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• W2069809595 abstract "Legionella pneumophila proliferates within alveolar macrophages as a central property of Legionnaires' disease. Intracellular growth involves formation of a replicative phagosome, which requires the bacterial Dot/Icm system, a multiprotein secretion apparatus that translocates proteins from the bacterium across the macrophage plasma membrane. Two components of this system, IcmR and IcmQ, are proposed to exhibit a chaperone/substrate relationship similar to that observed in other protein translocation systems. We report here that IcmQ inserts into lipid membranes and forms pores that allow the efflux of the dye calcein but not Dextran 3000. Both membrane insertion and pore formation were inhibited by IcmR. Trypsin digestion mapping demonstrated that IcmQ is subdivided into two functional domains. The N-terminal region of IcmQ was necessary and sufficient for insertion into lipid membranes and calcein efflux. The C-terminal domain was necessary for efficient association of the protein with lipid bilayers. IcmR was found to bind to the N-terminal portion of the protein thus providing a mechanism for its ability to inhibit IcmQ pore-forming activity. Localization of IcmQ on the surface of the L. pneumophila shortly after infection as well as its pore-forming capacities suggest a role for IcmQ in forming a channel that leads translocated effectors out of the bacterium. Legionella pneumophila proliferates within alveolar macrophages as a central property of Legionnaires' disease. Intracellular growth involves formation of a replicative phagosome, which requires the bacterial Dot/Icm system, a multiprotein secretion apparatus that translocates proteins from the bacterium across the macrophage plasma membrane. Two components of this system, IcmR and IcmQ, are proposed to exhibit a chaperone/substrate relationship similar to that observed in other protein translocation systems. We report here that IcmQ inserts into lipid membranes and forms pores that allow the efflux of the dye calcein but not Dextran 3000. Both membrane insertion and pore formation were inhibited by IcmR. Trypsin digestion mapping demonstrated that IcmQ is subdivided into two functional domains. The N-terminal region of IcmQ was necessary and sufficient for insertion into lipid membranes and calcein efflux. The C-terminal domain was necessary for efficient association of the protein with lipid bilayers. IcmR was found to bind to the N-terminal portion of the protein thus providing a mechanism for its ability to inhibit IcmQ pore-forming activity. Localization of IcmQ on the surface of the L. pneumophila shortly after infection as well as its pore-forming capacities suggest a role for IcmQ in forming a channel that leads translocated effectors out of the bacterium. Legionella pneumophila is a Gram-negative bacterium that multiplies inside amoebae found in fresh water ecosystems (1Fields B.S. Trends Microbiol. 1996; 4: 286-290Abstract Full Text PDF PubMed Scopus (444) Google Scholar). It is believed that inhalation of aerosols containing either free-living L. pneumophila or amoeba laden with the bacteria leads to colonization of the lungs. Phagocytosed L. pneumophila then grow within alveolar macrophages in a fashion similar to that observed with amoeba (2Glavin F.L. Winn Jr., W.C. Craighead J.E. Ann. Intern. Med. 1979; 90: 555-559Crossref PubMed Scopus (44) Google Scholar, 3Katz S.M. Hashemi S. Lab. Invest. 1982; 46: 24-32PubMed Google Scholar), eventually causing a severe form of pneumonia known as Legionnaires' disease (4Fraser D.W. Tsai T.R. Orenstein W. Parkin W.E. Beecham H.J. Sharrar R.G. Harris J. Mallison G.F. Martin S.M. McDade J.E. Shepard C.C. Brachman P.S. N. Engl. J. Med. 1977; 297: 1189-1197Crossref PubMed Scopus (1307) Google Scholar). The ability of L. pneumophila to grow intracellularly can be recapitulated within cultured macrophages or cell lines (1Fields B.S. Trends Microbiol. 1996; 4: 286-290Abstract Full Text PDF PubMed Scopus (444) Google Scholar). Phagosomes harboring L. pneumophila avoid fusion with the endocytic network as early as 5-min postinfection (5Swanson M.S. Hammer B.K. Ann. Rev. Microbiol. 2000; 54: 567-613Crossref PubMed Scopus (303) Google Scholar, 17Roy C.R. Berger K.H. Isberg R.R. Mol. Microbiol. 1998; 28: 663-674Crossref PubMed Scopus (295) Google Scholar), as endosomal markers such as Rab5 or the transferrin receptor are excluded from the phagosome (6Horwitz M.A. J. Exp. Med. 1983; 158: 2108-2126Crossref PubMed Scopus (510) Google Scholar, 7Clemens D.L. Lee B.Y. Horwitz M.A. Infect. Immun. 2000; 68: 2671-2684Crossref PubMed Scopus (136) Google Scholar). Instead, the compartment appears to mature via a unique pathway that involves recruitment of components found in the early secretory pathway (8Tilney L.G. Harb O.S. Connelly P.S. Robinson C.G. Roy C.R. J. Cell Sci. 2001; 114: 4637-4650PubMed Google Scholar, 9Kagan J.C. Roy C.R. Nat. Cell Biol. 2002; 4: 945-954Crossref PubMed Scopus (363) Google Scholar). For example, the secretory pathway marker Arf1 has been recently localized on the L. pneumophila phagosome shortly after infection (10Nagai H. Kagan J.C. Zhu X. Kahn R.A. Roy C.R. Science. 2002; 295: 679-682Crossref PubMed Scopus (453) Google Scholar), and markers of early secretory vesicles are found surrounding the phagosome within 15 min after bacterial contact with macrophages (9Kagan J.C. Roy C.R. Nat. Cell Biol. 2002; 4: 945-954Crossref PubMed Scopus (363) Google Scholar). Within a few hours, rough endoplasmic reticulum accumulates and potentially docks with the phagosome (11Horwitz M.A. J. Exp. Med. 1983; 158: 1319-1331Crossref PubMed Scopus (496) Google Scholar, 12Swanson M.S. Isberg R.R. Infect. Immun. 1995; 63: 3609-3620Crossref PubMed Google Scholar). Inside this intracellular niche, the bacteria replicate, yielding dozens of bacteria before lysing out of the macrophage to initiate a new replication cycle. Several independent genetic screens have led to the identification of bacterial factors necessary for intracellular proliferation of L. pneumophila (13Berger K.H. Isberg R.R. Mol. Microbiol. 1993; 7: 7-19Crossref PubMed Scopus (496) Google Scholar, 14Brand B.C. Sadosky A.B. Shuman H.A. Mol. Microbiol. 1994; 14: 797-808Crossref PubMed Scopus (141) Google Scholar, 15Segal G. Shuman H.A. Infect. Immun. 1997; 65: 5057-5066Crossref PubMed Google Scholar, 16Vogel J.P. Andrews H.L. Wong S.K. Isberg R.R. Science. 1998; 279: 873-876Crossref PubMed Scopus (592) Google Scholar). Single mutations in the majority of the 25 dot/icm genes lead to complete inhibition of intracellular growth. Most dot/icm mutants fail to form a replicative phagosome and cannot avoid the endocytic pathway (17Roy C.R. Berger K.H. Isberg R.R. Mol. Microbiol. 1998; 28: 663-674Crossref PubMed Scopus (295) Google Scholar, 18Wiater L.A. Dunn K. Maxfield F.R. Shuman H.A. Infect. Immun. 1998; 66: 4450-4460Crossref PubMed Google Scholar). The majority of these genes show similarity to components of conjugative DNA transfer systems found on IncI plasmids R64 and Col1b-P9 (15Segal G. Shuman H.A. Infect. Immun. 1997; 65: 5057-5066Crossref PubMed Google Scholar, 16Vogel J.P. Andrews H.L. Wong S.K. Isberg R.R. Science. 1998; 279: 873-876Crossref PubMed Scopus (592) Google Scholar, 19Komano T. Yoshida T. Narahara K. Furuya N. Mol. Microbiol. 2000; 35: 1348-1359Crossref PubMed Scopus (112) Google Scholar). Consistent with this observation, L. pneumophila can mobilize the RSF1010 plasmid between bacterial strains in a dot/icm-dependent manner (16Vogel J.P. Andrews H.L. Wong S.K. Isberg R.R. Science. 1998; 279: 873-876Crossref PubMed Scopus (592) Google Scholar, 20Segal G. Purcell M. Shuman H.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1669-1674Crossref PubMed Scopus (454) Google Scholar). By analogy to other pathogens, it was hypothesized that the products of the dot/icm genes are the building blocks of a type IV secretion system that allows translocation of bacterial factors into the host cell. In agreement with this idea, RalF, which activates the cellular small GTPase protein Arf1, is a translocation substrate of the Dot/Icm system (10Nagai H. Kagan J.C. Zhu X. Kahn R.A. Roy C.R. Science. 2002; 295: 679-682Crossref PubMed Scopus (453) Google Scholar), as is LidA, a coiled-coil protein that associates with the phagosomal membrane (21Conover G.M. Derre I. Vogel J.P. Isberg R.R. Mol. Microbiol. 2003; 48: 305-321Crossref PubMed Scopus (201) Google Scholar). Wild-type L. pneumophila but not dot/icm mutants induce osmotic cell death of the macrophages at a high multiplicity of infection (22Kirby J.E. Vogel J.P. Andrews H.L. Isberg R.R. Mol. Microbiol. 1998; 27: 323-336Crossref PubMed Scopus (192) Google Scholar). It has been hypothesized that the Dot/Icm proteins allow the secretion of a pore-forming protein that inserts into the host cell membrane as part of the secretion channel, and the resulting pore is responsible for this cytotoxicity. In contrast with this hypothesis, however, a partial loss of function mutation in icmT depresses high multiplicity of infection cytotoxicity but does not affect intracellular proliferation in U937 cells. This indicates that the pore formation promoted by L. pneumophila may not be involved in translocation of effector molecules into host cells (23Molmeret M. Alli O.A. Radulic M. Susa M. Doric M. Kwaik Y.A. Mol. Microbiol. 2002; 43: 1139-1150Crossref PubMed Scopus (29) Google Scholar, 24Molmeret M. Alli O.A. Zink S. Flieger A. Cianciotto N.P. Kwaik Y.A. Infect. Immun. 2002; 70: 69-78Crossref PubMed Scopus (69) Google Scholar). Several of the Dot/Icm proteins have been studied in detail, although no biochemical activities have been demonstrated for any components of this apparatus, except for the ability of IcmR to inhibit self-association of IcmQ (25Coers J. Kagan J.C. Mathews M. Nagai H. Zuckman D.M. Roy C.R. Mol. Microbiol. 2000; 38: 719-736Crossref PubMed Scopus (140) Google Scholar, 26Dumenil G. Isberg R.R. Mol. Microbiol. 2001; 40: 1113-1127Crossref PubMed Scopus (61) Google Scholar). Analysis of icmR and icmQ deletion strains indicates that these genes encode proteins necessary for intracellular growth, proper intracellular trafficking as well as high multiplicity of infection cytotoxicity (25Coers J. Kagan J.C. Mathews M. Nagai H. Zuckman D.M. Roy C.R. Mol. Microbiol. 2000; 38: 719-736Crossref PubMed Scopus (140) Google Scholar, 26Dumenil G. Isberg R.R. Mol. Microbiol. 2001; 40: 1113-1127Crossref PubMed Scopus (61) Google Scholar). Although these phenotypes suggest participation in the secretion channel itself, either within the bacterial envelope or on the mammalian plasma membrane, subcellular fractionation studies indicate that both IcmR and IcmQ are found in soluble fractions (25Coers J. Kagan J.C. Mathews M. Nagai H. Zuckman D.M. Roy C.R. Mol. Microbiol. 2000; 38: 719-736Crossref PubMed Scopus (140) Google Scholar). The bulk of these two proteins is found in a complex likely to consist of one molecule of IcmQ and two or three molecules of IcmR (26Dumenil G. Isberg R.R. Mol. Microbiol. 2001; 40: 1113-1127Crossref PubMed Scopus (61) Google Scholar). In the absence of IcmR, however, IcmQ forms large soluble homopolymers having a wide range of molecular weights, a property reminiscent of the behavior of type III secretion substrates in the absence of their chaperone (27Wattiau P. Woestyn S. Cornelis G.R. Mol. Microbiol. 1996; 20: 255-262Crossref PubMed Scopus (187) Google Scholar, 28Hueck C.J. Microbiol. Mol. Biol. Rev. 1998; 62: 379-433Crossref PubMed Google Scholar). A chaperone/substrate interaction has also been observed in the virB secretion system of the plant pathogen Agrobacterium tumefaciens, suggesting that conjugative systems similar to Dot/Icm may use such chaperones (29Deng W. Chen L. Peng W.T. Liang X. Sekiguchi S. Gordon M.P. Comai L. Nester E.W. Mol. Microbiol. 1999; 31: 1795-1807Crossref PubMed Scopus (66) Google Scholar, 30Dumas F. Duckely M. Pelczar P. Van Gelder P. Hohn B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 485-490Crossref PubMed Scopus (77) Google Scholar). We report here that although the primary sequence of IcmQ indicates that it is a highly hydrophilic protein, purified soluble IcmQ efficiently inserts into lipid membranes allowing efflux of a fluor of defined molecular weight. This behavior is reminiscent of pore-forming toxins such as Staphylococcus aureus α-toxin (31Bhakdi S. Tranum-Jensen J. Microbiol. Rev. 1991; 55: 733-751Crossref PubMed Google Scholar). L. pneumophila Bacterial Strains and Cultures—L. pneumophila strains used in this study were derived from the wild-type strain LP02 (thyA, hsdR, rpsL) (32Berger K.H. Merriam J.J. Isberg R.R. Mol. Microbiol. 1994; 14: 809-822Crossref PubMed Scopus (191) Google Scholar). LP03 is a dotA mutant isogenic to LP02 (32Berger K.H. Merriam J.J. Isberg R.R. Mol. Microbiol. 1994; 14: 809-822Crossref PubMed Scopus (191) Google Scholar). GD59 is isogenic to LP02, and contains a ΔicmR mutation. Strains were passaged on CYET plates and AYE broth as described previously (13Berger K.H. Isberg R.R. Mol. Microbiol. 1993; 7: 7-19Crossref PubMed Scopus (496) Google Scholar). For L. pneumophila, chloramphenicol was used at 5 μg/ml. For all experiments, L. pneumophila strains were grown to post-exponential phase (OD600 of 3.3-3.5) using bacterial motility as a post-exponential phase marker. The broad host range plasmid pMMB207 (33Morales V.M. Backman A. Bagdasarian M. Gene (Amst.). 1991; 97: 39-47Crossref PubMed Scopus (427) Google Scholar) was used to overexpress IcmQ under the control of the Ptac promoter in L. pneumophila strains. An EcoRI and SalI digestion fragment of the plasmid pGD32 (26Dumenil G. Isberg R.R. Mol. Microbiol. 2001; 40: 1113-1127Crossref PubMed Scopus (61) Google Scholar) encoding the icmQ coding sequence was inserted into the EcoRI- and SalI-digested pMMB207, generating pGD41. Mouse bone marrow-derived macrophages were isolated from femurs of female A/J mice (Jackson Laboratories) and cultivated in L cell-conditioned media as described previously (34Celada A. Gray P.W. Rinderknecht E. Schreiber R.D. J. Exp. Med. 1984; 160: 55-74Crossref PubMed Scopus (357) Google Scholar). Production of the IcmR-IcmQ Complex—Plasmid pGD31 was used to express the GST-IcmR fusion protein in Escherichia coli (26Dumenil G. Isberg R.R. Mol. Microbiol. 2001; 40: 1113-1127Crossref PubMed Scopus (61) Google Scholar). Plasmid pGD41 (described above) was used to express IcmQ in the absence of any tag. To allow in vivo formation of the complex, pGD31 and pGD41 were cotransformed into E. coli XL-1 blue (35Bullock W.O. Fernandez J.M. Short J.M. BioTechniques. 1987; 5: 376-379Google Scholar), generating GD105. One-liter cultures of GD105 were grown to OD600 of 1.0 at 37 °C and induced with 0.1 mm IPTG 1The abbreviations used are: IPTGisopropyl-1-thio-β-d-galactopyranosidePLphospholipidPCphosphatidylcholinePEphosphatidylethanolaminePGphosphatidylglycerolPIphosphatidylinositolCAcardiolipinChcholesterolICDHisocitrate dehydrogenaseTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineGSTglutathione S-transferase. for 3 h. Bacteria were pelleted by centrifugation, resuspended in 40 ml of 20 mm Tris-HCl (pH 7.5) containing 100 mm NaCl (TN), and sonicated on ice for 2 min on 50% duty cycle with a Branson Sonifier 250. Samples were then cleared by centrifugation at 12,000 × g for 15 min. 1 ml of glutathione-agarose (Amersham Biosciences) slurry was incubated with the cleared bacterial lysates for a period of 1-2 h. After extensive washing of the beads with TN buffer, the IcmR-IcmQ complex was cleaved from GST by adding 80 units of thrombin to the slurry and incubating the samples overnight at 4 °C. The suspension was then centrifuged briefly to pellet the beads, and the supernatant was collected. Thrombin was then removed by mixing the cleavage product with ∼30 μl of benzamidine agarose slurry (Sigma) and incubating for 15 min. The agarose was then pelleted, and the supernatant was collected. Protein concentration was determined using the Bradford assay (BioRad), and the sample was analyzed by SDS-PAGE followed by Coomassie Blue staining. isopropyl-1-thio-β-d-galactopyranoside phospholipid phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol phosphatidylinositol cardiolipin cholesterol isocitrate dehydrogenase N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine glutathione S-transferase. Purification of His-tagged Proteins—The open reading frame of IcmQ was inserted into the pQE70 plasmid (Qiagen) to allow expression of IcmQ fused to a C-terminal histidine tag, generating pGD37. Oligonucleotides 5′-CATGAAAGATCAACTCTCGGATGAAC-3′ and 5′-CGGGATCCTGCATTTTTAATTAGACGGCCATTG-3′ (the BamHI site is underlined) were used to amplify a DNA fragment encoding the IcmQ open reading from LP02 genomic DNA. This fragment was digested by BamHI and cloned into pQE70 previously digested by SphI and BamHI (the SphI site overhang was blunted with mung bean nuclease). The C-terminal portion of IcmQ was cloned into the pQE70 plasmid to allow expression of amino acids 68-191 of IcmQ fused to a C-terminal histidine tag, generating pGD46. The DNA fragment corresponding to amino acids 68-191 of IcmQ was generated by PCR using oligonucleotides 5′-CATGATTGCCTTAAGGAGTGGTCAG-3′ and 5′-CGGGATCCTGCATTTTTAATTAGACGGCCATTG-3′ (the BamHI site is underlined) from LP02 genomic DNA. This fragment was digested by BamHI and cloned into pQE70 previously digested by SphI and BamHI (the SphI site overhang was blunted with mung bean nuclease). The plasmid pGD46 was electroporated in the XL-1 Blue containing pREP4 (Qiagen), generating GD 120. To construct IQN, the 5′-end of icmQ was inserted into pQE70 to allow expression of amino acids 1-72 of IcmQ fused to a C-terminal histidine tag. The DNA fragment corresponding to codons 1-72 of icmQ was generated by PCR using oligonucleotides 5′-CCCGAAAAGTGCCACCTG-3′ and 5′CGGGATCCATTTGCAAGATGAGACTCTGC-3′ (the BamHI site is underlined) from pGD46. This fragment was digested by BamHI and inserted into pQE70 previously digested by SphI and BamHI (the SphI site overhang was blunted with mung bean nuclease) generating pTIM1. The plasmid pTIM1 was electroporated in the XL-1 Blue containing pREP4 (Qiagen). One-liter cultures of the full-length or C-terminal portion of IcmQ were grown to an OD600 of 1.0 and induced with 0.1 mm IPTG for 3 h. Bacteria were pelleted, resuspended in 40 ml of 20 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 20 mm imidazole. The suspension was sonicated on ice for 2 min on 50% duty cycle with a Branson Sonifier 250, pelleted at 12,000 × g for 15 min, and lysates were loaded onto a 5-ml metal chelation Hi trap column (Amersham Biosciences) using a Amersham Biosciences FPLC system. After extensive washing of the column with TN buffer, His-tagged proteins were eluted using 50 ml of a 0-200 mm imidazole gradient. Fractions containing full-length IcmQ or IQC were pooled and dialyzed overnight against TN buffer. IcmQ Membrane Insertion Assay—All lipids used in this study were purchased from Avanti Polar Lipids Inc. E. coli PL from this supplier represents a crude extract from bacteria with the following composition: 67% PE, 23% PG, and 10% CA. Lipids stored in chloroform at –80 °C were dried down under a nitrogen stream in a 15-ml Corex tube and then dessicated overnight. Unless otherwise stated, 5 mg of lipids were used. Large unilamellar vesicles were prepared by two different techniques, either by detergent dilution (36Mimms L.T. Zampighi G. Nozaki Y. Tanford C. Reynolds J.A. Biochem. 1981; 20: 833-840Crossref PubMed Scopus (557) Google Scholar) or by extrusion (37Hope M.J. Bally M. Webb G. Cullis P.R. Biochim. Biophys. Acta. 1985; 812: 55-65Crossref PubMed Scopus (2019) Google Scholar). For the detergent-based approach, lipids were resuspended in 0.5 ml of 20 mm Tris-HCl (pH 7.5), 100 mm NaCl, and 5% n-octylglucoside in the presence or absence of protein. The mixtures were then dialyzed against TN buffer for 24 h. For the extrusion-based approach, the lipid cake was hydrated at room temperature for 30 min in 0.5 ml of 20 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 1 mm EDTA (TNE). Four cycles of freeze and thaw were performed between an ethanol/dry ice mix and a water bath either at room temperature or at 60 °C depending on whether the transition temperature of the lipids was above or below 50 °C. The suspension was then extruded by several passes through a 0.1 μm pore polycarbonate membrane using a mini extruder (Avanti Polar Lipids). Certain lipid mixtures such as the PC 18:0 first required passage through a 1 μm polycarbonate membrane to prevent clogging of the filter. To assess association of proteins with preformed lipid vesicles, 20 μg of protein was mixed with 5 mg of lipid vesicles in TNE buffer and incubated at room temperature for 30 min prior to sucrose gradient centrifugation. Sucrose in TNE buffer was added to the samples to a final concentration of 45% (w/v) and 1 ml of the mixture was placed at the tube bottom (bottom fraction). A 7-ml 30% (w/v) sucrose solution in TNE buffer was layered on the samples (middle fraction) and finally 1 ml of TNE was layered on top (top fraction). The samples were then subjected to ultracentrifugation at 100,000 × g in a Beckman SW41 rotor for 15 h. The contents of the different fractions were analyzed by SDS-PAGE follow by Coomassie staining of the gel or by Western blot analysis. To generate lipid-associated IcmQ, the top fraction including the interface with the 30% (w/v) sucrose layer was dialyzed against TNE, prior to treatment with various reagents as described in Fig. 2. Subcellular Fractionation—A 50-ml culture in AYET broth was grown until motility was observed (OD ∼3.5). The bacteria were pelleted, the supernatant was precipitated with trichloroacetic acid and spheroplasts were made from the pelleted bacteria (38Roy C.R. Isberg R.R. Infect. Immun. 1997; 65: 571-578Crossref PubMed Google Scholar). Subcellular fractionation was then performed following the procedure described by Coers et al. (25Coers J. Kagan J.C. Mathews M. Nagai H. Zuckman D.M. Roy C.R. Mol. Microbiol. 2000; 38: 719-736Crossref PubMed Scopus (140) Google Scholar) except that Tris-HCl was used as a buffer instead of Hepes. Briefly, sonicated spheroplasts were pelleted, resuspended in 20 mm Tris-HCl, pH 7.5, and loaded on a 3-step sucrose gradient. Membranes were collected at the 25-60% sucrose interphase, washed with a high salt solution containing 20 mm Tris-HCl, pH 7.5, and 0.5 m NaCl solution and then solubilized with 2% Triton X-100. Equivalent volumes of the different fractions were loaded on SDS-PAGE and analyzed by Western blot using antibodies directed against L. pneumophila proteins. Calcein Release Assay—Lipid vesicles were generated by extrusion as described above except that lipids were resuspended at 10 mg/ml in TNE containing 80 mm calcein (pH 7.5) (39Kayalar C. Duzgunes N. Biochim. Biophys. Acta. 1986; 860: 51-56Crossref PubMed Scopus (48) Google Scholar). Free calcein was then removed using a 10-ml G-75 column (Amersham Biosciences) equilibrated with TNE. The lipid vesicles were diluted in TNE to a final concentration of 60 μg/ml in cuvettes having four clear sides, and subjected to constant agitation. Fluorescence emission was monitored at 520 nm after excitation at 490 nm using an SLM Aminco spectrofluorometer. As an alternative to calcein, Dextran 3000 tetramethyl rhodamine (Molecular Probes) was resuspended at 20 mg/ml in TNE, added to the dried lipids and extruded lipid vesicles were prepared as above. Fluorescence was monitored at 580 nm after excitation at 530 nm. Sonication Release Assay—PC vesicles containing IcmQ derivatives were prepared using the OG dialysis technique described above. As a control, vesicles were prepared in the absence of protein but in the presence of 80 mm calcein. Following purification, the lipid vesicles were sonicated using a Bransen Sonifier 250. To determine the efficiency of vesicle disruption, the vesicles were diluted in TNE and fluorescence emission was monitored as above. Sonication conditions were found such that there was 50% of the maximum fluorescence of the calceinladen vesicles (30 pulses, output level 3, 50% duty cycle), indicating at least 50% of the vesicle contents had been released, relative to treatment with 0.1% Triton X-100. These conditions were then applied to lipid vesicles incubated with protein. SDS-PAGE and Western Blot Analysis—Tris-Tricine gels (15% acrylamide) were performed as described (40Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10503) Google Scholar) except that the sample buffer contained phenol red dye. For Tris glycine gels, samples were boiled for 5 min in Laemmli buffer, resolved on 15% SDS-PAGE and transferred onto Immobilon-P membranes (Millipore). Immunoblotting was then performed as described (26Dumenil G. Isberg R.R. Mol. Microbiol. 2001; 40: 1113-1127Crossref PubMed Scopus (61) Google Scholar). Membranes were processed with the Renaissance detection kit (PerkinElmer Life Science Products), and blots were either exposed to film (Kodak) or the image was directly acquired on a Kodak Image Station to allow quantitation of the signal. Immunofluorescence—For IcmQ localization studies, GD132 (LP02/pGD32, Ptac-icmQ) was grown to an OD600 of 2.5 in AYET broth before adding IPTG at 0.1 mm for about 2 h until the culture reached post-exponential phase. Bacteria were then either fixed in periodate-lysine-paraformaldehyde (PLP) (41McLean I.W. Nakane P.K. J. Histochem. Cytochem. 1974; 22: 1077-1083Crossref PubMed Scopus (3200) Google Scholar) containing 5% sucrose for 30 min or used to infect macrophages. Bacteria were deposited on bone marrow-derived macrophages at an multiplicity of infection of 20 by centrifugation at 150 × g for 5 min at room temperature and then incubated at 37 °C for various times. Cells were fixed for 30 min at 37 °C in PLP containing 5% sucrose. After removal of the fixative by washing with phosphate-buffered saline, infected cells were permeabilized with –20 °C methanol for 10 s. To analyze broth grown bacteria in the absence of mammalian cells, bacteria were pelleted onto coverslips previously treated with 0.1% poly-l-lysine. Coverslips with fixed infected macrophages or broth grown bacteria were then treated in parallel. Coverslips were blocked and stained with anti-L. pneumophila antibody as described (42Swanson M.S. Isberg R.R. Infect. Immun. 1996; 64: 2585-2594Crossref PubMed Google Scholar). Samples were then stained with biotinylated affinity-purified anti-IcmQ polyclonal antibody (1:200) (26Dumenil G. Isberg R.R. Mol. Microbiol. 2001; 40: 1113-1127Crossref PubMed Scopus (61) Google Scholar). Biotinylation was performed according to the manufacturer (Pierce). After extensive washing, coverslips were then stained with streptavidin-Alexa-488 (1:2000, Molecular Probes). IcmQ Associates with Lipid Vesicles—Several substrates found in type III and IV secretion systems interact with lipid membranes when released from their chaperone (43Cornelis G.R. Van Gijsegem F. Annu. Rev. Microbiol. 2000; 54: 735-774Crossref PubMed Scopus (648) Google Scholar). To address whether IcmQ exhibits such properties we tested whether purified recombinant His-tagged IcmQ (“Materials and Methods”) could associate with and/or insert into phospholipid (PL) bilayers by incubating purified IcmQ with PL extracts in the presence of 5% n-octylglucoside (OG; “Materials and Methods”). Glutathione S-transferase (GST) was also added to the mixture as a negative control. The detergent was removed by dialysis to allow the formation of PL vesicles and the preparation was analyzed on a three-step sucrose gradient to determine the amount of PL-associated IcmQ. About 30% of the total IcmQ added in the reaction floated with E. coli PL to the top step of the gradient (Fig. 1A, E. coli). In the same sample, less than 10% of GST copurified with the PL, presumably due to the trapping of the protein within the internal volume of the vesicles. When liver PL extracts were used to make the vesicles, almost 90% of total IcmQ was found to be PL-associated (Fig. 1A, Liver). In the absence of PL, IcmQ was found entirely in the bottom fraction of the gradient (data not shown). IcmQ is thus able to associate with lipids and the efficiency of association depended on the composition of the PL vesicles. We next tested whether soluble IcmQ could associate with preformed lipid vesicles (Fig. 1B). PL vesicles were first formed by incubation with OG followed by dialysis to remove the detergent. IcmQ was added to the vesicles in the absence of OG prior to loading onto a sucrose gradient and collecting fractions. Quantitative Western blot analysis revealed that under these conditions, 60% of total IcmQ associated with E. coli PL vesicles and 100% of IcmQ was associated with liver PL vesicles (Fig. 1B, IcmQ). In contrast, little or no GST was found associated with the preformed vesicles (Fig. 1B, GST). Therefore, soluble IcmQ appears to associate with PL vesicles in the absence of detergent. Phospholipid Association Is Due to Insertion—The copurification of IcmQ with PC vesicles could be the result of peripheral association or integration into the bilayer. To determine the nature of the association of IcmQ with lipids, membrane-associated IcmQ was exposed to buffers containing 1 m NaCl, or 1% Triton X-100 and subjected to sucrose gradient fractionation(Fig. 2A). Western blot analysis allowed visualization of a major band migrating at 22 kDa corresponding to IcmQ and a minor band migrating at 50 kDa that could correspond to an SDS-resistant dimer of IcmQ, High salt did not affect the association of IcmQ with lipid vesicles indicating that IcmQ is not peripherally associated with the lipid bilayer. In contrast, IcmQ could be released by addition of Triton X-10" @default.
• W2069809595 created "2016-06-24" @default.
• W2069809595 creator A5010198377 @default.
• W2069809595 creator A5040829444 @default.
• W2069809595 creator A5070494018 @default.
• W2069809595 creator A5076382250 @default.
• W2069809595 date "2004-02-01" @default.
• W2069809595 modified "2023-10-16" @default.
• W2069809595 title "IcmR-regulated Membrane Insertion and Efflux by the Legionella pneumophila IcmQ Protein" @default.
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