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

Investigation the Expression of Virulence Genes (hlyA) 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: Haemolysin (HlyA) is another critical toxin secreted by most Vibrio cholerae O1 and non-O1 strains. HlyA induces enterotoxicity, cytotoxicity, lysis of erythrocytes from several species, and cellular vacuolation. In the present study, we examined the expression of virulence genes (hlyA) of V. cholerae classic biotype ATCC14035 and medical eltor biotype in BHI broth medium and the Caco-2 cell line.
Methods: The expression of hlyA gene of ATCC14035 classic biotype and medical strain eltor biotype investigated everyone in two conditions, including Caco-2 cell interaction and BHI broth medium. The untreated population of Caco-2 cells used as control. The MTT test done to obtain the best MOI then adhesion and invasion test capacity were done 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. The RNA extraction and cDNA synthesis done with the specific kit. HlyA gene primers are confirmed by the PCR method. MRNA concentration of hlyA gene assessed using a Real-time PCR method on the using SYBR Green. Bacterial 16S rRNA gene used as the internal control. All tests done three-time repeat to reach the logical result and decrease the effect of errors due to unwanted mistakes.
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%. Calculation of fold change in hlyA gene transcription shows an increase of about 14.9 and 8.39 in classic biotype and eltor biotype, respectively, with Caco-2 cell intraction versus untreated Caco-2 cell (1.0) as control. In contrast, this result in BHI broth medium was 2.11 and 2.02 in classic and eltor biotypes. HlyA gene had different transcription in different conditions; we also observed the increase of transcription in interaction with Caco-2 cell in both the ATCC14035 classic biotype and medical strain eltor biotype.
Conclusion: In conclusion, results showed that the expression of virulence genes (hlyA) was different in Caco-2 cell line interaction and the BHI broth medium. We also observed the increase of transcription in interaction with Caco-2 cells. This study may be valuable for understanding the relation of adenocarcinoma colorectal with the severity of cholera. More analyses 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, HlyA, Real Time PCR, Vibrio cholerae