Browsing by Subject "ABC transporter"

Sort by: Order: Results:

Now showing items 1-4 of 4
  • Xiao, Kun (Helsingin yliopisto, 2015)
    Lactobacillus rhamnosus GG is a lactic acid bacterium that is widely used as probiotic products in the dairy industry. To gain insights into the genome stability of the L. rhamnosus GG in the human gastrointestinal tract and the possible adaption mechanism under different stresses, we first examined the genotype and phenotype of the L. rhamnosus GG grown over 1000 generations under various stresses, including bile salts, osmotic stress or shearing forces. Immunoblotting analysis of L. rhamnosus grown over 1000 generations showed that the production of mucus-binding pili by L. rhamnosus GG was the most impaired when exposed to bile salts. Complementary PCR screening of 13 highly variable chromosomal regions in GG confirmed that the pilus gene cluster had been lost when exposed to bile stress over time. In vitro bile-induced genomic changes observed in GG possibly reflects the genome plasticity and stability of GG in the human intestinal tract. Still, we showed that these changes only occurred after more than 100 generations, a period of time relatively long compared to the observed time of persistence and transit of GG in the intestinal tract. Although damages and stresses may be caused by bile salts, L. rhamnosus GG still has the ability to tolerate bile salts. The resistance mechanism is still unclear but, based on previous studies, we identified one ABC transporter encoded by the gene operon called tauABC that may be involved in bile resistance. In an effort to demonstrate its function, a tauB-null mutant derivative was generated and phenotypically characterized in terms of metabolic, signaling and functional properties. The data revealed that the tauB-null mutant significantly grow slower than L. rhamnosus GG wild-type strain in the presence of ox bile extracts. Additional screenings using various bile conjugates specifically revealed that two compounds of bile salts, i.e. taurodeoxycholic and taurochenodeoxycholic acid, may be processed by the TauABC transporter, contributing at least partially to the tolerance of GG to bile salts. Overall, we showed that bile salts constitute an important stress factor for GG that causes genomic alterations, although it has bile tolerance mechanisms to bile, such as the newly-characterized tauABC operon.
  • Sjöstedt, Noora; Salminen, Tiina A.; Kidron, Heidi (2019)
    Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda-derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,' 7'-dichlorofluorescein (CDCF), estradiol-17 beta-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 +/- 1.11 mu M. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.
  • Karlsson, Magnus; Durling, Mikael Brandstrom; Choi, Jaeyoung; Kosawang, Chatchai; Lackner, Gerald; Tzelepis, Georgios D.; Nygren, Kristiina; Dubey, Mukesh K.; Kamou, Nathalie; Levasseur, Anthony; Zapparata, Antonio; Wang, Jinhui; Amby, Daniel Buchvaldt; Jensen, Birgit; Sarrocco, Sabrina; Panteris, Emmanuel; Lagopodi, Anastasia L.; Poeggeler, Stefanie; Vannacci, Giovanni; Collinge, David B.; Hoffmeister, Dirk; Henrissat, Bernard; Lee, Yong-Hwan; Jensen, Dan Funck (2015)
  • Cai, Yida (Helsingin yliopisto, 2018)
    Bacteriocins are ribosomally synthesized antimicrobial proteins. They can be applied as biopreservatives in food processing for extending the shelf-life of food. Lactic acid bacterium Leuconostoc carnosum 4010 is Generally Recognized As Safe strain, which can be used as a protective culture in meat products. The strain 4010 produces three bacteriocins: leucocins A (LcnA), B (LebB) and C (LecC). For the secretion of bacteriocin out from the cell, bacteria usually use an ABC transporter, which is often dedicated to secrete only one bacteriocin. The leucocin operons in Ln. carnosum 4010 plasmids include genes for only one ABC transporter, namely LecXTS. The fact that Ln. carnosum 4010 produces three bacteriocins but only carries one bacteriocin transporter, raises a question, which leucocin(s) is/are translocated via LecXTS transporter. Therefore, the first aim of this study was to determine which bacteriocin(s) is/are secreted by LecXTS in Ln. carnosum 4010. Ln. carnosum 4010 carries at least two plasmids. Leucocin A gene lcnA is located on the plasmid pLC4010-2, and leucocin B and C with transporter genes (lebB, lecC, lecXTS) are located on the plasmid pLC4010-1. In a previous work, two plasmid cured derivatives of Ln. carnosum 4010 have been made: the plasmid-free strain PCS-10, and the strain PCS-11 carrying only pLC4010-2. Neither of the derivatives secrete bacteriocins. In this study, the idea was to construct five recombinant plasmids containing the pLC4010-1 replication gene repB and a gene for erythromycin resistance ErmR. They were ligated with different sets of leucocin and transporter genes (repB-lebB-lecXTS-lecC-ErmR, repB-lebB-lecXTS-ErmR, repB-lebB-ErmR, repB-lecC-ErmR, and a vector control with only repB-ErmR). The constructs were aimed to be introduced into the two Ln. carnosum 4010 mutant strains PCS-10 and PCS-11. However, after several attempts of electroporation, no colonies were obtained. To acquire a testing plasmid for optimization of transformation, the ligation mixture for the smallest plasmid repB-ErmR was electroporated into another strain, Lactococcus lactis N8. The plasmid repB-ErmR was successfully obtained from Lc. lactis N8. For improving the efficiency of transformation, the plasmid repB-ErmR was isolated from Lc. lactis N8, and the plasmid was used in optimization of electroporation. The copy number of the plasmid was shown to be very low, as only a little amount of plasmid could be isolated from large culture volume. Even with optimized electroporation method, the repB-ErmR could not be electroporated into Ln. carnosum 4010. This indicates that the larger constructions are nearly impossible to be transferred into the strain Ln. carnosum 4010. In conclusion, it was confirmed that the plasmid replication gene repB of Ln. carnosum 4010 is functional in Lc. lactis. Due to the low copy number of the plasmid repB-ErmR, the amount of plasmid was definitely a problem in electroporation. Therefore, for studying the efficiency of electroporation, the plasmid amount needs to be increased. Although the electroporation of repB-ErmR into Lc. lactis was successful, the results from Ln. carnosum electroporation after optimization indicate that the strain Ln. carnosum 4010 is difficult to be transformed.