Investigation the Expression of Virulence genes (ctxA) of Clinical Strain Vibrio cholerae in BHI Broth Medium and the Caco-2 Cell Line

Investigation the Expression of Virulence genes (ctxA) of Clinical Strain Vibrio cholerae in BHI Broth Medium and the Caco-2 Cell Line
Mohammad Reza Kheradmand,¹ Sareh Bagheri-Josheghani,² Shahin Najar-Peerayeh,³ Bita Bakhshi,^4,*
1. Tarbiat Modares University Tehran, Iran
2. Tarbiat Modares University Tehran, Iran
3. Tarbiat Modares University Tehran, Iran
4. Tarbiat Modares University Tehran, Iran
Introduction: Cholera is an infection of the small intestines caused by the bacterium Vibrio cholerae. It is a significant cause of an acute intestinal infection characterized via watery diarrhea that may cause dehydration and death, contaminating thousands of people each year worldwide and especially in developing countries. The major virulence factor produced by V. cholerae during infection is the cholera toxin. We examined the ctxA gene expression in medical eltor biotype and standard classic biotype ATCC14035 of V. cholerae in BHI broth medium and the Caco-2 cell line in the present study.
Methods: This study investigated the mRNA concentration of ctxA genes in four qualitative samples, including the ATCC14035 classic biotype and medical strain eltor biotype in two conditions: BHI broth medium and Caco-2 cell line interaction. The untreated population of Caco-2 cells used as control. The best MOI (Multiplicity of Infection) of two strain V. cholerae assessed by MTT assay, then adhesion and invasion test capacity determined to calculate the suitable population of bacteria. We analyze the number of adhesive bacteria in each biotype. Then we added such a number of bacterial populations in BHI broth and Caco-2 cell to having similar conditions based on this result. Total mRNA extraction and cDNA synthesis was performed with a specific kit. MRNA concentration calculated by quantitative Real-time PCR method on the using SYBR Green. Bacterial 16S rRNA used gene as internal control. All tests performed three-time repeat to reach the logical outcome.
Results: The best results with Caco-2 cell interaction were obtained at the MOI of 10:1.The adhesion rate of ATCC14035 classic biotype and eltor biotype to Caco-2 cells was 53% and 44%. Transcription of ctxA gene in eltor biotype and classic biotype interaction with Caco-2 cells was 10.4 and 10.5, respectively, that show a tenfold increase versus untreated Caco-2 cell (1.0) as control. In contrast, transcription of this gene in these two biotypes (eltor and classic) was 6.06 and 1.3 in the BHI broth medium. CtxA gene had different transcription in different conditions; we also observed the increase of transcription in interaction with Caco2 cell in both the ATCC14035 classic biotype and medical strain eltor biotype.
Conclusion: In conclusion, our study proposes that the expression of virulence genes (ctxA) was different in the BHI broth medium and the Caco-2 cell line interaction, also observed the increase of transcription in interaction with Caco-2 cell. This study may be valuable for understanding the relation of adenocarcinoma colorectal with the severity of cholera. More reviews on factors of these cells involved in different gene expression of bacteria and hence the severity of disease are needed, especially in cancerous eukaryotic cells.
Keywords: BHI broth, Caco2 cell line, CtxA, Real-time PCR, Vibrio cholerae