Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2.

Animals; Bacterial Proteins/genetics/metabolism; Brucella abortus/pathogenicity; Cell Line; Epithelial Cells/microbiology; Gene Deletion; Host-Pathogen Interactions; Humans; Macrophages/microbiology; Mice; Phagosomes/metabolism/microbiology; Protein Binding; Protein Interaction Mapping; Two-Hybrid System Techniques; Virulence; Virulence Factors/genetics/metabolism; rab2 GTP-Binding Protein/metabolism

Abstract :

[en] Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-beta-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a DeltavirB mutant. In HeLa cells, a DeltaricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortusDeltaricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus.

Disciplines :

Biochemistry, biophysics & molecular biology

Author, co-author :

de Barsy, Marie

Jamet, Alexandre

Filopon, Didier

Nicolas, Cecile

Laloux, Geraldine

Rual, Jean-Francois

Muller, Alexandre

Twizere, Jean-Claude ; Université de Liège - ULiège > Chimie et bio-industries > Biologie cell. et moléc.

Nkengfac, Bernard

Vandenhaute, Jean

More authors (5 more)

Language :

English

Title :

Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2.

Publication date :

2011

Journal title :

Cellular Microbiology

ISSN :

1462-5814

eISSN :

1462-5822

Publisher :

Blackwell, Oxford, United Kingdom

Volume :

Issue :

Pages :

1044-58

Peer reviewed :

Peer Reviewed verified by ORBi

Additional URL :

http://onlinelibrary.wiley.com/doi/10.1111/j.1462-5822.2011.01601.x/pdf

Commentary :

Available on ORBi :

since 05 March 2014

Statistics

Number of views

207 (0 by ULiège)

Number of downloads

221 (0 by ULiège)

More statistics

Scopus citations^®

106

Scopus citations^®
without self-citations

OpenCitations

101

Bibliography

Anderson, T.D., and Cheville, N.F. (1986) Ultrastructural morphometric analysis of Brucella abortus-infected trophoblasts in experimental placentitis. Bacterial replication occurs in rough endoplasmic reticulum. Am J Pathol 124: 226-237.
Antoine, R., and Locht, C. (1992) Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol Microbiol 6: 1785-1799.
Bohlin, J., Snipen, L., Cloeckaert, A., Lagesen, K., Ussery, D., Kristoffersen, A.B., and Godfroid, J. (2010) Genomic comparisons of Brucella spp. and closely related bacteria using base compositional and proteome based methods. BMC Evol Biol 10: 249.
Brandner, C.J., Maier, R.H., Henderson, D.S., Hintner, H., Bauer, J.W., and Onder, K. (2008) The ORFeome of Staphylococcus aureus v 1.1. BMC Genomics 9: 321.
Braun, P., Tasan, M., Dreze, M., Barrios-Rodiles, M., Lemmens, I., Yu, H., etal. (2009) An experimentally derived confidence score for binary protein-protein interactions. Nat Methods 6: 91-97.
Brumell, J.H., and Scidmore, M.A. (2007) Manipulation of rab GTPase function by intracellular bacterial pathogens. Microbiol Mol Biol Rev 71: 636-652.
Brymora, A., Valova, V.A., and Robinson, P.J. (2004) Protein-protein interactions identified by pull-down experiments and mass spectrometry. Curr Protoc Cell Biol Chapter 17, Unit 17.5.
Caldwell, R.B., and Lattemann, C.T. (2004) Simple and reliable method to precipitate proteins from bacterial culture supernatant. Appl Environ Microbiol 70: 610-612.
Celli, J., and Gorvel, J.P. (2004) Organelle robbery: Brucella interactions with the endoplasmic reticulum. Curr Opin Microbiol 7: 93-97.
Celli, J., de Chastellier, C., Franchini, D.M., Pizarro-Cerda, J., Moreno, E., and Gorvel, J.P. (2003) Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J Exp Med 198: 545-556.
Celli, J., Salcedo, S.P., and Gorvel, J.P. (2005) Brucella coopts the small GTPase Sar1 for intracellular replication. Proc Natl Acad Sci USA 102: 1673-1678.
Cloeckaert, A., Zygmunt, M.S., Dubray, G., and Limet, J.N. (1993) Characterization of O-polysaccharide specific monoclonal antibodies derived from mice infected with the rough Brucella melitensis strain B115. J Gen Microbiol 139: 1551-1556.
Colicelli, J. (2004) Human RAS superfamily proteins and related GTPases. Sci STKE 2004: RE13.
Comerci, D.J., Martinez-Lorenzo, M.J., Sieira, R., Gorvel, J.P., and Ugalde, R.A. (2001) Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole. Cell Microbiol 3: 159-168.
Delrue, R.M., Martinez-Lorenzo, M., Lestrate, P., Danese, I., Bielarz, V., Mertens, P., etal. (2001) Identification of Brucella spp. genes involved in intracellular trafficking. Cell Microbiol 3: 487-497.
Dricot, A., Rual, J.F., Lamesch, P., Bertin, N., Dupuy, D., Hao, T., etal. (2004) Generation of the Brucella melitensis ORFeome version 1.1. Genome Res 14: 2201-2206.
Ensminger, A.W., and Isberg, R.R. (2009) Legionella pneumophila Dot/Icm translocated substrates: a sum of parts. Curr Opin Microbiol 12: 67-73.
Fretin, D., Fauconnier, A., Kohler, S., Halling, S., Leonard, S., Nijskens, C., etal. (2005) The sheathed flagellum of Brucella melitensis is involved in persistence in a murine model of infection. Cell Microbiol 7: 687-698.
Fugier, E., Salcedo, S.P., de Chastellier, C., Pophillat, M., Muller, A., Arce-Gorvel, V., etal. (2009) The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication. PLoS Pathog 5: e1000487.
Garin, J., Diez, R., Kieffer, S., Dermine, J.F., Duclos, S., Gagnon, E., etal. (2001) The phagosome proteome: insight into phagosome functions. J Cell Biol 152: 165-180.
Hardt, W.D., Chen, L.M., Schuebel, K.E., Bustelo, X.R., and Galan, J.E. (1998) S. yphimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell 93: 815-826.
Hemsath, L., and Ahmadian, M.R. (2005) Fluorescence approaches for monitoring interactions of Rho GTPases with nucleotides, regulators, and effectors. Methods 37: 173-182.
de Jong, M.F., Sun, Y.H., den Hartigh, A.B., van Dijl, J.M., and Tsolis, R.M. (2008) Identification of VceA and VceC, two members of the VjbR regulon that are translocated into macrophages by the Brucella type IV secretion system. Mol Microbiol 70: 1378-1396.
Kovach, M.E., Phillips, R.W., Elzer, P.H., Roop, R.M., 2nd, and Peterson, K.M. (1994) pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16: 800-802.
Labaer, J., Qiu, Q., Anumanthan, A., Mar, W., Zuo, D., Murthy, T.V., etal. (2004) The Pseudomonas aeruginosa PA01 gene collection. Genome Res 14: 2190-2200.
Lamesch, P., Li, N., Milstein, S., Fan, C., Hao, T., Szabo, G., etal. (2007) hORFeome v3.1: a resource of human open reading frames representing over 10, 000 human genes. Genomics 89: 307-315.
Lin, M., den Dulk-Ras, A., Hooykaas, P.J., and Rikihisa, Y. (2007) Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection. Cell Microbiol 9: 2644-2657.
Machner, M.P., and Isberg, R.R. (2006) Targeting of host Rab GTPase function by the intravacuolar pathogen Legionella pneumophila. Dev Cell 11: 47-56.
Mangahas, P.M., Yu, X., Miller, K.G., and Zhou, Z. (2008) The small GTPase Rab2 functions in the removal of apoptotic cells in Caenorhabditis elegans. J Cell Biol 180: 357-373.
Mignolet, J., Van der Henst, C., Nicolas, C., Deghelt, M., Dotreppe, D., Letesson, J.J., and De Bolle, X. (2010) PdhS, an old-pole-localized histidine kinase, recruits the fumarase FumC in Brucella abortus. J Bacteriol 192: 3235-3239.
Moreno, E., and Moriyon, I. (2006) The Genus Brucella. Prokaryotes 5: 315-456.
Murata, T., Delprato, A., Ingmundson, A., Toomre, D.K., Lambright, D.G., and Roy, C.R. (2006) The Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange factor. Nat Cell Biol 8: 971-977.
Nijskens, C., Copin, R., De Bolle, X., and Letesson, J.J. (2008) Intracellular rescuing of a B. elitensis 16M virB mutant by co-infection with a wild type strain. Microb Pathog 45: 134-141.
Ninio, S., and Roy, C.R. (2007) Effector proteins translocated by Legionella pneumophila: strength in numbers. Trends Microbiol 15: 372-380.
O'Callaghan, D., Cazevieille, C., Allardet-Servent, A., Boschiroli, M.L., Bourg, G., Foulongne, V., etal. (1999) A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis. Mol Microbiol 33: 1210-1220.
Ostrowski, M., Carmo, N.B., Krumeich, S., Fanget, I., Raposo, G., Savina, A., etal. (2010) Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol 12: 19-30. sup pp. 11-13.
Pizarro-Cerda, J., Meresse, S., Parton, R.G., van der Goot, G., Sola-Landa, A., Lopez-Goni, I., etal. (1998) Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect Immun 66: 5711-5724.
Qin, Q.M., Pei, J., Ancona, V., Shaw, B.D., Ficht, T.A., and de Figueiredo, P. (2008) RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication. PLoS Pathog 4: e1000110.
Quandt, J., and Hynes, M.F. (1993) Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene 127: 15-21.
Raetz, C.R., and Roderick, S.L. (1995) A left-handed parallel beta helix in the structure of UDP-N-acetylglucosamine acyltransferase. Science 270: 997-1000.
Raffatellu, M., Sun, Y.H., Wilson, R.P., Tran, Q.T., Chessa, D., Andrews-Polymenis, H.L., etal. (2005) Host restriction of Salmonella enterica serotype Typhi is not caused by functional alteration of SipA, SopB, or SopD. Infect Immun 73: 7817-7826.
Rual, J.F., Hirozane-Kishikawa, T., Hao, T., Bertin, N., Li, S., Dricot, A., etal. (2004) Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res 14: 2128-2135.
Rual, J.F., Venkatesan, K., Hao, T., Hirozane-Kishikawa, T., Dricot, A., Li, N., etal. (2005) Towards a proteome-scale map of the human protein-protein interaction network. Nature 437: 1173-1178.
Sambrook, J., and Russel, D.W. (2001) Molecular Cloning, A Laboratory Manual Cold Spring Harbour. New York: Cold Spring Harbour Laboratory Press, pp. 2, 344.
Seabra, M.C., Mules, E.H., and Hume, A.N. (2002) Rab GTPases, intracellular traffic and disease. Trends Mol Med 8: 23-30.
Sherman, F. (1991) Getting started with yeast. Methods Enzymol 194: 3-21.
Shohdy, N., Efe, J.A., Emr, S.D., and Shuman, H.A. (2005) Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking. Proc Natl Acad Sci USA 102: 4866-4871.
Simon, R., Priefer, U., and Pühler, A. (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat Biotechnol 1: 784-791.
Smith, A.C., Heo, W.D., Braun, V., Jiang, X., Macrae, C., Casanova, J.E., etal. (2007) A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium. J Cell Biol 176: 263-268.
Stuart, L.M., Boulais, J., Charriere, G.M., Hennessy, E.J., Brunet, S., Jutras, I., etal. (2007) A systems biology analysis of the Drosophila phagosome. Nature 445: 95-101.
Takai, Y., Sasaki, T., and Matozaki, T. (2001) Small GTP-binding proteins. Physiol Rev 81: 153-208.
Thery, C., Duban, L., Segura, E., Veron, P., Lantz, O., and Amigorena, S. (2002) Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes. Nat Immunol 3: 1156-1162.
Tisdale, E.J., Bourne, J.R., Khosravi-Far, R., Der, C.J., and Balch, W.E. (1992) GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. J Cell Biol 119: 749-761.
Tisdale, E.J., Azizi, F., and Artalejo, C.R. (2009) Rab2 utilizes glyceraldehyde-3-phosphate dehydrogenase and protein kinase C(iota) to associate with microtubules and to recruit dynein. J Biol Chem 284: 5876-5884.
Vergunst, A.C., van Lier, M.C., den Dulk-Ras, A., Stuve, T.A., Ouwehand, A., and Hooykaas, P.J. (2005) Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium. Proc Natl Acad Sci USA 102: 832-837.
Vidal, M., Brachmann, R.K., Fattaey, A., Harlow, E., and Boeke, J.D. (1996) Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proc Natl Acad Sci USA 93: 10315-10320.
Vidalain, P.O., Boxem, M., Ge, H., Li, S., and Vidal, M. (2004) Increasing specificity in high-throughput yeast two-hybrid experiments. Methods 32: 363-370.
Walhout, A.J., and Vidal, M. (2001) High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 24: 297-306.
Walhout, A.J., Temple, G.F., Brasch, M.A., Hartley, J.L., Lorson, M.A., van den Heuvel, S., and Vidal, M. (2000) Gateway recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328: 575-592.
Wang, Y., Cui, T., Zhang, C., Yang, M., Huang, Y., Li, W., etal. (2010) Global protein-protein interaction network in the human pathogen Mycobacterium tuberculosis H37Rv. J Proteome Res 9: 6665-6677.
Young, G.M., Schmiel, D.H., and Miller, V.L. (1999) A new pathway for the secretion of virulence factors by bacteria: the flagellar export apparatus functions as a protein-secretion system. Proc Natl Acad Sci USA 96: 6456-6461.