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Applied and Environmental Microbiology, September 2000, p. 3931-3938, Vol. 66, No. 9
UPRES EA-1318, LTAB
Received 1 May 2000/Accepted 5 July 2000
A competitive reverse transcription-PCR method was developed for
the semiquantitation of the expression of genes encoding bicomponent
leucotoxins of Staphylococcus aureus, e.g.,
Panton-Valentine leucocidin (lukPV), gamma-hemolysin
(hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA
preparation; reverse transcription; the use of various amounts of
enzymes, antisense primer, and RNA; and the final amplification chain
reaction. Reproducible results were obtained, with sensitivity for
detection of cDNA within the range of 1 mRNA/104 CFU to
102 mRNA/CFU, depending on the gene. Both specific mRNAs
were more significantly expressed at the late-exponential phase of
growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the
widely distributed gamma-hemolysin locus in the NTCC 8178 strain was
around 10-fold diminished compared with that in the ATCC 49775 strain.
Because of the lower level of hlgA expression, the
corresponding protein, which is generally not abundant in culture
supernatant, should be investigated for its contribution to the
leucotoxin-associated virulence. The agr, sar,
and agr sar mutant strains revealed a great dependence with
regard to leucotoxin expression on the global regulatory system in
S. aureus, except that expression of hlgA was
not affected in the agr mutant.
Staphylococcal bicomponent
leucotoxins are exotoxins consisting of two nonassociated but synergic
class S (31 to 32 kDa) and class F (35 kDa) proteins. Among this family
of toxins, the Panton-Valentine leucocidin (PVL) is encoded by two
contiguous and cotranscribed genes, lukFPV and
lukS-PV (31). Another locus encodes Leucotoxin production was associated with infections resulting in
furuncles, community pneumonia, and some antibiotic-associated diarrhea
(11, 15, 24). These infection-related leucotoxins act as
activators of human neutrophils before creating lytic pores sensitive
to monovalent cations (34). The leucotoxins were shown to
induce an important inflammatory response in vivo in rabbit skin and in
rabbit vitreous humor (16, 31, 32). Therefore, to determine
their respective roles in pathogenicity and whether expression might
influence these roles, it is important to semiquantify, at least, the
expression of loci within the leucotoxin family. RNA methods, and
reverse transcription (RT)-PCR in particular, were shown to be more
sensitive than antibody-based detection methods (3). In
contrast to Northern blotting, which has a lower sensitivity
(35), RT-PCR allows multiple and simultaneous detection of
mRNAs contained in limited amounts of total RNA prepared from tissues
or sample volumes. This method is widely used to quantify viruses
(human immunodeficiency virus and hepatitis B and C viruses) and
cytokine expression in different systems (12, 20). RNA
methods are still underutilized for detection (19) or
quantitative analysis (14, 18) of bacterial gene expression.
The aim of this work was to develop a semiquantitative and competitive
RT-PCR method to compare the expression of the bicomponent leucotoxins
under different culture conditions and to explore the dependence of
these leucotoxins on the global accessory gene regulator
(agr) and staphylococcal accessory regulator
(sar) systems (8).
Bacterial strains, plasmids, and culture media.
The S. aureus V8 strain (ATCC 49775) produces both
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Variable Expressions of Staphylococcus
aureus Bicomponent Leucotoxins Semiquantified by Competitive
Reverse Transcription-PCR
Institut de
Bactériologie de la Faculté de Médecine,
Université Louis PasteurHôpitaux Universitaires de
Strasbourg, Strasbourg, France
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-hemolysin and comprises three genes: the first two encode class S proteins (HlgA
and HlgC), and the third one encodes a class F protein (HlgB). hlgA constitutes an upstream open reading frame, whereas
hlgC and hlgB (hlgCB in this text) are
cotranscribed (10). Another locus was recently characterized
as two cotranscribed class S and class F protein-encoding genes,
lukE and lukD (lukED in the text),
respectively (16). Production of leucotoxins among
Staphylococcus aureus strains was studied by radial gel
immunoprecipitation (16, 30), but quantitation of leucotoxin
expression by enzyme-linked immunosorbent assay remains difficult
because of cross-reactivity due to sequence identity between class S
components (55 to 72%) and class F components (71 to 79%),
respectively (29). There may be S. aureus strains
that produce -hemolysin, PVL, and/or LukE-LukD.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-hemolysin (HlgA,
-B, and -C) and PVL. S. aureus Newman strain (NTCC 8178) produces -hemolysin (HlgA, -B, and -C) and LukE-LukD but does not
produce PVL. The agr, sar, and agr sar
mutant strains derived from Newman were kindly provided by A. L. Cheung (Rockefeller University, New York, N.Y.) (7). The
agr, sar, and agr sar mutant strains
derived from ATCC 49775 were obtained by transduction with
bacteriophage 85 of the Newman agr and sar mutant
strains (28). The agr and sar mutant
strains were selected for their resistance to tetracycline and
erythromycin, respectively, and the disruptions of the agr
and sar loci were verified by Southern blotting
(33) with specific probes (data not shown).
M15
Tn10(tet)]; Stratagene, Amsterdam, The
Netherlands} were recipients for the competitor plasmids listed in
Table 1.
TABLE 1.
Plasmid competitors used as internal standards for RT-PCR
and related materials
[wt/vol] Na2HPO4, 0.4 [wt/vol] KH2HPO4; pH 7.0) or in 2.5% (wt/vol) heart
infusion (HI) medium (pH 7.4; Difco Laboratories).
Primers.
Specific primers were deduced from the
corresponding nucleotide sequences (Table
2) and were synthesized by Life
Technologies (Gaithersburg, Md.).
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Total RNA extractions.
The usual care was taken in handling
RNA. Cultures were started with 100 µl of inoculum, grown at 37°C
to stationary phase, and dropped in 2-liter Erlenmeyer flasks filled
with 120 ml of YCP or HI medium for incubation at 37°C and 180 rpm.
Volumes generally corresponding to a range of 5 × 107
to 5 × 109 CFU/ml (1 to 20 ml, depending on growth
phase) were serially harvested, and the bacteria were pelleted at 4°C
and washed with 1 ml of diethyl pyrocarbonate (DEPC;
Sigma-Aldrich)-treated H2O. The washed cell pellet was
resuspended in 200 µl of DEPC-treated H2O plus 40 U of
RNase OUT (Life Technologies). RNA extraction was achieved with the
FastRNA Blue Kit (Bio101, Inc., Vista, Calif.) (6); cells
were ground by centrifugation with silica beads associated with
chaotropic agents and phenol, according to the manufacturer's
recommendations. After isopropanol precipitation, the dried pellet was
solubilized in 100 µl of SAFEE (DEPC-treated H2O, 0.5 mM
EDTA) and an additional LiCl precipitation step was performed to
minimize carbohydrate contamination. The carbohydrate-free pellet was
washed twice with 250 µl of SEWS-BLUE (Bio101, Inc.), and the RNA
preparation was resuspended in 0.5 mM EDTA (pH 7.5) plus 40 U of RNase
OUT and stored in aliquots at 
80°C before use.
80°C. The quantity and purity of total RNA were deduced from
absorbances, gauged by the optical density at 260 nm
(OD260) and the OD280. DNA contamination was
only detected by RT-PCR with RNA preparations (data not shown) not
having an LiCl precipitation prior to DNase treatment, indicating that
mucopolysaccharides, which constitute powerful inhibitors for enzymatic
reaction (9), have to be eliminated.
Reverse transcription of RNA templates.
cDNA synthesis was
performed after optimization of the RT procedure, and the reverse
transcription was carried out using Moloney murine leukemia virus
(M-MuLV) from Perkin-Elmer (Foster City, Calif.). RNA sample was added
to a solution containing 80 pmol of antisense 3' primer (AP), 20 U of
RNase OUT, 1 mM deoxynucleoside triphosphate (dNTP)
(Boehringer-Mannheim, Germany), 2 µl of 10× GeneAmp PCR buffer II, 5 mM MgCl2, and 50 U of M-MuLV reverse transcriptase
(Perkin-Elmer), and the volume was adjusted to 20 µl by adding
DEPC-treated H2O. After a 15-min incubation at 42°C, the
reaction was stopped by heating for 5 min at 95°C. The resulting cDNA
was stored at 20°C. Parallel incubations were performed without the
addition of reverse transcriptase in order to alternatively verify the
DNA removal.
PCR.
The amplification step was performed by using
previously aliquoted reagents. One microliter of cDNA or RNA solution
incubated without RT was added to a 49-µl volume containing 1×
Taq buffer (3 mM MgCl2), 0.2 mM dNTP
(Boehringer-Mannheim), 0.2 pM concentrations of specific primers, and
2.5 U of Taq DNA polymerase (Life Technologies). The RNA-DNA
heteroduplex was denatured for 3.5 min at 94°C. Amplification was
achieved by 35 cycles of denaturation for 1.5 min at 92°C, annealing
for 1.5 min (at 52°C for lukPV and hlgCB, at
54°C for hlgA, and at 56°C for lukED), and
polymerization for 1.5 min at 72°C in a thermocycler (Perkin-Elmer
model 9700), and it was ended with 8 min of incubation at 72°C.
Samples were stored at 20°C prior to electrophoresis on a 1.2%
(wt/vol) small fragment agarose gel (Quantum Biotechnologies, Inc.) in
0.5× TEB buffer (45 mM Tris, 0.6 mM EDTA, 45 mM boric acid [pH 8.3])
and staining with ethidium bromide. The 35 cycles chosen for PCR turned
out to be sufficient. In every PCR assay, 15 initial copies of plasmid
competitors always provided visible DNA bands on agarose gels.
Specific mRNA quantitation and RT-PCR optimization.
Constant
amounts of cDNA corresponding to a determined quantity of CFU were
coamplified by PCR with a dilution series of the corresponding
competitor (Table 1), ranging from 100 ng to 0.01 fg. Bands of equal
intensity on the agarose gel corresponding to amplified cDNA and to the
amplified internal standard were assumed to reflect equal amounts of
cDNA and the known concentration of competitor DNA from the plasmid. A
visual observation of the equivalence of DNA bands was made on agarose
gels. When no equivalence was visualized, secondary PCR using
middle-range intermediate concentrations of competitors was achieved.
Thus, the true equivalence ± 25% is given. For each sample, we
performed three competitive PCRs on cDNAs to assess reproducibility.
Punctual controls performed on independent RNA extractions and cDNA
synthesis corroborated the results. Determinations of the initial
number of specific mRNA copies/CFU could then be obtained with the
following equation:
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Sensitivity of RT-PCR. Amplifications of cDNA and competitive internal standard were found to proceed with equal efficiency, independently of the PCR phase, up to the plateau (13). To determine the sensitivity of the RT-PCR, a culture in YCP medium of the Newman strain (NTCC 8178) was harvested at 9 × 108 CFU/ml, and total RNA was purified. Competitive RT-PCR allowed quantification of 1 hlgCB mRNA/25 CFU. Then, serial dilutions of DNA-free RNA preparation, which corresponded to concentrations of from 1.3 × 108 to 1.3 × 104 CFU/50 µl, were carried out and were submitted to hlgCB-specific reverse transcriptions. PCR on cDNA equivalent to from 5.2 × 106 to 5.2 × 102 CFU per 2-µl aliquot was performed with 100-µl reaction mixtures. Positive signals for the hlgCB amplified fragments were visualized for 15-µl aliquots, corresponding to a sensitivity of 2.5 × 105 CFU. Taking into account the initial titer of 1 mRNA/25 CFU, the results corresponded to a sensitivity at least as low as 1 hlgCB mRNA/60 CFU for the whole RT-PCR.
Expression in YCP and HI media of lukPV,
hlgA, and hlgCB from S. aureus V8
(ATCC 49775).
Iterative RNA preparations at chosen times of the
growth in YCP medium of ATCC 49775 were submitted to lukPV,
hlgA, and hlgCB RT-PCR. The semiquantitative
curves show the number of mRNA per CFU relative to the passage of time
(Fig. 1 and
2). Expression at 37°C of
lukPV mRNAs increased 103-fold (Fig. 1), from
40 ± 12 mRNA/600 CFU at a bacterial density of 6 × 107 CFU/ml (2.5 h of culture) to 90 ± 30 mRNA/CFU at a bacterial density of 4.2 × 109 CFU/ml
(5.5 h of culture). Concomitantly, expression of hlgA increased 30-fold to reach 25 ± 5 mRNA/100 CFU and the expression of hlgCB increased 300-fold to reach 23 ± 7 mRNA/CFU.
Transcription of the lukP and lukV genes reached
the highest level at the late-exponential growth phase, as was observed
for the two other transcriptional units. Expression of hlgCB
was close to lukPV, except for a significant difference in
its optimal expression, which was threefold lower. The abundance of
hlgA transcripts was low compared to hlgCB and lukPV transcripts, with an optimal expression 300-fold lower
than that of lukPV and 100-fold lower than that of
hlgCB. During 2 h of the stationary-growth phase, where
the bacterial density remained almost constant, the expression of the
transcription units decreased to a limited extent, not more than
10-fold.
|
), hlgA (
), and hlgCB
(
) in wild-type S. aureus ATCC 49775 grown in YCP medium.
The expression of the genes encoding staphylococcal leucotoxins was
semiquantified by RT-PCR. (A) Growth curve. (B) Semiquantitation curves
of the expression of lukPV, hlgA, and
hlgCB. The data represent the means ± standard
deviations (error bars) from three RT-PCRs.
|
Expression in YCP and HI media of lukED, hlgA, and hlgCB from S. aureus Newman (NTCC 8178). In YCP medium, expression of hlgA was detected at a bacterial density of 9 × 107 CFU/ml, with 40 ± 12 mRNA observed per 105 CFU (Fig. 2A), which increased 100-fold until a bacterial density of 2.7 × 108 CFU/ml was reached and remained stable at the beginning of the stationary-growth phase. Expression of hlgCB varied by 100-fold and appeared to be quite delayed, compared to that of hlgA, being optimal at a bacterial density of 4 × 109 CFU/ml. Finally, the Newman strain appeared to be a better producer of lukED mRNA than of those specific for hlgCB and hlgA, with 20 ± 6 mRNA/CFU at a density of 4 × 109 CFU/ml, though expression decreased threefold for the stationary-growth phase.
In HI medium, expression of leucotoxins remained very low, never exceeding 100 ± 25 mRNA/104 CFU, and hlgCB appeared to be strongly repressed compared to its expression in the YCP medium. Leucotoxin expression remained detectable from the mid-log phase on but did not increase more than 10-fold.Comparison of leucotoxin expression in agr,
sar, and agr sar mutant strains derived from
ATCC 49775 and Newman and grown in YCP medium.
To assess the
regulation of leucotoxins by the global agr sar regulatory
system, wild-type and agr, sar, and agr
sar mutant ATCC 49775 and NTCC 8178 strains were grown in YCP
medium at 37°C before leucotoxin mRNAs were semiquantified. The
semiquantitative curves show the dependence of the number of mRNA per
CFU on bacterial density (Fig. 3 and
4). The bacterial density of the ATCC
49775 agr sar mutant strain was quite inferior to those of
the other mutant or wild-type strains during the stationary phase.
Semiquantitative analysis for bacterial densities of 
109
CFU/ml was not obtained.
|
)
and agr (
), agr sar
(
) mutants of the same strain grown in YCP medium. (A) Growth
curves. (B, C, and D) Semiquantitative curves specific for
lukPV, hlgA, and hlgCB, respectively.
The data represent means ± standard deviations (error bars) from
three RT-PCR.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Compared to the cumulative detection of protein expression and production by radial immunoprecipitation or Northern blotting, RT-PCR methods constitute sensitive and real-time alternatives. A competitive RT-PCR has been optimized for the detection of staphylococcal leucotoxin mRNAs. Because of the short half-life of bacterial RNAs (6) and the need for reproducibility, a standardized procedure for the preparation of total RNAs was used. In this work, the competitive RT-PCR test using plasmid competitors provided a semiquantitative approach, with a sensitivity of 60 CFU expressing one specific mRNA, which is in the range reported (14) in a study that used the gyr invariant reporter gene and provided relative variations of mRNA expression compared to the reporter.
Expression of the staphylococcal leucotoxins from S. aureus grown in YCP medium was detected at very low yields at the beginning of the exponential-growth phase but increased by about 3 log units from the mid-log phase to the late-exponential growth phase. Then expression remain significant, but at a lower level, at the beginning of the stationary growth. The possible increase of ribonucleases, the nonsaturating conditions of enzymes for gene transcription at the stationary phase, or the privileged efficiency of expression for other genes might explain such observations (17). However, the expression of hlgA appeared to be 100-fold lower than that of lukPV, hlgCB, or lukED, which effectively results in a lower yield of protein recovered from bacterial cultures (31). Similar results were obtained whatever the two strains tested.
When the bacteria were grown in HI medium, expression of the toxins remained basal, not exceeding 10 ± 2.5 mRNA/3,000 CFU, showing that expression depends on regulation factors which are themselves dependent on environment conditions. Similar observations were made for the control of the expression of hla (alpha-hemolysin gene) with variations of salt concentrations (NaCl and KCl concentrations) of the medium (26). This observation may also suggest the presence of antagonist molecules in growth media or the presence of regulatory mechanisms in the bacteria.
To illustrate this observation, dependence of the expression of the leucotoxins on the global agr sar accessory system (7, 21) was investigated with agr- and sar-defective mutants of ATCC 49775 and NTCC 8178 S. aureus strains. The results (Fig. 3 and 4) showed that expression of lukPV, lukED, hlgA, and hlgBC depends on the sar locus for the two strains, whereas hlgA seemed to be the only locus not sensitive to the agr mutation. For the sensitive loci, the control exerted by both the agr and sar loci results in a 10- to 100-fold increase of expression that is maximal at the late-exponential growth phase. In the sar- and agr-defective mutants, the lack of control by sarA and sarB expression products (4, 23) and by agrA, -B, -C, -D, and RNA III expression products, via P2 and P3 promoters (2, 23), led to the detection of an early and significant expression of leucotoxins. The sar locus was demonstrated to control partially and positively the agr locus by the DNA binding protein SarA (8), but it may also directly control genes encoding exoproteins (7). The lack of sar and agr expression products may effectively result in the absence of control of toxin expression. A recently characterized sigma factor in S. aureus (36) acts on shock proteins (22) but was also reported to act on the sar locus, by increasing sarA expression and, consequently, the expression of alpha-hemolysin (5). Therefore, absence of both the agr and sar controls would result in a lack of regulation, in the case of independence of leucotoxin genes with another mechanism. Differences in expression of staphylococcal leucotoxins in the agr sar double mutants may reflect differences in the control by other mechanisms, e.g., the sigma factor. Only hlgA in the ATCC 49775 agr sar mutant did not show a strong increase compared to that in the NTCC 8178 agr sar mutant. Differences in the sequences of the corresponding promoters (11, 31) and genes may be responsible for such discrepancies; for the loci encoding the leucotoxins discussed, promoter regions do not show great sequence identity, except for a GNA(T)TAAA sequence located 35 to 31 bases upstream of the ATG codons (29). Unfortunately, the molecular mechanisms of the regulation of staphylococcal exoprotein expression remain poorly studied (25). Goerke et al. (14) recently showed that the expression of alpha-toxin (hla) was independent of the agr locus in vitro and in vivo and that the agr activity seemed to be nonessential in cystic fibrosis lung infections. The authors postulated that in vivo, other regulatory mechanisms apart from agr were involved. A potential element in the regulation of gene expression is the variable stability of mRNAs according to the locus and to the growth rate of the bacteria (17). The half-life of the ompA transcript in E. coli is reduced from 17 min in fast-growing cells to 4 min in slow-growing cells, due to the variable stability of the 5' end of the ompA mRNA. This could affect the regulation rate of the regulatory system.
In conclusion, as clinical S. aureus strains may encode from two (hlgA and hlgCB) to four (hlgA, hlgCB, lukPV, and lukED) leucotoxin loci and combinations of the corresponding class S and class F proteins result in leucotoxins with specific biological activities (16, 29), semiquantitation of their expression provides insights concerning their respective and possible involvement in bacterial virulence. hlgA appears to be the least expressed of the proteins studied. As toxin concentrations result from their cumulative expression, it may be hypothesized that HlgA is not as abundant as the other leucotoxin components. This is regularly the case upon purification of this protein (31). However, expression remains highly dependent on environmental conditions (e.g., growth media) and on the strain genetic equipment. Sensitivity to antibiotics of the gene encoding alpha-toxin secreted by S. aureus was reported in in vitro studies (27). Inappropriate antimicrobial therapy might, therefore, result in a temporal upregulation of virulence factors, leading to undesirable lesions. Semiquantitation of virulence factors in in vivo experimental models would be a suitable tool for the evaluation of antimicrobial therapeutics.
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ACKNOWLEDGMENTS |
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We thank A. L. Cheung (Rockefeller University) for the kind gift of the S. aureus Newman agr, sar, and agr sar mutant strains. We appreciated the excellent technical assistance of V. Wattelet. We thank N. Boord for English improvement.
This work was supported by grant EA 1318 from the French "Direction de la Recherche et des Etudes Doctorales" (DRED).
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FOOTNOTES |
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*
Corresponding author. Mailing address: UPRES EA-1318,
LTABInstitut de Bactériologie de la Faculté de
Médecine, Université Louis PasteurHôpitaux
Universitaires de Strasbourg, 3 rue Koeberlé, F-67000 Strasbourg,
France. Phone: (33) 3 88 21 23 87. Fax: (33) 3 88 25 11 13. E-mail:
gilles.prevost{at}medecine.u-strasbg.fr.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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