First, the organism is very scarce in the environment, except where large numbers of infected rats are present (Vargas et al., 1995
; Wakefield, 1996
). Second, outside of the rat, P. carinii propagates very poorly (Sloand et al., 1993
; Cushion, 1998b
; Merali et al., 1999
). Third, P. carinii appears to be specific for rats, and not able to proliferate in other host species. This host-specificity phenotype is also exhibited by Pneumocystis from other host species, such as humans, mouse, rabbit and ferret (Gigliotti et al., 1993
; Sidman & Roths, 1994
; Mazars et al., 1997
; Wakefield et al., 1998
; Demanche et al., 2001
; Durand-Joly et al., 2002
).
While the different Pneumocystis species and presumptive species exhibit many genetic differences, they all share the life style of habitation within the alveolar spaces of a mammalian lung (Stringer, 2002). The MSG system of P. carinii presumably plays a role in this lifestyle. As far as is known, all species in the genus Pneumocystis, including that found in humans, have families of genes that encode various forms of surface proteins (Stringer et al., 1993
; Garbe & Stringer, 1994
; Kutty et al., 2001
). Understanding the MSG system in P. carinii should serve as a model for understanding the role of surface variation in other hosts, including humans.
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ACKNOWLEDGEMENTS |
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Received 4 June 2003;
revised 4 December 2003;
accepted 16 December 2003.
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