Abstract
The AcrAB-TolC multidrug efflux pump confers resistance to Escherichia coli against many antibiotics and toxic compounds. The TolC protein is an outer membrane factor that participates in the formation of type I secretion systems. The genome of Vibrio vulnificus encodes two proteins homologous to the E. coli TolC, designated TolCV1 and TolCV2. Here, we show that both TolCV1 and TolCV2 partially complement the E. coli TolC function and physically interact with the membrane fusion protein AcrA, a component of the E. coli AcrAB-TolC efflux pump. Using site-directed mutational analyses and an in vivo cross-linking assay, we demonstrated that the α-barrel tip region of TolC homologs plays a critical role in the formation of functional AcrAB-TolC efflux pumps. Our findings suggest the adapter bridging model as a general assembly mechanism for tripartite drug efflux pumps in Gram-negative bacteria.
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Akama, H., Matsuura, T., Kashiwagi, S., Yoneyama, H., Narita, S., Tsukihara, T., Nakagawa, A., and Nakae, T. 2004. Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa. J. Biol. Chem. 279, 25939–25942.
Bavro, V.N., Pietras, Z., Furnham, N., Perez-Cano, L., Fernandez-Recio, J., Pei, X.Y., Misra, R., and Luisi, B. 2008. Assembly and channel opening in a bacterial drug efflux machine. Mol. Cell. 30, 114–121.
Elkins, C.A. and Nikaido, H. 2003. Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efflux pump. J. Bacteriol. 185, 5349–5356.
Federici, L., Du, D., Walas, F., Matsumura, H., Fernandez-Recio, J., McKeegan, K.S., Borges-Walmsley, M.I., Luisi, B.F., and Walmsley, A.R. 2005. The crystal structure of the outer membrane protein VceC from the bacterial pathogen Vibrio cholerae at 1.8 Å resolution. J. Biol. Chem. 280, 15307–15314.
Fernandez-Recio, J., Walas, F., Federici, L., Venkatesh, Pratap, J., Bavro, V.N., Miguel, R.N., Mizuguchi, K., and Luisi, B. 2004. A model of a transmembrane drug-efflux pump from Gram-negative bacteria. FEBS Lett. 578, 5–9.
Ge, Q., Yamada, Y., and Zgurskaya, H. 2009. The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efflux pump AcrAB-TolC. J. Bacteriol. 191, 4365–4371.
Gerken, H. and Misra, R. 2004. Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli. Mol. Microbiol. 54, 620–631.
Hobbs, E.C., Yin, X., Paul, B.J., Astarita, J.L., and Storz, G. 2012. Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance. Proc. Natl. Acad. Sci. USA 109, 16696–16701.
Husain, F., Humbard, M., and Misra, R. 2004. Interaction between the TolC and AcrA proteins of a multidrug efflux system of Escherichia coli. J. Bacteriol. 186, 8533–8536.
Kim, H.M., Xu, Y., Lee, M., Piao, S., Sim, S.H., Ha, N.C., and Lee, K. 2010. Functional relationships between the AcrA hairpin tip region and the TolC aperture tip region for the formation of the bacterial tripartite efflux pump AcrAB-TolC. J. Bacteriol. 192, 4498–4503.
Koronakis, V., Li, J., Koronakis, E., and Stauffer, K. 1997. Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two-dimensional crystals. Mol. Microbiol. 23, 617–626.
Koronakis, V., Sharff, A., Koronakis, E., Luisi, B., and Hughes, C. 2000. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405, 914–919.
Koronakis, V., Eswaran, J., and Hughes, C. 2004. Structure and function of TolC: the bacterial exit duct for proteins and drugs. Annu. Rev. Biochem. 73, 467–489.
Krishnamoorthy, G., Tikhonova, E.B., Dhamdhere, G., and Zgurskaya, H.I. 2013. On the role of TolC in multidrug efflux: the function and assembly of AcrAB-TolC tolerate significant depletion of intracellular TolC protein. Mol. Microbiol. 87, 982–997.
Lee, M., Jun, S.Y., Yoon, B.Y., Song, S., Lee, K., and Ha, N.C. 2012. Membrane fusion proteins of type I secretion system and tripartite efflux pumps share a binding motif for TolC in Gram-negative bacteria. PLoS One 7, e40460.
Lee, M., Kim, H.L., Song, S., Joo, M., Lee, S., Kim, D., Hahn, Y., Ha, N.C., and Lee, K. 2013. The α-barrel tip region of Escherichia coli TolC homologs of Vibrio vulnificus interacts with the MacA protein to form the functional macrolide-specific efflux pump MacAB-TolC. J. Microbiol. 51, 154–159.
Lewis, K. 2000. Translocases: a bacterial tunnel for drugs and proteins. Curr. Biol. 10, R678–681.
Lobedanz, S., Bokma, E., Symmons, M.F., Koronakis, E., Hughes, C., and Koronakis, V. 2007. A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps. Proc. Natl. Acad. Sci. USA 104, 4612–4617.
Meacock, P.A. and Cohen, S.N. 1980. Partitioning of bacterial plasmids during cell division: a cis-acting locus that accomplishes stable plasmid inheritance. Cell 20, 529–542.
Murakami, S., Nakashima, R., Yamashita, E., and Yamaguchi, A. 2002. Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419, 587–593.
Murakami, S., Nakashima, R., Yamashita, E., Matsumoto, T., and Yamaguchi, A. 2006. Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443, 173–179.
Murakami, S. 2008. Multidrug efflux transporter, AcrB-the pumping mechanism. Curr. Opin. Struct. Biol. 18, 459–465.
Nikaido, H. and Pages, J.M. 2012. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiol. Rev. 36, 340–363.
Park, J.H., Cho, Y.J., Chun, J., Seok, Y.J., Lee, J.K., Kim, K.S., Lee, K.H., Park, S.J., and Choi, S.H. 2011. Complete genome sequence of Vibrio vulnificus MO6-24/O. J. Bacteriol. 193, 2062–2063.
Paulsen, I.T., Park, J.H., Choi, P.S., and Saier, M.H. Jr. 1997. A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from gram-negative bacteria. FEMS Microbiol. Lett. 156, 1–8.
Pei, X.Y., Hinchliffe, P., Symmons, M.F., Koronakis, E., Benz, R., Hughes, C., and Koronakis, V. 2011. Structures of sequential open states in a symmetrical opening transition of the TolC exit duct. Proc. Natl. Acad. Sci. USA 108, 2112–2117.
Piao, S., Xu, Y., and Ha, N.C. 2008. Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans. Acta. Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 64, 391–393.
Seeger, M.A., Schiefner, A., Eicher, T., Verrey, F., Diederichs, K., and Pos, K.M. 2006. Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313, 1295–1298.
Sennhauser, G., Amstutz, P., Briand, C., Storchenegger, O., and Grutter, M.G. 2007. Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol. 5, e7.
Thanabalu, T., Koronakis, E., Hughes, C., and Koronakis, V. 1998. Substrate-induced assembly of a contiguous channel for protein export from E. coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore. EMBO J. 17, 6487–6496.
Tikhonova, E.B., Yamada, Y., and Zgurskaya, H.I. 2011. Sequential mechanism of assembly of multidrug efflux pump AcrAB-TolC. Chem. Biol. 18, 454–463.
Wang, B., Weng, J., Fan, K., and Wang, W. 2012. Interdomain flexibility and pH-induced conformational changes of AcrA revealed by molecular dynamics simulations. J. Phys. Chem. B. 116, 3411–3420.
Xu, Y., Sim, S.H., Song, S., Piao, S., Kim, H.M., Jin, X.L., Lee, K., and Ha, N.C. 2010. The tip region of the MacA alpha-hairpin is important for the binding to TolC to the Escherichia coli MacAB-TolC pump. Biochem. Biophys. Res. Commun. 394, 962–965.
Xu, Y., Song, S., Moeller, A., Kim, N., Piao, S., Sim, S.H., Kang, M., Yu, W., Cho, H.S., Chang, I., Lee, K., and Ha, N.C. 2011a. Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-TolC. J. Biol. Chem. 286, 13541–13549.
Xu, Y., Lee, M., Moeller, A., Song, S., Yoon, B.Y., Kim, H.M., Jun, S.Y., Lee, K., and Ha, N.C. 2011b. Funnel-like hexameric assembly of the periplasmic adapter protein in the tripartite multidrug efflux pump in Gram-negative bacteria. J. Biol. Chem. 286, 17910–17920.
Xu, Y., Moeller, A., Jun, S.Y., Lee, M., Yoon, B.Y., Kim, J.S., Lee, K., and Ha, N.C. 2012. Assembly and channel opening of outer membrane protein in tripartite drug efflux pumps of Gram-negative bacteria. J. Biol. Chem. 287, 11740–11750.
Yeom, J.H. and Lee, K. 2006. RraA rescues Escherichia coli cells over-producing RNase E from growth arrest by modulating the ribonucleolytic activity. Biochem. Biophys. Res. Commun. 345, 1372–1376.
Yu, E.W., McDermott, G., Zgurskaya, H.I., Nikaido, H., and Koshland, D.E. Jr. 2003. Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump. Science 300, 976–980.
Yum, S., Xu, Y., Piao, S., Sim, S.H., Kim, H.M., Jo, W.S., Kim, K.J., Kweon, H.S., Jeong, M.H., Jeon, H., and et al. 2009. Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump. J. Mol. Biol. 387, 1286–1297.
Zgurskaya, H.I. and Nikaido, H. 1999. Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli. Proc. Natl. Acad. Sci. USA 96, 7190–7195.
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Lee, S., Song, S., Lee, M. et al. Interaction between the α-barrel tip of Vibrio vulnificus TolC homologs and AcrA implies the adapter bridging model. J Microbiol. 52, 148–153 (2014). https://doi.org/10.1007/s12275-014-3578-2
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DOI: https://doi.org/10.1007/s12275-014-3578-2