Download Quantitative expression of cholera toxin mRNA in Vibrio cholerae

Journal of Medical Microbiology (2012), 61, 1071–1073
DOI 10.1099/jmm.0.038752-0
Quantitative expression of cholera toxin mRNA in
Vibrio cholerae isolates with different CTX cassette
arrangements
Seyed Mahmoud Amin Marashi,1,2 Bita Bakhshi,3
Abbas Ali Imani Fooladi,4 Akbar Tavakoli,5 Afsoon Sharifnia6
and Mohammad R. Pourshafie2
Correspondence
1
Mohammad R. Pourshafie
2
Department of Microbiology & Immunology, Babol University of Medical Sciences, Babol, Iran
[email protected]
Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran
3
Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
4
Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
5
Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, Iran
6
Science and Research Branch, Islamic Azad University, Tehran, Iran
Received 30 September 2011
Accepted 17 April 2012
Cholera toxin (CT) is the major virulence factor produced by Vibrio cholerae. Several genomic
arrangements within the CTX cassette have been elucidated in V. cholerae. Previously, it was
shown that three different CTX cassette arrangements, one complete CTX cassette (arrangement
A), one complete and two incomplete CTX cassettes (arrangement B), and two complete CTX
cassettes (arrangement C), exist within V. cholerae isolates. In the present study, the level of CT
expression by V. cholerae isolates carrying different CTX cassette arrangements was evaluated.
Real-time quantitative PCR analysis showed unequal production of CT mRNA in V. cholerae
isolates with different CTX arrangements. V. cholerae with the CTX arrangement C expressed
more CT mRNA than isolates with the other CTX arrangements. In addition, CT mRNA was
expressed more in the isolates with CTX arrangement B than in those with arrangement A. Overall,
these results suggest that the arrangement and number of regulatory elements (rstA) within the
CTX cassette could affect the level of expression of CT.
INTRODUCTION
Cholera remains a major health problem in developing and
underdeveloped countries. Today, we are facing the
continuation of the seventh cholera pandemic, which has
been spread from South-east Asia across the Middle East
into Central America and Africa (Pourshafie et al., 2000).
Cholera toxin (CT) is a potent enterotoxin that plays a
major role in the pathogenesis of Vibrio cholerae through a
complex cooperation with CTXW phage and other phages
(Bakhshi et al., 2008). Environmental factors also affect
expression of CT through a number of regulatory genes such
as tcpP, tcpH and toxT. ToxT attaches to the ToxT-binding
motif toxbox within the CTX cassette, located upstream of
the ctxAB genes, which encode CT (Withey & DiRita, 2006).
The production of ToxT protein is also affected by the
products of the toxR gene and the quorum-sensing system
(Klose, 2001; Tsou et al., 2009). The RS1 element, containing
Abbreviations: CT, cholera toxin; Ct, cycle threshold; qPCR, real-time
quantitative PCR; RT-PCR, reverse transcriptase PCR.
038752 G 2012 SGM
rstA, rstB, rstR and rstC, is located upstream or downstream
adjacent to the CTX cassette (Davis & Waldor, 2003). These
genes encode proteins for replication and activation (RstA),
integration (RstB), regulation of gene expression (RstR) and
anti-repression (RstC). It has been shown that RS1 has a
positive effect on the ctxAB genes, resulting in increased
production of CT by modulation of the RS1 genomic
segment (Davis & Waldor, 2003). A previous study showed
that each CTX cassette contains an RS2 element and ctxAB
genes. The RS2 element is like RS1 but lacks the rstC ORF;
thus, the CTX cassette contains only the rstA, rstB and rstR
ORFs (Davis & Waldor, 2003). A previous report by this
laboratory showed that, among V. cholerae isolates obtained
from various parts of Iran, three distinct CTX cassettes with
different arrangements existed (Bakhshi et al., 2008) (Fig. 1).
We showed that incomplete CTX cassettes contained all
elements of the CTX cassette except the ctxAB genes. The
present study was designed to determine the level of CT
mRNA production in V. cholerae isolates with different CTX
arrangements and to explore potential associations of
specific arrangements and levels of CT production.
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Printed in Great Britain
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S. M. Amin Marashi and others
CTX genomic
arrangement A
CTX genomic
arrangement B
CTX genomic
arrangement C
CTX genomic
arrangement
classical strain
RS1
TLC
TLC
TLC
TLC
RS1
CTX
CTX(t)
CTX(t)
CTX
RS1
CTX
RS1
RTX
CTX
CTX
RTX
RTX
RTX
METHODS
Fig. 1. Proposed schematic of the genomic
organization of the CTX elements. The three
possible arrangements found in V. cholerae
isolates are indicated as A, B and C. Filled
boxes, complete CTX genetic element;
hatched boxes, toxin-linked cryptic (TLC)
element; open boxes, RTX cluster; spotted
boxes, incomplete (truncated) CTX genetic
element [CTX(t)]; checked boxes, RS1 element. Reproduced from Bakhshi et al. (2008),
with permission from John Wiley & Sons.
determine the specificity of the qPCR using genomic DNA for each
gene studied to confirm that the primers amplified at the same rate
and to validate the experiment (55–95 uC, increasing by 0.2 uC s21).
In the qPCR, a negative control of distilled water was included in each
run. The qPCR was performed twice in triplicate. Classical V. cholerae
O1 ATCC 14035 was used as a standard control.
Bacterial strains and growth conditions. In total, 94 clinical
isolates of V. cholerae O1 El Tor obtained from different outbreaks
between 2004 and 2009 were studied. A previous study in this
laboratory showed that these isolates were Inaba and Ogawa serotypes
(Bakhshi et al., 2008). In the present study, three strains were selected
randomly, one from each of the three different arrangements of the
CTX cassette. All strains were cultured in synthetic AKI media with
shaking and aeration for 15–18 h. The synthetic AKI medium was
prepared according to a protocol described previously (Iwanaga &
Kuyyakanond, 1987).
RESULTS AND DISCUSSION
The amplicons obtained for the recA (106 bp) and ctxAB
(115 bp) genes were verified by sequencing. The presence
of a single PCR product was confirmed by melting-curve
analysis, which resulted in a single product-specific melting
curve. PCR efficiencies were determined as between 1.90
and 1.94. The relative expression ratio was calculated for
each gene of interest by a mathematical model using the
22DDCt method (Livak & Schmittgen, 2001). This model
allows for relative changes in gene expression in qPCR
experiments and relates the PCR signal of the target
transcript in the treatment to that of the untreated control.
RT-PCR. For each isolate, 26108 c.f.u. ml21 was used for total RNA
extraction using an RNeasy Protect Bacteria Mini kit (Qiagen).
Separately, an equivalent concentration of total RNA was selected
from each sample as template for RT-PCR. cDNA synthesis and PCR
amplification were performed using a QuantiTect reverse transcription kit (Qiagen). RT-PCR was performed in the presence of random
primers at 42 uC for 10 min. Amplification of recA was used as the
internal control. The amplified products were verified by sequencing.
Real-time quantitative PCR (qPCR). Prepared cDNA was quan-
tified using SYBR Green I dye. The amount of cDNA used for qPCR
from each sample was 50 ng ml21. Four primers were designed: recA-f
(59-ATTGAAGGCGAAATGGGCGATAG-39), recA-r (59-TACACATACAGTTGGATTGCTTGAG-39), ctxAB-f (59-TATGCCAAGAGGACAGAGTGAG-39) and ctxAB-r (59-AACATATCCATCATCGTGCCTAAC-39). In the present study, the recA gene was considered a
housekeeping gene. A SYBR Green qPCR assay was performed in a
20 ml volume containing 26 QuantiTect SYBR Green PCR Master
Mix (Qiagen), 0.25 mM (each) specific primer set and 2 ml of cDNA
sample. Amplification of the primers, data acquisition and relative
expression analysis were carried out in a Chromo4 real-time detector
(Bio-Rad). qPCRs were performed as follows: one cycle of 95 uC for
5 min, followed by 40 cycles of 95 uC for 15 s and 60 uC for 30 s, and
a final extension at 72 uC for 2 min. Following amplification,
melting-curve analysis of the PCR products was performed to
Table 1 shows the results of cycle threshold (Ct) analysis
for the CTX arrangements A, B and C and the classical
strain. The mean ratios were also calculated as 0.05, 0.11
and 0.25 for the CTX arrangements A, B and C and were
significantly different from that of the classical strain,
which was given a value of 1 (P,0.05).
The results showed a significant difference between the
clinical isolates, regardless of their arrangement, and the
classical strain (ATCC 14035) (P,0.05 for each). Kaper
et al. (1995) previously showed that CT production in the
classical strain was significantly higher than that in the El
Tor strain of V. cholerae. In addition, DiRita et al. (1996)
Table 1. Cycle threshold (Ct) results for the A, B and C arrangements and the classical V. cholerae O1 strain ATTC 14035
DDCt was calculated as: DCt (test)2DCt (calibrator). Ratio5efficiency
Arrangement
A
B
C
Classical
1072
–DDC t
.
Mean Ct recA±SD
Mean Ct ctxAB±SD
Mean DDCt
22.74±0.19
23.61±0.31
23.34±0.30
25.58±0.14
25.88±0.22
25.78±0.37
24.26±0.30
24.35±0.39
4.37
3.41
2.16
1.23
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Mean ratio
0.05
0.11
0.25
1.0
Journal of Medical Microbiology 61
Evaluation of cholera toxin expression
showed that the level of CT in the classical strain was
higher than that in the El Tor strain because of the
influence of toxR on CT production in the classical strain.
Both classical and El Tor strains have been shown to
express equivalent levels of ToxR. In contrast, the classical
strain expresses more ToxT, which has a higher binding
affinity to toxbox, resulting in higher expression of CT
(González-Bonilla et al., 1994; DiRita et al., 1996).
In this study, we compared the Ct value of classical V.
cholerae with El Tor strains with three different CTX
arrangements, A, B and C. The results demonstrated the
production of lower levels of CT mRNA in the El Tor
strains with the various CTX arrangements. The Ct data
also suggested that ctxAB gene expression was approximately fourfold and twofold higher in the CTX arrangement C compared with the arrangements A and B,
respectively. The differences between arrangements C and
A are: (i) the presence of two copies of the complete CTX
in arrangement C, and (ii) the position of RS1 downstream
of CTXW in arrangement C compared with upstream in
arrangement A. The data support the suggestion that the
position of RS1 may have a direct effect on expression of
the ctxAB genes (Davis & Waldor, 2003). This difference in
positioning of RS1 could result in enhanced attachment of
the RNA polymerase enzyme leading to higher expression
of the ctxAB genes (Faruque et al., 2002, 2003). In contrast,
the twofold increase in the production of CT in
arrangement C compared with arrangement B could be
the result of: (i) the presence of one complete CTX and two
incomplete CTX cassettes in arrangement B, and (ii) the
upstream positioning of RS1. Arrangement B produced
twice as much CT mRNA as arrangement A, which could be
explained by the presence of three copies of the rstA gene in
arrangement B compared with one in arrangement A.
Although the CTX arrangement (CTX cassette and RS1) in
the classical strain was similar to arrangement A of the El
Tor strain, the results showed that the mean production of
CT mRNA was 1.0 in the classical strain compared with
0.05 in arrangement A of the El Tor strain, indicating 20
times less CT production in the strain with arrangement A.
In conclusion, the results from this study suggest that other
factors, besides the genomic arrangement within the CTX
cassette, modulate the production of CT by regulating the
CTX cassette, supporting the idea that CT production in V.
cholerae is a multi-factorial phenomenon.
http://jmm.sgmjournals.org
ACKNOWLEDGEMENTS
The authors are grateful to the Molecular Biology Research Center,
Baqiyatallah University of Medical Sciences, Tehran, Iran, for
support.
REFERENCES
Bakhshi, B., Pourshafie, M. R., Navabakbar, F. & Tavakoli, A. (2008).
Genomic organisation of the CTX element among toxigenic Vibrio
cholerae isolates. Clin Microbiol Infect 14, 562–568.
Davis, B. M. & Waldor, M. K. (2003). Filamentous phages linked to
virulence of Vibrio cholerae. Curr Opin Microbiol 6, 35–42.
DiRita, V. J., Neely, M., Taylor, R. K. & Bruss, P. M. (1996). Differential
expression of the ToxR regulon in classical and El Tor biotypes of
Vibrio cholerae is due to biotype-specific control over toxT expression.
Proc Natl Acad Sci U S A 93, 7991–7995.
Faruque, S. M., Asadulghani, B., Kamruzzaman, M., Nandi, R. K.,
Ghosh, A. N., Nair, G. B., Mekalanos, J. J. & Sack, D. A. (2002). RS1
element of Vibrio cholerae can propagate horizontally as a filamentous
phage exploiting the morphogenesis genes of CTXW. Infect Immun 70,
163–170.
Faruque, S. M., Kamruzzaman, M., Asadulghani, B., Sack, D. A.,
Mekalanos, J. J. & Nair, G. B. (2003). CTXW-independent production
of the RS1 satellite phage by Vibrio cholerae. Proc Natl Acad Sci U S A
100, 1280–1285.
González-Bonilla, C., Gutiérrez-Cogco, L., Moguel-Pech, L. &
Villanueva-Zamudio, A. (1994). [Evaluation of the ELISA method
for cholera toxin determination in Vibrio cholerae cultures]. Rev
Latinoam Microbiol 36, 273–276 (in Spanish).
Iwanaga, M. & Kuyyakanond, T. (1987). Large production of cholera
toxin by Vibrio cholerae O1 in yeast extract peptone water. J Clin
Microbiol 25, 2314–2316.
Kaper, J. B., Morris, J. G., Jr & Levine, M. M. (1995). Cholera. Clin
Microbiol Rev 8, 48–86.
Klose, K. E. (2001). Regulation of virulence in Vibrio cholerae. Int J
Med Microbiol 291, 81–88.
Livak, K. J. & Schmittgen, T. D. (2001). Analysis of relative gene
expression data using real-time quantitative PCR and the 22DDCt
method. Methods 25, 402–408.
Pourshafie, M. R., Grimont, F., Saifi, M. & Grimont, P. A. D. (2000).
Molecular epidemiological study of Vibrio cholerae isolates from
infected patients in Tehran, Iran. J Med Microbiol 49, 1085–1090.
Tsou, A. M., Cai, T., Liu, Z., Zhu, J. & Kulkarni, R. V. (2009). Regulatory
targets of quorum sensing in Vibrio cholerae: evidence for two distinct
HapR-binding motifs. Nucleic Acids Res 37, 2747–2756.
Withey, J. H. & DiRita, V. J. (2006). The toxbox: specific DNA sequence
requirements for activation of Vibrio cholerae virulence genes by
ToxT. Mol Microbiol 59, 1779–1789.
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