* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Quantitative expression of cholera toxin mRNA in Vibrio cholerae
Gene therapy of the human retina wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Short interspersed nuclear elements (SINEs) wikipedia , lookup
RNA silencing wikipedia , lookup
Non-coding RNA wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Public health genomics wikipedia , lookup
Minimal genome wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Primary transcript wikipedia , lookup
Genome (book) wikipedia , lookup
Microevolution wikipedia , lookup
Ridge (biology) wikipedia , lookup
Genome evolution wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Designer baby wikipedia , lookup
Epitranscriptome wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Genomic imprinting wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Mir-92 microRNA precursor family wikipedia , lookup
Gene expression programming wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Pathogenomics wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 05:36:03 Printed in Great Britain 1071 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 05:36:03 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 05:36:03 1073