Abstract

Background: Outer membrane vesicles (OMVs) are proteoliposomes release naturally from Gram negative bacteria (GNB), no compromise membrane integrity or viability of bacteria. These nanospheres has a specific cargo defined since their biogenesis. Where its functionality focusses principality at intercellular communication and molecules transport. For default OMVs are constituted principally by outer membrane and periplasm components, however, sometimes it possible find other inner molecules, like virulence factors (e.g. HlyE). An important case is the discovering bacterial DNA into OMVs, who associated to horizontal gene transference between bacteria, these find out suggest that OMVs are capable transport DNA inside and endure onto medium. We hypothesize that an exogen DNA can be incorporated into OMVs and kept inside. Methods: Each strain was grown in liquid culture using Luria Bertani Lennox broth at 37ºC shaking, until reach approximately to OD600 1.1. Culture was centrifugated 15 min at 5400 x g, then supernatant filtrated at Nalgene® rapid-flow™ (0.45 μm), ultrafiltered with membrane disks 100 kDa (Amicon Bioseparations), ultracentrifugated 3 h at 150000 g at 4ºC. Finally, supernatant was discarted, pellet was resuspended at 1 ml DPBS, obtained raw OMVs. Strains of S. Typhi with mutation in genes involved in biogenesis of OMVs were transformed with empty pTOPO 2.1, to incorporate pDNA into OMVs. All raw OMVs were treated with DNase I for eliminate possible DNA present at their surface and the same time confirm protective capacity to OMVs over luminal DNA. OMV was lysed, and extracts were running at agarose 2% to 100V during 45 min. Results: Of all OMVs generated by different mutants of S. Typhi, only ΔdegS had DNA cargo. All of strains of S. Typhi it be transformed with pTOPO 2.1, generated OMVs with pDNA cargo, even ΔdegS founded both DNA (pDNA and bacterial DNA), same result it obtains using simple technique shaking between OMVs and pTOPO 2.1. Conclusion: Our study indicates that OMVs generated by mutants of S. Typhi STH2370 can be loaded with exogen DNA through two ways. Suggesting that OMVs would be a DNA intentional vehicle by carrying DNA with biotechnology applications.