Vibrio spp. detection using groEL gene-based loop-mediated isothermal amplification (LAMP) method and determination of FklB-GroEL interaction in Vibrio anguillarum

Abstract

Bacteria under the genus Vibrio are the natural inhabitants of marine and estuarine environment and are responsible for various diseases to aquatic animals and humans as well. Among more than 100 Vibrio spp., V. parahaemolyticus (human pathogenic) is the major cause of seafood-borne infection and V. anguillarum (major fish pathogenic) is the causal agent of classical vibriosis which is a fatal haemorrahagic septicemia; whereas, V. alginolyticus has been implicated for diseases to both aquatic animals and humans. Beside this, the bacteria are always encountering temperature, pH, salinity and other stresses from the marine and host environment and few proteins are serving them to survive against harsh situations. In this context, the development of rapid, reliable and sensitive molecular diagnostic method, such as loop-mediated isothermal amplification (LAMP) assay, and understanding of the basic physiological phenomenon inside any bacteria, are of great importance. . Hence, in this present study, standard LAMP assay has been developed for detecting V. parahaemolyticus using groEL gene (encodes molecular chaperon protein), then singlex and duplex LAMP have been developed for detecting V. anguillarum and V. alginolyticus separately and simultaneously using groEL gene and the fklB gene (encodes FklB protein which accelerate protein folding) and also observed the interaction of GroEL protein with FklB protein (FKBP-type 22 KDa peptidyl-prolyl cis/trans isomerase) in V. anguillarum. The groEL gene based LAMP assay for V. parahaemolyticus was optimized and conducted at 63°C for 40 min using Bst DNA polymerase large fragment and found species-specific, highly sensitive and efficient both for pure culture and environmental samples. Moreover, the LAMP assay was 100 times and 1000 times more sensitive than the conventional polymerase chain reaction (PCR) method in case of pure culture and environmental samples, respectively. Again, the groEL LAMP for V. anguillarum, fklB LAMP for V. alginolyticus and groEL-fklB based duplex LAMP were also found cost-effective, rapid, specific and highly sensitive than conventional PCR assay. The reactions were conducted incorporating Bst 2.0 DNA polymerase at 65°C for 30 min, 63°C for 30 min and 63°C for 40 min in case of groEL LAMP, fklB LAMP and duplex LAMP assays, respectively. The amplification results were interpreted on 2% agarose gel and found specific and consistent band patterns for each type of LAMP assay. As a detection method, Xho-I restriction enzyme digestion was performed and revealed reliable results. The limit of detection (LoDs) of the assays for DNA template from pure culture and artificially contaminated seawater were 10 fg and 14 fg, 12.5 fg and 17 fg, 50 fg and 70 fg per reaction utilizing groEL LAMP, fklB LAMP and duplex LAMP assay, respectively, which were found much higher than those of conventional polymerase chain reaction (PCR). Hence, these LAMP assays could be applied for laboratory and field diagnosis. Finally, the interaction of FklB (member of FK506 binding protein family) with GroEL (member of molecular chaperonin GroE system) was observed through bacterial two hybrid system where pBT-fklB (bait plasmid containing fklB gene) and pTRG-groEL (target plasmid containing groEL gene) were transformed into Bacterio-Match II validation reporter-competent cells and transformants were screened on selective screening media containing 3-amino-1,2,4-triazole, a HIS3 inhibitor and positives are verified by using the aadA gene (grown on dual selective screening medium), which confers streptomycin resistance, as a secondary reporter. The results obtained from two hybrid system were further analyzed using various computational methods. The 3D structures of FklB and GroEL were obtained through M4T and RaptorX online software programs and docking were done using ZDOCK server, ClusPro 2.0 and PRISM. The interacting complex protein were further analyzed by PDBsum generate and Rosetta online programs. A total of 18 hydrogen bonds (H-bonds) were observed in interacting protein complex which indicate the presence of interaction between FklB and GroEL proteins. Therefore, it could be concluded that 22 Kda FklB protein has interaction with GroEL protein and helps in folding and stabilization via its peptidyl-prolyl cis/trans isomerization activity. As future perspective of this protein-protein interaction study, site-directed mutagenesis or domain-based analyses could be done for characterizing the interaction.