Shigella Invasion of Macrophage Requires the Insertion of IpaC into the Host Plasma Membrane
2001; Elsevier BV; Volume: 276; Issue: 34 Linguagem: Inglês
10.1074/jbc.m103831200
ISSN1083-351X
AutoresAsaomi Kuwae, Sei Yoshida, Koichi Tamano, Hitomi Mimuro, Toshihiko Suzuki, Chihiro Sasakawa,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoShigella infects residential macrophages via the M cell entry, after which the pathogen induces macrophage cell death. The bacterial strategy of macrophage infection, however, remains largely speculative. Wild type Shigella flexneri (YSH6000) invaded macrophages more efficiently than the noninvasive mutants, where YSH6000 induced large scale lamellipodial extension including ruffle formation around the bacteria. When macrophages were infected with the noninvasive ipaC mutant, the invasiveness and induction of membrane extension were dramatically reduced as compared with that of YSH6000. J774 macrophages infected with YSH6000 showed tyrosine phosphorylation of several proteins including paxillin and c-Cbl, and this pattern was distinctive from those stimulated by Salmonella typhimurium or phorbol ester. Upon addition of IpaC into the external medium of macrophages, membrane extensions were rapidly induced, and this promoted uptake ofEscherichia coli. The exogenously added IpaC was found to be integrated into the host cell membrane as detected by immunostaining. The IpaC domain required for the induction of membrane extension from J774 was narrowed down within the region of residues 117–169, which contains a putative membrane-spanning sequence. Our data indicate that Shigella directs its own entry into macrophages, and the IpaC domain which is required for the association with its host membrane is crucial. Shigella infects residential macrophages via the M cell entry, after which the pathogen induces macrophage cell death. The bacterial strategy of macrophage infection, however, remains largely speculative. Wild type Shigella flexneri (YSH6000) invaded macrophages more efficiently than the noninvasive mutants, where YSH6000 induced large scale lamellipodial extension including ruffle formation around the bacteria. When macrophages were infected with the noninvasive ipaC mutant, the invasiveness and induction of membrane extension were dramatically reduced as compared with that of YSH6000. J774 macrophages infected with YSH6000 showed tyrosine phosphorylation of several proteins including paxillin and c-Cbl, and this pattern was distinctive from those stimulated by Salmonella typhimurium or phorbol ester. Upon addition of IpaC into the external medium of macrophages, membrane extensions were rapidly induced, and this promoted uptake ofEscherichia coli. The exogenously added IpaC was found to be integrated into the host cell membrane as detected by immunostaining. The IpaC domain required for the induction of membrane extension from J774 was narrowed down within the region of residues 117–169, which contains a putative membrane-spanning sequence. Our data indicate that Shigella directs its own entry into macrophages, and the IpaC domain which is required for the association with its host membrane is crucial. polymorphonuclear leukocyte human monocyte-derived macrophage multiplicity of infection phosphate-buffered saline monoclonal antibody complement-opsonized zymosan IgG-opsonized zymosan bovine serum albumin-opsonized zymosan phorbol myristate acetate phosphotyrosine glutathioneS-transferase polyacrylamide gel electrophoresis lipopolysaccharide Shigella are highly adapted human pathogens that cause bacillary dysentery, a disease provoking severe bloody and mucus diarrhea. When Shigella reach the colon, the bacteria are translocated through the epithelial barrier by way of the M cells which overlay the solitary lymphoid nodules (1Wassef J.S. Keren D.F. Mailloux J.L. Infect. Immun. 1989; 57: 858-863Crossref PubMed Google Scholar, 2Sansonetti P.J. Arondel J. Fontaine A. d'Hauteville H. Bernardini M.L. Vaccine. 1991; 9: 416-422Crossref PubMed Scopus (131) Google Scholar, 3Sansonetti P.J. Arondel J. Cantey J.R. Prevost M.C. Huerre M. Infect. Immun. 1996; 64: 2752-2764Crossref PubMed Google Scholar). Once they reach the underlying M cells, Shigella infect the resident macrophages and induce cell death (4Zychlinsky A. Prevost M.C. Sansonetti P.J. Nature. 1992; 358: 167-169Crossref PubMed Scopus (824) Google Scholar). Meanwhile the pathogens released from the killed macrophages enter into enterocytes from the basolateral surface by inducing membrane ruffles and macropinocytosis. Once the bacterium is surrounded by a membrane vacuole, it immediately disrupts the vacuole to escape into the cytoplasm (5Sansonetti P.J. Ryter A. Clerc P. Maurelli A.T. Mounier J. Infect. Immun. 1986; 51: 461-469Crossref PubMed Google Scholar). Within the cytoplasm,Shigella can multiply and move by inducing actin polymerization at one pole of the bacterium, which allows intracellular spreading of the bacterium within the cytoplasm as well as into the adjacent epithelial cells (6Makino S. Sasakawa C. Kamata K. Kurata T. Yoshikawa M. Cell. 1986; 46: 551-555Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 7Bernardini M.L. Mounier J. d'Hauteville H. Coquis-Rondon M. Sansonetti P.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3867-3871Crossref PubMed Scopus (546) Google Scholar). Thus, the predominant feature of theShigella pathogenicity is the ability to infect macrophage and epithelial cells, resulting in dissemination to adjacent epithelial cells. A genetic study revealed that Shigella invasion of epithelial cells requires the numerous genes encoded by the 31-kilobase pair pathogenicity island on the large 230-kilobase pair plasmid (8Sasakawa C. Kamata K. Sakai T. Makino S. Yamada M. Okada N. Yoshikawa M. J. Bacteriol. 1988; 170: 2480-2484Crossref PubMed Google Scholar). The pathogenicity island of Shigella flexneri contains 28 invasion-associated genes arranged in several transcribed regions (9Buchrieser C. Glaser P. Rusniok C. Nedjari H. D'Hauteville H. Kunst F. Sansonetti P. Parsot C. Mol. Microbiol. 2000; 38: 760-771Crossref PubMed Scopus (307) Google Scholar). One of these contains the ipa genes whose products such as IpaA, IpaB, IpaC, and IpaD are required for bacteria-cell interactions including induction of macropinocytosis from epithelial cells, whereas the mxi and spa regions are mostly involved in the formation of the type III secretion machinery (10Sasakawa C. Adler B. Tobe T. Okada N. Nagai S. Komatsu K. Yoshikawa M. Mol. Microbiol. 1989; 3: 1191-1201Crossref PubMed Scopus (69) Google Scholar, 11Sasakawa C. Buysse J.M. Watanabe H. Curr. Top. Microbiol. Immunol. 1992; 180: 21-44Crossref PubMed Google Scholar, 12Parsot C. Curr. Top. Microbiol. Immunol. 1994; 192: 217-241PubMed Google Scholar). Studies have indicated that Shigella-induced macropinocytosis in epithelial cells occurs through a complicated process requiring the interaction of the type III-secreted effector proteins and host factors. In this process, the Ipa proteins (IpaA–D) are thought to play the most important roles. However, the involvement of Ipa proteins in the Shigella entry into epithelial cells is complicated and still unclear. Some of the Ipa proteins act both as regulators of the type III secretion system as well as effector proteins within the host cells. For example, IpaB and IpaD act as the molecular plug for regulating the type III secretion system (13Menard R. Sansonetti P. Parsot C. Vasselon T. Cell. 1994; 79: 515-525Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 14Menard R. Prevost M.C. Gounon P. Sansonetti P. Dehio C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1254-1258Crossref PubMed Scopus (174) Google Scholar, 15Tran Van Nhieu G. Sansonetti P.J. Curr. Opin. Microbiol. 1999; 2: 51-55Crossref PubMed Scopus (133) Google Scholar). In addition, IpaB and IpaC, as well as Salmonella SipB and SipC (16Collazo C.M. Galan J.E. Mol. Microbiol. 1997; 24: 747-756Crossref PubMed Scopus (257) Google Scholar) and Yersinia YopB and YopD (17Hakansson S. Schesser K. Persson C. Galyov E.E. Rosqvist R. Homble F. Wolf-Watz H. EMBO J. 1996; 15: 5812-5823Crossref PubMed Scopus (318) Google Scholar), serve as a membrane pore in part of the type III machinery of the host cell plasma membrane, allowing the translocation of secreted effector proteins into the host cells (18Blocker A. Gounon P. Larquet E. Niebuhr K. Cabiaux V. Parsot C. Sansonetti P. J. Cell Biol. 1999; 147: 683-693Crossref PubMed Scopus (394) Google Scholar). Upon contact to epithelial cells, a massive amount of IpaB and IpaC proteins is also secreted into the external medium from Shigella and promotes bacterial invasion by interacting with putative host receptors such as α5β1 integrin and CD44 (19Watarai M. Funato S. Sasakawa C. J. Exp. Med. 1996; 183: 991-999Crossref PubMed Scopus (182) Google Scholar, 20Skoudy A. Mounier J. Aruffo A. Ohayon H. Gounon P. Sansonetti P. Nhieu G.T. Cell Microbiol. 1999; 2: 19-33Crossref Scopus (105) Google Scholar). However, these interactions would still be insufficient to elicit the macropinocytic events in epithelial cells required for bacterial entry. Further large scale rearrangement of actin dynamism is assumed to require a second signaling event within the host cytoplasm through the delivery of IpaA, IpaC, and IpgD via the type III secretion machinery (21Tran Van Nhieu G. Bourdet-Sicard R. Dumenil G. Blocker A. Sansonetti P.J. Cell Microbiol. 2000; 2: 187-193Crossref PubMed Scopus (117) Google Scholar, 22Sansonetti P.J. FEMS Microbiol. Rev. 2001; 25: 3-14PubMed Google Scholar). IpaA binds to vinculin, and the IpaA-vinculin complex together with F-actin promotes depolymerization of actin filaments required for modification of Shigella-induced membrane protrusions (23Tran Van Nhieu G. Ben-Ze'ev A. Sansonetti P.J. EMBO J. 1997; 16: 2717-2729Crossref PubMed Scopus (173) Google Scholar, 24Bourdet-Sicard R. Rudiger M. Jockusch B.M. Gounon P. Sansonetti P.J. Nhieu G.T. EMBO J. 1999; 18: 5853-5862Crossref PubMed Scopus (126) Google Scholar). IpaC somehow modulates actin dynamism, since formation of filopodia and lamellipodia can be induced when purified IpaC protein is added to semipermeabilized Swiss 3T3 cells or aipaC clone is transfected into HeLa cells. The IpaC-induced membrane protrusions have been implicated in the activation of Cdc42 which in turn activates Rac1 (25Tran Van Nhieu G. Caron E. Hall A. Sansonetti P.J. EMBO J. 1999; 18: 3249-3262Crossref PubMed Scopus (205) Google Scholar). Although the mechanisms of IpaC-induced membrane protrusions are unknown, these studies have suggested that IpaC can somehow act as the effector for promotingShigella invasion of epithelial cells. Many invasive pathogenic bacteria have a specific strategy to resist the bactericidal activities of macrophages. For example, upon contact to macrophages, Yersinia activates the type III secretion system and delivers a set of Yop proteins into the cell (26Rosqvist R. Magnusson K.E. Wolf-Watz H. EMBO J. 1994; 13: 964-972Crossref PubMed Scopus (482) Google Scholar, 27Sory M.P. Cornelis G.R. Mol. Microbiol. 1994; 14: 583-594Crossref PubMed Scopus (452) Google Scholar, 28Lee V.T. Anderson D.M. Schneewind O. Mol. Microbiol. 1998; 28: 593-601Crossref PubMed Scopus (119) Google Scholar). The injected Yop proteins such as YopE, YopH, YopT, YopO, and YopP act in various ways to prevent phagocytosis and kill the macrophages, such as inducing destruction of actin filaments, interfering with cell signal transduction, or provoking apoptosis (29Lee V.T. Schneewind O. Immunol. Rev. 1999; 168: 241-255Crossref PubMed Scopus (38) Google Scholar). Salmonellainduces a macropinocytic event from epithelial cells and macrophages and delivers a subset of effector proteins from the type III secretion system encoded by the Salmonella pathogenicity island I, and then the pathogen can enter both types of cells. In the infection of macrophages, however, the pathogen eventually induces apoptosis to kill the cells (30Chen L.M. Kaniga K. Galan J.E. Mol. Microbiol. 1996; 21: 1101-1115Crossref PubMed Scopus (340) Google Scholar, 31Alpuche-Aranda C.M. Racoosin E.L. Swanson J.A. Miller S.I. J. Exp. Med. 1994; 179: 601-608Crossref PubMed Scopus (275) Google Scholar), for which SipB encoded by Salmonellapathogenicity island I is delivered into macrophages (32Hersh D. Monack D.M. Smith M.R. Ghori N. Falkow S. Zychlinsky A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2396-2401Crossref PubMed Scopus (604) Google Scholar, 33Monack D.M. Raupach B. Hromockyj A.E. Falkow S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9833-9838Crossref PubMed Scopus (526) Google Scholar).Salmonella typhimurium possesses an additional type III secretion system encoded by the Salmonella pathogenicity island II, which is activated inside the infected macrophage and delivers proteins such as SpiC and SifA that are required for bacterial proliferation in phagosomes and their maintenance (34Uchiya K. Barbieri M.A. Funato K. Shah A.H. Stahl P.D. Groisman E.A. EMBO J. 1999; 18: 3924-3933Crossref PubMed Scopus (295) Google Scholar, 35Brumell J.H. Rosenberger C.M. Gotto G.T. Marcus S.L. Finlay B.B. Cell Microbiol. 2001; 3: 75-84Crossref PubMed Scopus (145) Google Scholar).Shigella infects macrophages and disrupts the phagocytic vacuoles to escape into the cytoplasm, from which the bacterium later induces the cell death (36Clerc P.L. Ryter A. Mounier J. Sansonetti P.J. Infect. Immun. 1987; 55: 521-527Crossref PubMed Google Scholar). A previous study (4Zychlinsky A. Prevost M.C. Sansonetti P.J. Nature. 1992; 358: 167-169Crossref PubMed Scopus (824) Google Scholar) indicated that internalized Shigella induced macrophage apoptosis after 1–2 h postinfection, in which IpaB protein secreted via the type III secretion system within the macrophage cytoplasm played a crucial role. However, some other studies indicated that macrophage killing by apoptosis could only be induced if the cells were activated such as by stimulating with interferon-γ (37Nonaka T. Kuwae A. Sasakawa C. Imajoh-Ohmi S. FEMS Microbiol. Lett. 1999; 174: 89-95Crossref PubMed Google Scholar, 38Fernandez-Prada C.M. Hoover D.L. Tall B.D. Venkatesan M.M. Infect. Immun. 1997; 65: 1486-1496Crossref PubMed Google Scholar). Whenever Shigellaelicits different killing systems from the infected macrophages, the cell death results in release of massive amounts of interleukin-1β, thus triggering a strong inflammatory response (39Zychlinsky A. Fitting C. Cavaillon J.M. Sansonetti P.J. J. Clin. Invest. 1994; 94: 1328-1332Crossref PubMed Scopus (195) Google Scholar), and leads to an increase in the permeability of the epithelial barrier toShigella entry and the migration of PMN1 (40Sansonetti P.J. Arondel J. Cavaillon J.M. Huerre M. J. Clin. Invest. 1995; 96: 884-892Crossref PubMed Scopus (119) Google Scholar, 41Arondel J. Singer M. Matsukawa A. Zychlinsky A. Sansonetti P.J. Infect. Immun. 1999; 67: 6056-6066Crossref PubMed Google Scholar). Therefore, elucidation of the bacterial strategy for infection of macrophages is an important issue for understanding the pathogenicity of Shigella and for the development of novel attenuated vaccines that are still invasive but do not induce macrophage cell death. Nevertheless, the molecular basis for infection of macrophages by Shigella still remains poorly understood. Hence, we decided to investigate whether the pathogen had the ability to direct its own internalization into macrophages and induce the activation of macrophage functions. Our data strongly indicate thatShigella directs its own phagocytic events in macrophages by exploiting the ability of IpaC to be integrated into the host membrane. In this study, we provide direct evidence for the first time indicating that the membrane-spanning IpaC domain is critical for the inducing macropinocytic event in macrophages. S. flexneri 2a YSH6000 is the wild type strain, and S325 is a mxiA::Tn5 derivative of YSH6000 used as the negative control deficient in the type III secretion activity (42Sasakawa C. Makino S. Kamata K. Yoshikawa M. Infect. Immun. 1986; 54: 32-36Crossref PubMed Google Scholar). S. typhimurium SB300 was obtained from J. E. Galan (Yale School of Medicine, New Haven, CT).Escherichia coli MC1061 was used as the host for constructing various plasmids. pBluescript SK+ (Stratagene), pGEX-2T (Amersham Pharmacia Biotech), pCACTUS-Tpr (43Tamano K. Aizawa S. Katayama E. Nonaka T. Imajoh-Ohmi S. Kuwae A. Nagai S. Sasakawa C. EMBO J. 2000; 19: 3876-3887Crossref PubMed Scopus (197) Google Scholar), and pMW119Tp (44Durand J.M. Okada N. Tobe T. Watarai M. Fukuda I. Suzuki T. Nakata N. Komatsu K. Yoshikawa M. Sasakawa C. J. Bacteriol. 1994; 176: 4627-4634Crossref PubMed Google Scholar) were used for genetic engineering experiments. E. coli JM109 was used as the host for constructing pQE-30 (Qiagen)-borne plasmids. All primers used for construction of the various plasmids are listed in Table I. Strains containing pMW119Tp- or pCACTUS-Tpr-based plasmids were grown in Muller-Hinton broth (Difco) when selection for trimethoprim resistance was necessary. For all macrophage infection experiments, overnight cultures of the bacterial strains were diluted 50-fold in brain-heart infusion broth (Difco) and incubated at 37 °C for 2 h. J774 cells (ATCC TIB-67) and THP-1 cells (ATCC TIB-202) were maintained in RPMI 1640 (Sigma), and RAW264.7 cells (ATCC TIB-71) were maintained in Dulbecco's modified Eagle's medium (Sigma). For the preparation of HMDM, monocytes were isolated from the peripheral blood of healthy donors using Ficoll-Paque (Amersham Pharmacia Biotech) following the manufacturer's protocol. The separated mononuclear cells were plated on coverslips, and nonadherent cells were removed after 1 h of incubation. The medium was then replaced by fresh RPMI 1640 containing 10% FBS and 50 ng/ml human recombinant granulocyte-macrophage colony-stimulating factor (PeproTech). Monocytes were cultivated for 5–7 days, and the adherent population was shown to be >95% macrophages as determined morphologically by Giemsa's stain.Table IOligonucleotides used in this studyPrimersSequenceicm-15′-TGGGGGCCCAGATTCTAAAATAAAAGACCicm-25′-TCCCCCGGGTGTGGTTGTTAATACGGGATicm-35′-TCCCCCGGGAATATAATTGACAGCATCicm-45′-CGCGGATCCATTTGAAAATTCGAGAATCicc-15′-TCGTAGCCACTGTTGGTAAACAGGCicc-25′-CGGGATCCGTTGAGCATAGTAAGGGipaD-15′-CGGGATCCATGAATATAACAACTCTipaD-25′-CGGAATTCAGAAATGGAGAAAAAGvirA-15′-CGGGATCCATGCAGACATCAAACATvirA-25′-TCCCCCGGGTTAAACATCAGGAGATAipaC-15′-TAAGGATCCATGTTGCAAAAGCAATTTTGCipaC-25′-TAAGTCGACTTAAGCTCGAATGTTACCAGCNC-15′-CGGGATCCGTGATGGTGATGGTGATGCGNC-25′-CGGGATCCGATATTTCCAGTCTTTCTTCTACTM-15′-ACGCGTCGACTGGAAATATCCAGAGTGCTM-25′-ACGCGTCGACAAGGCCTGGCAGCCCC8–15′-TAAGTCGACCAGAGTGTTCTCTGGAGTAAGC8–25′-TAAGTCGACCTGCAGCCAAGCTTAATTAG Open table in a new tab The antibodies specific for IpaB, IpaC, and IpaD used for the immunoblots have been described previously (45Tobe T. Yoshikawa M. Sasakawa C. Mol. Microbiol. 1994; 12: 267-276Crossref PubMed Scopus (19) Google Scholar). A fusion protein of IpaC tagged with six histidine residues at the N terminus was constructed using the QIAexpress system with a pQE-30 plasmid. A DNA fragment including the ipaC gene was amplified by PCR using an ipaC-1 primer containing aBamHI site and an ipaC-2 primer containing a SalI site together with pMYSH6000, the large virulence plasmid in YSH6000 as the template. The amplified BamHI-SalI fragment was cloned into pQE-30, resulting in the plasmid pQE-ipaC-fl. The fusion IpaC protein purified by nickel-nitrilotriacetic acid chromatography (Qiagen) was used to immunize rabbits. The anti-IpaC whole molecule antiserum was incubated with nitrocellulose membrane containing transferred fusion IpaC protein, and the Ig fraction specific for the whole molecule of IpaC (anti-IpaC-wm) was eluted. IpaC peptides named IpaC-n, encompassing amino acid residues 35–55 (ISTKQTQSSSETQKSQNYQQI), IpaC-m, encompassing amino acid residues 140–169 (RTAETKLGSQLSLIAFDATKSAAENIVRQG), and IpaC-c, encompassing amino acid residues 228–247 (KQIDTNITSPQTNSSTKFLG), were synthesized. The anti-IpaC whole molecule antiserum was incubated with IpaC-n-, IpaC-m- or IpaC-c-conjugated, epoxy-activated Sepharose 6B (Amersham Pharmacia Biotech) to obtain an Ig fraction specific for each peptide; the antibodies obtained were named anti-IpaC-n, anti-IpaC-m, and anti-IpaC-c, respectively. The invasion efficiency of bacteria was measured with a gentamicin protection assay (46Small P.L. Isberg R.R. Falkow S. Infect. Immun. 1987; 55: 1674-1679Crossref PubMed Google Scholar). Briefly, a 24-well tissue culture plate (Costar) was seeded with 2 × 105 cells/well and incubated overnight at 37 °C under 5% CO2. Each well was then infected at an m.o.i. of ∼500 for 30 min, before prewarmed tissue culture medium containing gentamicin was added at a final concentration of 200 µg/ml. After a 15-min incubation, the medium was removed, and the macrophages were washed three times with PBS. PBS containing 0.5% Triton X-100 was then added to each well in order to lyse the cells. After an appropriate dilution with saline, the lysed solution was plated onto L agar, and the number of viable bacteria was counted. Immunostaining was performed essentially as described previously (47Suzuki T. Miki H. Takenawa T. Sasakawa C. EMBO J. 1998; 17: 2767-2776Crossref PubMed Scopus (200) Google Scholar). Mac-1 and FcγR were labeled with the anti-Mac-1 mAb M1/70 (BIOSOURCEInternational, Inc.) or the anti-FcγR mAb 2.4G2 (PharMingen), respectively. The anti-rat IgG antibody conjugated with fluorescein isothiocyanate (Sigma) was used as the secondary antibody. In order to measure the invasive capacity of the bacteria by immunostaining, the number of bacteria internalized in the macrophage cytoplasm was counted as follows: J774 cells seeded on coverslips were infected at an m.o.i. of ∼30 and centrifuged for 10 min at 900 × g. The infected J774 cells were then incubated for 15 min at 37 °C under 5% CO2. Next the cells were washed with PBS and then fixed for 15 min with PBS containing 4% paraformaldehyde. The fixed cells were incubated in PBS containing anti-Shigella LPS antiserum for 1 h. To visualize extracellular bacteria, the cells were washed and then incubated with anti-rabbit IgG conjugated to fluorescein isothiocyanate (Sigma). To visualize intracellular bacteria, the cells were washed again and permeabilized by incubation with PBS containing 0.2% Triton X-100 for 20 min at room temperature. The cells were then treated once more with the anti-ShigellaLPS antiserum and incubated with anti-rabbit IgG conjugated to Cy5 (Amersham Pharmacia Biotech). Immunostained samples were examined by confocal laser scanning microscopy (Radiance Plus, Bio-Rad). J774 cells seeded on coverslips were infected at an m.o.i. of ∼500 before being centrifuged for 10 min and incubated for 30 min at 37 °C under 5% CO2. The cells were then washed with PBS and fixed in PBS containing 4% paraformaldehyde. The fixed cells were stained with Giemsa's solution. Photographs of the cells were taken with a CCD camera on the microscope, and the cell areas were measured using IPLab Spectrum software (Signal Analytics Corp.). Macrophages were grown on 12-mm round coverslips (Matsunami) before being infected by centrifugation as described above. For further fixation, the washed and fixed cells were treated with 2% OsO4 and then processed for electron microscopy as described by Ginocchio et al. (48Ginocchio C.C. Olmsted S.B. Wells C.L. Galan J.E. Cell. 1994; 76: 717-724Abstract Full Text PDF PubMed Scopus (219) Google Scholar). Visualization of samples was carried out by an S800 scanning electron microscope (Hitachi). For construction of a nonpolar mutant of ipaC, theaphA-3 (kanamycin resistance gene) cassette was used (49Menard R. Sansonetti P.J. Parsot C. J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (616) Google Scholar). Purified pMYSH6000 was used as the template for PCR. Nonpolar mutants of ipaC were constructed as follows: a DNA fragment encompassing nucleotides from position 1226 upstream of the 5′ end of the ipaC gene to nucleotide 215 downstream from the 5′ end was amplified by PCR using primers icm-1 containing an ApaI site and icm-2 containing an SmaI site. TheApaI-SmaI fragment was cloned into pBluescript, resulting in the plasmid pipaC-u. Another DNA fragment encompassing nucleotides from position 1076 downstream from the 5′ end of theipaC gene to nucleotide 2494 downstream from the 5′ end was amplified using primers icm-3 containing an SmaI site and icm-4 containing a BamHI site. TheSmaI-BamHI fragment was then cloned into pipaC-u, yielding the plasmid pipaC-ud. The aphA-3 cassette was cloned at the SmaI site of pipaC-ud in the correct orientation, resulting in the plasmid pBSipaC::Kmr. The inactivatedipaC gene digested from pBSipaC::Kmr at ApaI andNotI sites was subcloned into pCACTUS-Tpr, followed by introduction of the resultant plasmid named pipaC::Kmr into YSH6000 by electroporation. Integration and selection of nonpolar ipaCmutants were achieved as described previously (43Tamano K. Aizawa S. Katayama E. Nonaka T. Imajoh-Ohmi S. Kuwae A. Nagai S. Sasakawa C. EMBO J. 2000; 19: 3876-3887Crossref PubMed Scopus (197) Google Scholar), and the obtained mutant strain, ΔipaC, was named TK001. For complementation of the ipaC gene defect in TK001, pIpaC was constructed by cloning the DNA fragment containing the ipaC gene into pMW119Tp. This fragment was amplified by PCR using primer icc-1 and icc-2 with pMYSH6000 as the template. The effector protein solution released in the Shigella culture supernatants following stimulation with Congo red and the whole bacterial lysate were prepared as described previously (43Tamano K. Aizawa S. Katayama E. Nonaka T. Imajoh-Ohmi S. Kuwae A. Nagai S. Sasakawa C. EMBO J. 2000; 19: 3876-3887Crossref PubMed Scopus (197) Google Scholar). Each protein sample from the same number of bacteria was separated by 12% SDS-PAGE and immunoblotted with anti-IpaB, anti-IpaC, and anti-IpaD antibody. J774 cells were seeded on 6-well tissue culture plates (Costar) at a concentration of 4 × 105cells/well and incubated overnight at 37 °C under 5% CO2. Shigella and Salmonella strains were added at an m.o.i. of ∼500 and centrifuged for 10 min. COZ, IOZ, or BOZ was prepared as described previously (50Van Strijp J.A. Russell D.G. Tuomanen E. Brown E.J. Wright S.D. J. Immunol. 1993; 151: 3324-3336PubMed Google Scholar, 51Tapper H. Grinstein S. J. Immunol. 1997; 159: 409-418PubMed Google Scholar). Opsonized zymosan particles were centrifuged onto the macrophage (particle:cell ratio = 10:1) for 2 min. Treatment of macrophage with PMA (Sigma) was carried out at a final concentration of 100 ng/ml. After various incubation times, the cells were washed twice with ice-cold PBS and then lysed in 50 µl of lysis buffer (10 mm Tris-HCl, pH 7.6, 5 mm EDTA, 50 mm NaCl, 30 mmsodium pyrophosphate, 50 mm NaF, 1% Triton X-100, 100 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, 1 mg/ml pepstatin A, 10 mg/ml leupeptin) per well. The cell lysates were sonicated for 10 s and clarified by centrifugation at 15,000 × g for 15 min. The supernatants were then assayed for protein concentration with SDS-PAGE. The supernatants were incubated with anti-PY mAb 4G10 (Upstate Biotechnology Inc.), anti-paxillin mAb 349 (Transduction Laboratories), or anti-c-Cbl antibody C-15 (Santa Cruz Biotechnology). Immunoprecipitations were performed overnight at 4 °C. Protein G-Sepharose (Sigma) was used for precipitation of the immunocomplexes. The anti-PY antibody RC20 (Transduction Laboratories) was used for detection of phosphotyrosine proteins by immunoblot analysis. In the case of zymosan phagocytosis measured at 0, 15, 30, and 45 min, protein tyrosine phosphorylation was maximum at 15 min after addition (data not shown). J774 or HMDM were seeded on 35-mm dishes. The cells were washed twice with prewarmed RPMI medium without FBS, and then medium containing 1/10th volume of recombinant protein solution prepared at 5 µmwas added. Macrophage shape was observed with an Axiovert 135 microscope (Zeiss) equipped with a SenSys 1400 CCD camera (Roper Scientific) in chamber maintained at 37 °C with a 5% CO2 atmosphere. The bacterial pellet of E. coli HB101 grown to middle log phase was suspended in the FBS-free RPMI medium containing recombinant protein at a final concentration of 0.5 µm. Preparation of J774 cells and the phagocytosis assay were carried out under almost the same conditions as described for the gentamicin protection assay mentioned above. The cell medium was replaced with 0.5 ml/well RPMI without FBS prior to addition of the bacterial suspension. The bacterial pellet was added at an m.o.i. of ∼1000 for 15 min. After gentamicin treatment for 30 min, the internalized bacteria were recovered and spread onto L-agar plates. The DNA fragments containing virA oripaD gene amplified by PCR using primers virA-1 and virA-2 or ipaD-1 and ipaD-2, respectively, were cloned into pGEX-2T. After the purification under native conditions and digestion with thrombin according to the manufacturer's instructions, the GST fusion protein was dialyzed against Tris-buffered saline. pQE-ipaC-fl was digested with SacI and HindIII or StuI andHindIII in order to obtain the plasmids designated pQE-ipaC-ΔC1 (containing the coding region of IpaC amino acid residues 1–284) or pQE-ipaC-ΔC4 (containing the coding region of IpaC amino acid residues 1–169), respectively. After digestion, the DNA fragments containing the pQE-30 sequence were recovered from agarose gel and filled in using T4 DNA polymerase (Toyobo) followed by ligation. To obtain the plasmids named pQE-ipaC-ΔNC (containing the coding region of IpaC amino acid residues 117–284) and pQE-ipaC-ΔCTM (containing the ΔC1 deleted with coding region of IpaC amino acid residues 117–169), the DNA fragments were amplified by PCR using primers NC-1 and NC-2 or CTM-1 and CTM-2, respectively, together with pQE-ipaC-ΔC1 as a template. These DNA fragments were digested withBamHI (for construction of pQE-ipaC-ΔNC) orSalI (for construction of pQE-ipaC-ΔCTM) and
Referência(s)