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Antimicrobial Agents and Chemotherapy, March 2001, p. 776-780, Vol. 45, No. 3
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.3.776-780.2001
Rapid Detection of Mutations in the Human-Derived
Pneumocystis carinii Dihydropteroate Synthase Gene
Associated with Sulfa Resistance
Liang
Ma and
Joseph A.
Kovacs*
Critical Care Medicine Department, Warren
Grant Magnuson Clinical Center, National Institutes of Health,
Bethesda, Maryland
Received 27 September 2000/Returned for modification 27 November
2000/Accepted 11 December 2000
 |
ABSTRACT |
Recent studies have shown that point mutations in the
dihydropteroate synthase (DHPS) gene of human-derived
Pneumocystis carinii are related to exposure to sulfa drugs
and possibly represent the emergence of sulfa resistance. We developed
a simple single-strand conformation polymorphism (SSCP) method to
permit rapid detection of these mutations. With plasmid constructs,
SSCP was able to detect as little as 10% of a minority population. The
SSCP assay was compared to direct sequencing for typing the DHPS gene
by examining 37 clinical isolates with known DHPS sequences and 41 clinical isolates with unknown DHPS sequences. The typing results were
consistent between these two methods for all isolates except 11 in
which mutations were detected by SSCP but not by direct sequencing.
Sequencing of individual clones after subcloning confirmed the presence
of mutations in a minority population as determined by SSCP. SSCP is a
very simple and sensitive method for rapid identification of P. camii DHPS mutations.
| |
INTRODUCTION |
An increasing number of studies have
demonstrated that mutations at amino acids 55 (Thr
Ala) and 57 (ProSer) in the human-derived Pneumocystis carinii
dihydropteroate synthase (DHPS) gene are associated with prior exposure
to sulfa or dapsone, which target the DHPS (7, 10, 11, 13,
17; Q. Mei, S. Gurunathan, H. Masur, and J. A. Kovacs,
Letter, Lancet 351:1631, 1998). These mutations likely represent
emergence of sulfa resistance in P. carinii, since they are
present in one of the active sites of the enzyme (1) and
correlate with mutations that are shown to confer sulfa resistance in
other organisms, such as Streptococcus pneumoniae
(16), Plasmodium falciparum (4),
and Mycobacterium leprae (9). Because no
reliable culture system for human-derived P. carinii is
currently available, direct proof of sulfa resistance in P. carinii isolates with DHPS mutations cannot be determined by
traditional in vitro drug susceptibility testing. Evaluation of the
relationship between these mutations and clinical resistance to sulfa
drugs would be facilitated by a rapid method for identifying the
mutations. To date, detection of the P. carinii DHPS
mutations has relied on direct DNA sequencing of PCR-amplified
products. Although sequencing has the advantage of high accuracy and
possibly of identifying other mutations, it is not amenable to rapid
screening of large numbers of samples due to its technical complexity
and high cost. Furthermore, minority populations can be detected only if they represent 20 to 30% of the population.
Our goal in the present study was to develop a rapid, simple method for
detecting these mutations in clinical isolates that not only could be
utilized by other investigators but in the future could also be used by
clinical microbiology laboratories. Single-strand conformation
polymorphism (SSCP) is a relatively recently developed method for
detecting mutations that relies upon the ability of one or more
nucleotide changes to alter the electrophoretic mobility of
single-stranded DNA molecules under nondenaturing conditions (18). Because of its technical simplicity and relatively
high sensitivity, SSCP has become one of the most popular strategies for detection of genetic variations and mutations. Hauser et al. (6) have previously shown this method to be a promising
option for molecular typing of human-derived P. carinii
isolates. Here we describe a simple SSCP protocol using a compact
electrophoresis unit, small gel format, and nonradioactive staining to
detect the previously described point mutations (codons 55 and 57) in the DHPS gene of human-derived P. carinii.
(This work was presented in part at the 40th Interscience Conference on
Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 17 to
20 September 2000 [L. Ma and J. A. Kovacs, Abstr. 40th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. 1951, 2000].)
| |
MATERIALS AND METHODS |
Clinical P. carinii isolates and DNA extraction.
Two sets of clinical P. carinii isolates were used in this
study. The first consisted of 37 isolates for which the P. carinii DHPS nucleotide sequences had been determined previously
by direct sequencing and/or subcloning (17). The second
set consisted of 41 isolates (from 14 sputum samples and 27 bronchoalveolar lavage fluid samples) which were obtained from patients
diagnosed with P. carinii pneumonia between 1991 and 1995 but for which the DHPS sequence had not been previously determined. For
the first set of isolates, genomic DNA was extracted by treatment with
proteinase K followed by phenol-chloroform extraction as described
previously (17). For the second set, genomic DNA was extracted either by that method or by use of the NucliSens isolation kit (Organon Teknika, Durham, N.C.).
Experimentation guidelines of the U.S. Department of Health and Human
Services and the National Institutes of Health were followed in the
conduct of this research.
PCR.
On the basis of the full-length human-derived P. carinii DHPS gene (17), we designed two primers, A2
(5'-TTACTCCTGATTCTTTTTTCGATGGG-3') and PS308
(5'-GCCTTAATTGCTTGTTCTGCAACC-3'), which amplify a 259-bp fragment spanning the mutation sites (codons 55 and 57). For
determination of the basic SSCP patterns of the human-derived P. carinii DHPS variants and for optimization of the assay
conditions, we used plasmids containing wild-type or mutant (either
single or double mutation) sequences of the human-derived P. carinii DHPS gene. The wild-type plasmid contained a DHPS sequence
with nucleotides A and C at positions 163 and 169, respectively
(17). In the single mutant plasmids, a G was at position
163 instead of A (resulting in amino acid change Thr-55Ala-55) or a
T was at position 169 instead of C (Pro-57Ser-57), and in the double
mutant plasmid, nucleotides G and T were present at positions 163 and
169 (Thr-55Ala-55 and Pro-57Ser-57, respectively). These plasmids
were obtained by subcloning of the PCR products from several clinical
isolates in our previous study (17) and in the present
study. PCR amplification with plasmids was performed in a 50-µl
reaction volume containing 10 ng of plasmid DNA, 0.4 µM (each)
primers A2 and PS308, 0.2 mM deoxynucleoside triphosphates, 1× native
Pfu buffer [20 mM Tris-HCl (pH 8.0), 10 mM KCl, 2 mM
MgCl2, 6 mM (NH4)2SO4,
0.1% Triton X-100, and 10 µg of bovine serum albumin per ml), and
2.5 U of native Pfu DNA polymerase (Stratagene, La Jolla,
Calif.). The thermal cycling conditions included an initial incubation at 95°C for 45 s, followed by 35 cycles of 95°C for 45 s,
60°C for 1 min, and 72°C for 1 min, with a final extension at
72°C for 10 min. PCR products were examined on a 3% agarose gel and then used in the SSCP assay described below.
To amplify the DHPS fragments from clinical isolates, a nested PCR
protocol was performed using the previously described primer
pair PK95
and PS876 (
17) for the first round and the primer
pair A2
and PS308 (described above) for the second round. All
PCRs were carried
out in a total volume of 50 µl. The first round
of PCR was carried
out with a touchdown protocol, which consisted
of 10 cycles of 45 s at 94°C and 2 min at 65°C, with a decrease
by 1°C every cycle
to reach 55°C in the last cycle of the period,
and 2 min at 72°C,
followed by 25 cycles of 45 s at 94°C, 1 min
at 55°C, and 2 min at 72°C. The thermal cycling parameters for
the second round of
PCR were the same as for the PCR with the
plasmid DNA as described
above. Each experiment included a negative
control without template DNA
and a positive control containing
100 ng of human-derived
P. carinii genomic DNA. The amplified
products from the second-round
PCR were used in the SSCP assay
after examination by 3% agarose
electrophoresis.
SSCP.
The SSCP conditions were optimized using plasmids
containing either wild-type or mutant human-derived P. carinii DHPS sequences and the GeneGel SSCP Starter Kit according
to the instructions of the manufacturer (Amersham Pharmacia Biotech,
San Francisco, Calif.). Four-microliter aliquots of PCR products were
mixed with 4 µl of loading buffer containing 90% formamide, 0.025%
(wt/vol) bromophenol blue, 0.025% (wt/vol) xylene cyanol, 3%
(vol/vol) glycerol, and 0.5 µM (each) primers A2 and PS308. The
addition of the primers has been shown to improve the resolution of the single-stranded DNA bands in the SSCP gel, presumably by preventing reannealing of denatured DNA fragments, thus decreasing the amount of
double-stranded DNA and increasing the concentration of single-stranded DNA (2). This mixture was heated at 95°C for 5 min and
then chilled in an ice water bath. Five microliters was loaded on a precast GeneGel SSCP gel (122 by 110 by 0.5 mm; Amersham Pharmacia Biotech). Electrophoresis was performed using the
temperature-controlled GenePhor Electrophoresis System (Amersham
Pharmacia Biotech) under the conditions recommended by the
manufacturer. The gels were stained by using the PlusOne DNA Silver
Staining Kit (Amersham Pharmacia Biotech).
DNA sequencing.
PCR products were purified by use of the
StrataPrep PCR Purification Kit (Stratagene) and sequenced either by
direct sequencing or after subcloning. Subcloning was performed using
the PCR-Script Amp Cloning Kit (Stratagene) according to the
manufacturer's instructions. Inserts were screened by PCR with primers
A2 and PS308 followed by SSCP. Inserts which showed SSCP patterns
different from the wild-type pattern were sequenced using universal or
sequence-specific primers. DNA sequencing was carried out by the
dideoxy chain termination reaction method using the ABI PRISM 377 automated DNA sequencer (Perkin-Elmer, Foster City, Calif.) as
described previously (17). Nucleic acid sequences were
analyzed using MacVector 6.5.3 software (Oxford Molecular Ltd., Oxford, England).
| |
RESULTS |
Optimization of assay conditions.
We initially performed the
SSCP analysis using plasmids containing either wild-type or mutant
sequences of the human-derived P. carinii DHPS gene and the
GeneGel SSCP Starter Kit, and we examined the four key factors, i.e.,
fragment length, electrophoresis buffer, temperature, and gel matrix,
which can affect the resolution of SSCP. Although PCR products ranging
in size from 189 to 596 bp were evaluated, optimal separation of wild
type (T-55 and P-57), single mutation (A-55 or S-57), and double
mutation (A-55 and S-57) was obtained with a 259-bp fragment generated
by primers A2 and PS308. Additionally, use of the GeneGel SSCP Clean
gel, a running temperature of 12°C, running buffer C (pH 8.3), and a
running voltage at 80 V for 20 min followed by 510 V for 140 min
provided optimal conditions. Moreover, the addition of primers to PCR
products before loading onto the gel increased the yield of single
strands (data not shown), as has been described previously (2).
Under these optimal assay conditions, we could distinguish the SSCP
patterns of different types of
P. carinii DHPS sequences.
As
shown in Fig.
1, mobility differences
among different genotypes
can be seen in at least one strand. Compared
to the wild type
(T-55 and P-57), the single A-55 mutation and the
double mutation
(A-55 and S-57) cause mobility shifts in the upper
band, and the
single S-57 mutation causes a shift in both bands. The
mobility
shift of the upper band in the single A-55 mutation is
different
from that in the double mutation as clearly shown in the
sample
containing a mixture of these two types of mutation. In the
samples
containing a mixture of two different sequences, there are
three
or four bands corresponding to individual bands seen in the
samples
with one homozygous sequence. SSCP is capable of distinguishing
different mixed forms, including the mixture of wild type and
single
A-55 mutant (A/T-55, P-57), the mixture of wild type and
single S-57
mutant (T-55, S/P-57), the mixture of wild type and
double mutant
(A/T-55, S/P-57), and the mixture of single A-55
mutant and double
mutant (A-55, S/P-57). Except for the mixed
form of wild type and
single S-57 mutant sequences, all forms
shown have been detected in
clinical samples in previous studies
from our group (
17)
and/or others (
3,
7,
10,
11).
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FIG. 1.
SSCP patterns of the human-derived P. carinii
DHPS gene. The templates used were plasmid DNAs containing different
human-derived P. carinii DHPS sequences. Above each lane is
the amino acid sequence at codons 55 and 57. The genotype A/T-55 plus
S/P-57 represents a mixture of wild-type (T-55, P-57) and double mutant
(A-55, S-57) sequences. The mobility difference is clearly seen in at
least one strand among the different sequences.
|
|
Sensitivity of SSCP for detection of one allele in a mixture of two
alleles.
Because multiple alleles may be present in a single
clinical isolate, we wanted to examine the ability of SSCP to detect a minority population. For these studies, plasmids containing wild-type or mutant (either a single mutation at codon 55 or double mutations at
codons 55 and 57) P. carinii DHPS sequences were mixed, with mutant concentrations ranging from 5 to 95%, and evaluated by SSCP.
Under the previously optimized conditions, SSCP was able to detect the
minority population (either wild type or mutant) when it was present in
as little as 10% of the total population (Fig.
2).
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FIG. 2.
Sensitivity of SSCP for detection of minority
populations of the P. carinii DHPS gene. Plasmids containing
wild-type or mutant DHPS sequences were mixed in various
concentrations, amplified by PCR, and analyzed by SSCP. The percentage
of mutant DNA template is indicated above each lane. (A) Serial
mixtures of wild-type (T-55, P-57) and single A-55 mutant sequences.
(B) Serial mixtures of wild-type (T-55, P-57) and double mutant (A-55
and S-57) sequences.
|
|
Evaluation of clinical isolates by SSCP.
To validate the SSCP
method for detection of DHPS mutations, we first examined 37 clinical
isolates for which the DHPS sequences had been determined previously by
direct sequencing (17). The results of one representative
SSCP assay are shown in Fig. 3. The
typing results of the SSCP assay were very consistent with those of
direct sequencing (Table 1). However, in
three samples the results of these two methods were discordant. By
direct sequencing of the PCR product (without subcloning), two samples
(samples a and b in Table 2) were
determined to be wild type (T-55 and P-57) and one sample (sample c in
Table 2) was a mixture of wild-type and single A-55 mutant (A/T-55 and
P-57) sequences. Based on the SSCP patterns, all three samples (Fig. 3)
appeared to be a mixture of wild-type and double mutant sequences
(A/T-55 and S/P-57). To clarify the discrepant results in these three
samples, we performed subcloning of the PCR products followed by
sequencing of multiple clones per sample. As shown in Table 2, in all
three samples the presence of double mutant clones as a minority
population (9.5 to 36.4%) was verified, although the majority of the
clones were wild type. These findings confirmed that the SSCP typing results were correct and suggested that SSCP is more sensitive than
direct sequencing for detection of a minority population.
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FIG. 3.
Representative SSCP analysis of P. carinii
DHPS mutations in clinical isolates. Above each lane is the amino acid
sequence at codons 55 and 57. The genotype A/T-55 plus S/P-57
represents a mixture of wild-type (T-55, P-57) and double mutant (A-55,
S-57) sequences. Plasmids containing DHPS sequences were used as the
standard control (double-underlined lanes). Samples a, b, and c showed
discordant results between SSCP and direct sequencing but were
confirmed by sequencing of individual clones to be mixtures of wild
type and double mutation as determined by SSCP.
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TABLE 1.
Comparison of SSCP assay and direct sequencing for
detection of P. carinii DHPS mutations in 37 clinical
isolates
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TABLE 2.
Subcloning of PCR products from five P. carinii isolates which showed different DHPS genotypes in direct
sequencing and SSCP
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|
Notably, sample c contained four different DHPS sequences, including
wild type, double mutation, and single mutation at codon
55 or 57. The
single mutation at either codon 55 or 57 was not
detected by SSCP or
direct sequencing, presumably because each
of them accounted for only
5.9% of the total population and were
beyond the sensitivity of these
two
methods.
Following analysis of the 37 clinical samples described above, an
additional 41 samples with unknown DHPS sequence were examined
by the
SSCP assay. Based on the SSCP patterns, 22 samples had
wild-type
sequence, 17 samples contained either a homozygous or
heterozygous
mutation(s) at codon 55 alone or both codons 55 and
57, and 2 samples
showed unique SSCP patterns not previously identified.
To confirm the
SSCP typing results, direct sequencing was performed
on 7 samples with
a wild-type SSCP pattern and all 19 samples
showing patterns different
from those of wild-type samples. As
shown in Table
3, the SSCP typing results were verified
by direct
sequencing in most samples. However, six samples, which were
determined
by SSCP to be mixed forms of wild-type and mutant sequences,
either
at codon 55 alone (three samples) or at both codons 55 and 57
(three samples), were pure wild type by direct sequencing, and
two
samples, which were determined to be a mixture of wild-type
and double
mutant sequences by SSCP, were a mixture of wild-type
and single A-55
mutant sequences by direct sequencing. To determine
which was correct,
we performed subcloning of the PCR products
from two samples (samples
TH and KL in Table
2) which showed
inconsistent results between SSCP
and direct sequencing. Subsequent
sequencing of multiple individual
clones confirmed the presence
of both wild-type and double mutant
sequences in each sample,
as determined by SSCP.
For the two samples with unique SSCP patterns, direct sequencing showed
that both samples had wild-type sequence at both codons
55 and 57 but
contained novel synonymous nucleotide changes at
other codons that did
not result in a change in the predicted
amino acid. One sample showed a
mixture of nucleotides T and C
at position 153 (codon 51), and the
other had a mixture of nucleotides
T and C at position 219 (codon 73).
The latter sample was further
examined by subcloning of the PCR
product, and we obtained six
clones containing T and six clones
containing C at nucleotide
219.
| |
DISCUSSION |
This study has shown that single or double mutations in the DHPS
gene can be easily detected by SSCP, even when they represent as little
as 10% of the population. The reliability of SSCP for detection of the
DHPS mutations was confirmed by examining 78 clinical isolates of
human-derived P. carinii. The results from the present study
suggest that the SSCP assay we have developed will prove to be highly
useful for detection of the DHPS mutations in clinical P. carinii isolates.
The system we have described has advantages in simplicity and speed,
thus permitting rapid detection of DHPS mutations. In this study, we
used the small precast gel (12.2 by 11 cm) and semidry running system,
which requires no liquid running buffer and allows fast and easy
electrophoresis set-up and clean-up. For sample processing, the only
step after PCR is a heat denaturation in formamide. For detection of
the DNA bands, we used silver staining instead of radioactive labeling.
The silver staining method is easy, fast, and highly sensitive. The
total time needed for performing this assay starting from PCR was about
10 h, including 6 h for nested PCR, 2.5 h for
electrophoresis, and 1.5 h for silver staining.
Another major advantage of the SSCP method is its high sensitivity.
Under optimized conditions, a mixed population was detected when the
minority sequence represented as little as 10% of the total
population. When clinical isolates were studied, SSCP detected heterozygous sequences in 11 isolates, which were not detected by
direct sequencing but were documented by sequencing of individual clones after subcloning. These findings indicate that the SSCP assay
has higher sensitivity than direct sequencing. In automated direct
sequencing with fluorescent terminators, a heterozygous sequence is
identified as a mixture of two signals at one position, and it is
sometimes difficult to differentiate a weak signal from background
noise. More recently, another method based on restriction fragment
length polymorphism analysis has been shown to be useful for detection
of the P. carinii DHPS mutations (8). However, in preliminary experiments evaluating this methodology, we found that,
like direct sequencing, this method has low sensitivity for detection
of a minority population.
The ability to detect a minor population in mixed populations will be
important to help understand the prevalence as well as the kinetics of
the development of DHPS mutations in patients. There have been a number
of reports demonstrating a high rate of coinfection with multiple
P. carinii strains in clinical isolates. Studies using DNA
sequencing-based methods for typing human-derived P. carinii
isolates revealed that coinfection rates are seen in 10 to 30% of
isolates at several different genetic loci (12, 14, 15). A
much higher coinfection rate (69%) has been reported with SSCP
analysis of similar loci (5). The reported coinfection rates at the DHPS locus varied from 1 to 11% in studies utilizing direct sequencing of the PCR products (3, 7, 10, 11, 17).
The relatively low sensitivity of direct sequencing as shown in the
present study suggests that the coinfection rate has been substantially
underestimated. In the present study, the coinfection rate determined
by SSCP assay was 28% (21 of 78).
Another advantage of the method is its high reproducibility. Once the
optimal conditions have been established, this method gives
reproducible SSCP patterns. Since all of the materials, including the
electrophoresis unit, the gel, and the buffer are commercially
available, the results should be reproducible in different laboratories.
SSCP does have disadvantages compared to sequencing. First, this method
is unable to detect other mutations that have been reported at codons
23, 111, and 248 (13), because they are located outside
the region being investigated. Second, in a very few cases representing
novel nucleotide changes, the SSCP patterns may be difficult to
interpret. In this situation, sequencing is needed to identify the base
change. In the present study, novel nucleotide changes were detected at
positions 153 and 219 in two clinical samples. These changes are
synonymous and do not result in amino acid changes; thus, it is
unlikely that they represent a response to antibiotic pressure. Third,
there is a need to perform nested PCR before SSCP, since less than half
of clinical samples are positive after one round of PCR. Nested PCR is
time-consuming, taking about 6 h. However, nested PCR is also
needed for other mutation detection methods, including DNA sequencing.
In conclusion, the SSCP assay we have developed is a very simple and
highly sensitive method for rapid identification of P. carnii DHPS mutations.
| |
ACKNOWLEDGMENTS |
We thank Philippe Hauser of the Centre Hospitalier Universitaire
Vaudois, Division Autonome de Medecine Preventive Hospitaliere, Lausanne, Switzerland, for advice on SSCP methodology.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Building 10, Room 7D43, National Institutes of Health, 10 Center Dr. MSC 1662, Bethesda, MD 20892-1662. Phone: (301) 496-9907. Fax: (301) 402-1213. E-mail: jkovacs{at}nih.gov.
| |
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Antimicrobial Agents and Chemotherapy, March 2001, p. 776-780, Vol. 45, No. 3
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.3.776-780.2001
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Abstract
Introduction
