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1 Laboratory of Microbial Genetics, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea
2 Institute of Biotechnology, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea
3 Department of Microbiology and Immunology, Chosun University School of Dentistry, Gwang ju 501-759, Republic of Korea
4 Department of Biological Science, Hanseo University, Seosan 356-706, Republic of Korea
5 Department of Microbiology, Hannam University, DaeJeon 300-791, Republic of Korea
Correspondence
Yong Keun Park
ykpark{at}korea.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Galán & Collmer, 1999; Hueck, 1998; Kimbrough & Miller, 2002; Ramamurthi & Schneewind, 2005). The T3SS is composed of a basal body that crosses both bacterial membranes, and an external needle through which effector proteins are secreted (Blocker et al., 2001; Hueck, 1998; Kimbrough & Miller, 2002).
Various studies have shown that the Salmonella invasion proteins (Sips) encoded by the T3SS genes of Salmonella pathogenicity island 1 (SPI-1) are central to initiation of the entry process. For example, individual non-polar sipB/sipC/sipD deletion mutants have been found to be entry-deficient, due to their inability to deliver other effectors into cultured mammalian cells (Collazo & Galan, 1997). The effector proteins SipB and SipC form an extracellular complex following their secretion through the SPI-1 T3SS, and they are thought to assemble into a plasma membrane-integral structure (translocon) that mediates effector delivery (Hayward & Koronakis, 1999; Scherer et al., 2000). The IpaB and IpaC proteins in Shigella flexneri, BipB and BipD in Burkholderia pseudomallei, YopB and YopD in Yersinia enterocolitica, EspB and EspD in enteropathogenic Escherichia coli (EPEC), BopB and BopD in Bordetella bronchiseptica, and PopB and PopD in Pseudomonas aeruginosa are thought to be homologous components of the translocation apparatus formed by SipB and SipC in Salmonella. All of these secreted proteins have been shown to be delivered to the host-cell membrane, where they form pore complexes (Daniell et al., 2001; Davis et al., 1998; Hayward et al., 2000; Ide et al., 2001; Kuwae et al., 2003; Neyt & Cornelis, 1999; Nogawa et al., 2004; Scherer et al., 2000; Suparak et al., 2005; Faudry et al., 2006; Johnson et al. 2006). In addition to its role as a translocon, SipB can also reportedly be phagocytized by macrophages and subsequently translocated via SPI-1 T3SS into the cytoplasm of the macrophage, where it induces apoptosis of the host cell by associating with the proapoptotic protease caspase-1 (Hersh et al., 1999).
It has been reported that Shigella IpaB, a homologue of SipB, is associated with the bacterial envelope (Menard et al., 1994; Mills et al., 1988; Watarai et al., 1995) and can be localized to the Shigella cell surface in the presence of T3SS (Olive et al., 2007). Recently, Johnson et al. (2006) have shown that surface localization of IpaB is dependent on the presence of IpaD. Veenendaal et al. (2007) have also suggested that the interaction between IpaB and IpaD at the needle tip is a key to host-cell sensing, and allows insertion of the translocation pore into the host-cell membrane. Interestingly, Hayward et al. (2000) have shown that Salmonella SipB can also be localized to the cell surface of bacteria during invasion of mammalian target cells, and furthermore that the protein fractionated with outer-membrane proteins. However, other evidence on SipB localization is conflicting. SipB can be detected (1) in the culture medium, (2) associated with the bacterial outer membrane, and (3) inserted into the target-cell plasma membrane (Hayward & Koronakis, 1999; Hayward et al., 2000; Kubori & Galan, 2002; Scherer et al., 2000).
Here, we confirmed that wild-type SipB protein could be localized on the surface of Salmonella, and showed the essential domain of SipB for mediating outer-membrane localization.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Strains were grown on L-agar or in L-broth supplemented with 0.3 M NaCl to allow optimal expression of the components of the invasion-associated T3SS. When required, the following antibiotics were added at the indicated concentrations: kanamycin, 50 µg ml–1; ampicillin, 100 µg ml–1; streptomycin, 100 µg ml–1; tetracycline, 10 µg ml–1.
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). Restriction enzymes, T4 ligase and Taq polymerase were purchased from Boehringer Mannheim or Takara. General transduction was performed with P22 HT105/int, and non-lysogenic segregants were identified by sensitivity to P22 H5 (Davis et al., 1980). Bacterial mutant strains (
sipB and sipD) were constructed by the 
Red recombinase method (Datsenko & Wanner, 2000).
Plasmid construction.
All plasmids used in this study are listed in Table 1
. For expression of the 140 aa SipB and its derivatives, we used plasmid pSSV160 (Kim et al. 2007) as a template. To construct pSSV140, the region encoding the N-terminal 140 aa of SipB was amplified using appropriate primers (forward primer PlacL, 5'-ccgactagtaatacgcaaaccgcctct-3'; reverse primer Sip140R, 5'-ccgggtaccgagatccgtcgcctcct-3'). The underlined sequences indicate the restriction enzyme sites for SpeI and KpnI, respectively. The amplified fragments (Plac and SipB 140 aa) were digested with SpeI and KpnI, and then ligated with T4 ligase (Takara) to pSSV160, which had been pre-digested with SpeI and KpnI and eluted. The same strategy was used to construct plasmids pSSV120 (SipB1–120-LLO-M45), pSSV100 (SipB1–100-LLO-M45), pSSV72 (SipB1–72-LLO-M45), pSSV30 (SipB1–30-LLO-M45), pSSV15 (SipB1–15-LLO-M45), pSSV8 (SipB1–8-LLO-M45) and pSSV1 (SipB1-LLO-M45) (see Table 2). To construct pSSV2-30, the region encoding the N-terminal 31–160 aa of SipB was amplified as a template pSSV160 using appropriate primers (forward primer SipB31F, 5'-ccggatatcatgacggactttttaaaagcggcg-3'; reverse primer Sip160R, 5'-ccgggtaccttacgcagcgtcataaacact-3'). The underlined sequences indicate the restriction enzyme sites for EcoRV and KpnI, respectively. The amplified fragments (SipB 31–160 aa) were digested with EcoRV and KpnI, and then ligated with T4 ligase (Takara) to pSSV160, which had been pre-digested with EcoRV and KpnI and eluted. The DNA sequences of the ligated clones were confirmed by DNA sequencing (Macrogen).
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). Plasmid pSM was used as template, and the resulting mutagenized PCR products were digested with DpnI and transformed into E. coli DH5
cells. Plasmids obtained from these transformed colonies were sequenced to confirm the desired mutation (Macrogen).
Preparation of culture supernatant proteins and Western blotting.
For preparation of culture supernatant proteins, bacterial supernatants (10 ml) were passed through a 0.45 µm pore-size syringe filter in order to remove the bacteria. The proteins present in the bacteria-free medium were precipitated to 10 % (v/v) by the addition of cold TCA followed by incubation on ice for 2 h. The proteins were collected by centrifugation at 10 000 g for 20 min at 4 °C. The resulting pellets were washed in 0.8 ml cold acetone, dried, and resuspended in PBS buffered with 80 mM Tris/HCl, pH 8.0. Samples corresponding to 100 µl whole bacterial culture or 200 µl culture supernatant were separated by 12 % SDS-PAGE and transferred to nitrocellulose membranes. Wild-type SipB, truncated SipB or reporter-fused SipB proteins were detected by immunoblot analysis. Western blots were treated with a mAb against the M45 tag (a kind gift from P. Hearing, State University of New York, Stony Brook, NY) or a polyclonal anti-SipB antibody (Kim et al., 2007) separately, followed by incubation with horseradish peroxidase-labelled anti-mouse or anti-rabbit antibodies, respectively. The blots were developed using a chemiluminescence detection kit (Amersham ECL Western Blotting Detection Reagent).
Salmonella subcellular fractionation.
The outer-membrane fraction was prepared by a modification of the lysozyme–osmotic shock method (Osborn & Munson, 1974). Cultures were grown in LB broth to OD600 0.9 and then centrifuged at 7000 g for 10 min, and supernatants were saved for analysis of secreted proteins. The cell pellets were resuspended in 800 µl 100 mM Tris/HCl buffer (pH 8.6) containing 500 mM sucrose and 0.5 mM EDTA. Hen egg white lysozyme (40 µl of a 4 mg ml–1 stock solution) was added, followed immediately by the addition of 3.2 ml 50 mM Tris/HCl buffer (pH 8.6) containing 250 mM sucrose, 0.25 mM EDTA and 2.5 mM MgCl2. After gentle agitation, the suspension was incubated for 15 min on an ice bath. The resulting pellet was resuspended in 4 ml 20 mM Tris/HCl (pH 8.6), disrupted by two passages through a French pressure cell (82 800 kPa; American Instrument Company) and centrifuged at 7000 g for 6 min at 4 °C in order to remove unbroken cells. The supernatant was then centrifuged at 132 000 g for 1 h at 4 °C to separate the soluble fraction and insoluble cell envelopes. The soluble fraction was taken as containing the cytoplasmic proteins. For isolation of the outer-membrane fraction, total envelope pellets were suspended in 4 ml 20 mM Tris/HCl (pH 8.6) containing 1 % Sarkosyl and incubated for 30 min on ice. The outer-membrane fraction was obtained as a pellet after centrifugation at 132 000 g for 1 h at 4 °C. The pellet was resuspended in 20 mM Tris/HCl buffer (pH 8.6). The original culture supernatant was filtered (0.22 µm pore-size filter), and precipitated with 10 % TCA (2 h, 4 °C). Each fraction was separated by SDS-PAGE for Western blot analysis.
Separation of inner- and outer-membrane fractions by sucrose gradient.
Salmonella enterica serovar Typhimurium (S. typhimurium) stationary-phase cultures were diluted 1 : 20 into LB medium, and incubated to OD600 0.9 under type III secretion induction conditions (0.3 M NaCl) with mild aeration. Cells were harvested by centrifugation (8000 g, 10 min), resuspended in 0.03 culture volumes of 20 mM Tris/HCl (pH 7.4) containing 1 mM PMSF, and disrupted in a French press (82 800 kPa, American Instrument Company). The lysate was clarified (8000 g, 10 min, 4 °C) and the resulting supernatant was centrifuged again (132 000 g, 1 h, 4 °C) to separate the soluble cytosolic proteins (supernatant) from the whole membranes (pellet). Isolated whole membranes were resuspended in 20 mM Tris/HCl (pH 8.6) containing 5 mM EDTA and layered onto a six-step sucrose gradient (1.0–2.0 M sucrose, 5 mM EDTA). The gradient was centrifuged (100 000 g, 36 h, 4 °C) and fractionated into 14 fractions, and the proteins from each fraction were precipitated with 10 % (v/v) TCA and analysed by immunoblotting. The outer-membrane protein OmpW (a kind gift from H. Y. Kang, Pusan National University, Pusan, Korea) was detected as an outer-membrane marker by immunoblotting.
Immunofluorescence assay (IFA).
Cultures were grown in LB broth containing 0.3 M NaCl to OD600 0.9. In order to stain the cells without leakage of the cell envelope, Hoechst 33342 (Sigma) was added to the bacterial culture at a final concentration of 1.5 g ml–1, followed by incubation for 15 min as described by Walberg et al. (1999). Bacterial cells were harvested by centrifugation (3000 g, 10 min). The cell pellets were gently washed three times with PBS, and then the cells were fixed for 1 h with freshly prepared 4 % paraformaldehyde, washed twice with PBS, and blocked with PBS containing 3 % BSA for 1 h at room temperature. The blocked cells were incubated for 1 h at room temperature with anti-M45 mAb, gently washed seven times with PBS, and then incubated with FITC-conjugated goat anti-mouse IgG secondary antibodies (Sigma). The cells were washed five times with PBS and briefly rinsed with water before being mounted onto slides for observation under fluorescence microscopy. For confocal microscopy, bacterial cells were grown in LB broth containing 0.3 M NaCl or 2.5 mM sodium deoxycholate (DOC; Sigma) to OD600 0.9, fixed with 4 % paraformaldehyde, overlaid on gelatin-coated slides (0.1 % gelatin, 0.01 %, v/v) and then stained with polyclonal anti-SipB/AlexaFluor 488-conjugated anti-rabbit IgG.
In situ protease treatment.
Salmonella cells harbouring pSSV160 were grown to the late-exponential phase and pelleted by centrifugation for 5 min at 5000 r.p.m. The pellet was washed once with 1x PBS and resuspended in the same buffer to a final concentration of 2x1010 bacteria ml–1. The bacteria were then incubated at 37 °C with proteinase K (20 or 200 µg ml–1) for 10, 20, 40 or 60 min, whereupon digestion was terminated by addition of PMSF to a final concentration of 1.6 mg ml–1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), particularly during invasion of mammalian target cells, when it may be fractionated with outer-membrane proteins (Hayward et al., 2000). It has been also reported that Shigella IpaB, a homologue of Salmonella SipB, may be associated with the bacterial surface (Menard et al., 1994; Mills et al., 1988; Watarai et al., 1995; Veenendaal et al., 2007). Therefore, we first sought to confirm that SipB protein was present on the bacterial outer membrane.
Wild-type and sipB deletion mutants were cultured under SPI-1-inducing conditions (0.3 M NaCl), and total cell membranes were isolated and fractionated by sucrose density-gradient centrifugation. Consistent with earlier observations (Collazo & Galan 1996; Hayward et al., 2000), wild-type SipB co-migrated with the bacterial outer-membrane marker OmpW (data not shown). To further confirm that wild-type SipB could be localized to the Salmonella cell surface, bacterial cells were labelled with a polyclonal anti-SipB antibody, as described in Methods. As shown in Fig. 1(c), SipB was successfully localized to the bacterial cell surface.
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). Culture supernatants, cell lysates and outer-membrane fractions were prepared from the wild-type, sipB and invA strains, and Western blotting of the combined bottom fractions (fractions 1–6; see Fig. 1 legend) was used to observe the localization of the wild-type SipB proteins. As shown in Fig. 1(a), SipB was not secreted to the culture supernatant or detected in outer-membrane fractions from the invA strain. This supports our contention that the presence of SipB in the outer-membrane fractions of wild-type preparations (Fig. 1b) was not due to random association with the cell membrane during the preparation process.
Recent reports have shown that localization of Shigella IpaB to the bacterial cell surface is dependent on the presence of IpaD, a homologue of Salmonella SipD, and have further shown that interaction of IpaB and IpaD at the needle tip is necessary for their insertion into the host-cell membrane (Johnson et al., 2006; Veenendaal et al., 2007). Since no earlier study has examined whether outer-membrane localization of SipB is dependent on SipD, we prepared culture supernatants, cell lysates and outer-membrane fractions from a sipD deletion strain, and examined SipB localization by Western blotting. As shown in Fig. 1(b), SipB was detected in the outer-membrane fractions from the sipD deletion strain. Collectively, these results indicate that SipB is localized on the Salmonella surface in a SipD-independent manner.
The N-terminal 160 residues of SipB could direct localization to the bacterial cell surface
In a recent study, we were able to show that a recombinant protein containing the N-terminal 160 aa of SipB (SipB160) could be successfully secreted into the culture supernatant (Kim et al., 2007). We next examined whether the same fragment of SipB (SipB160) could direct localization to the outer membrane, by using a recombinant plasmid containing SipB160 protein fused to a C-terminal M45 epitope tag (pSSV160).
To investigate whether recombinant SipB160 fusion proteins were displayed on the bacterial surface in a manner similar to that seen for wild-type SipB, immunofluorescence microscopic analysis was carried out with a mouse anti-M45 mAb. Hoechst 33342 dye was added to the bacterial culture to facilitate cell staining without leakage of the cell envelope, as described in Methods, and stained cells expressing the recombinant protein were probed with an anti-M45 primary mAb followed by an FITC-conjugated anti-mouse IgG secondary antibody. As shown in Fig. 2(a), IFA showed that the M45 molecules were exposed to the cell surface by recombinant proteins harbouring the N-terminal 160 aa of SipB, but not by fusion proteins lacking this fragment. To further confirm the surface localization of the recombinant SipB160, we carried out a proteinase K assay followed by Western blotting with anti-M45 antibody. Treatment of the recombinant SipB160-expressing cells with proteinase K resulted in degradation of the recombinant protein (Fig. 2b, upper panel). To examine this digestion in more detail, we carried out time-dependent proteinase K treatment (Fig. 2b, lower panel), and found that the recombinant SipB160 protein was partially degraded after 20 min, and completely degraded after 90 min. These data confirm that M45 molecules were displayed on the surface of bacteria through localization by SipB160.
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). Although all tested strains could secrete M45 (Fig. 3a), only the strain containing parental pSSV160 directed M45 to the outer membrane (Fig. 3b). To further clarify the region necessary for localization to the outer membrane, we generated fusion vectors that included the first 72 (pSSV72), 100 (pSSV100), 120 (pSSV120) and 140 (pSSV140) N-terminal residues. As shown in Fig. 3(c), pSSV120 and pSSV140 could mediate localization to the outer membrane, but pSSV100 could not, indicating that the fragment between N-terminal amino acids 100 and 140 of SipB was essential to the outer-membrane localization of SipB.
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2-30, a derivative of pSSV160 that lacks the secretion signal of SipB (Kim et al., 2007). As shown in Fig. 3(d), recombinant SipB lacking a secretion signal could not be localized to the outer membrane. Furthermore, native SipB was not localized to the outer membrane in an invA mutant strain lacking SPI-1-dependent T3SS (Fig. 1b). Taken together, these findings suggest that SipB localizes to the outer membrane following its secretion outside the cell.
To further elucidate the residues responsible for the outer-membrane localization of SipB, we first generated a template plasmid for alanine mutagenesis. We cloned a full-length sipB gene into a low-copy plasmid pMW118 (pSM), and introduced the pSM plasmid into the sipB strain. Preparation and analysis of culture supernatants, cell lysates and outer-membrane fractions from the sipB mutant strain containing plasmid pSM confirmed that, consistent with wild-type SipB, recombinant SipB was expressed in the cytosol, secreted into the culture supernatant, and localized on the bacterial outer membrane (Fig. 4). We then used an oligo cassette mutagenesis approach (see Methods) to construct eight variant plasmids in which blocks of 4 aa were replaced with alanine, stepwise from amino acids 111 to 142. Since a secondary-structure prediction of the N-terminal 160 aa of SipB revealed that amino acids 111–118 represented a putative coiled-coil domain (J net secondary structure prediction; http://www.compbio.dundee.ac.uk/
www-jpred/), we hypothesized that the putative coiled-coil domain of SipB (residues 111–118) was associated with the interaction with the bacterial cell surface, and first selected the region between amino acids 111 and 142 (pSA1–pSA8; see Table 1) for alanine substitution mutagenesis. SipBAla135-138 (pSA7) and wild-type SipB (pSM) were both expressed and secreted to culture supernatants at wild-type levels, whereas the levels of SipBAla135-138 in the outer-membrane fraction were dramatically reduced (Fig. 4b), demonstrating that amino acids 135–138 of SipB are required for SipB localization in the outer membrane.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Veenendaal et al., 2007). However, the surface localization of PopB and PopD of Pseudomonas, BopB and BopD of Bordetella, and BipB and BipD of Burkholderia have not yet been characterized. In the case of Salmonella, the surface localization of SipD has not been characterized before.
It was recently suggested that the cell-surface localization of Shigella IpaB, which is homologous to SipB, could be dependent on the presence of IpaD, and furthermore that an interaction between IpaB and IpaD is required for insertion of the translocation pore into the host-cell membrane (Johnson et al., 2006; Veenendaal et al., 2007). In Salmonella, sipD mutants have been shown to be deficient for invasion of epithelial cells (Kaniga et al., 1995; Collazo & Galan, 1997), even though these sipD mutants have increased secretion of Sip proteins (Kaniga et al., 1995). Here, we showed that SipB could be localized to the bacterial outer membrane of a sipD strain (Fig. 1). Therefore, we suggest that unlike IpaD (Veenendaal et al., 2007), SipD does not participate in localizing SipB to the bacterial cell surface.
SipB in S. typhimurium has been shown to have a few discrete functional domains, including a large C-terminal domain that contains two -helical transmembrane regions essential to its integration into the host-cell plasma membrane (McGhie et al., 2002). However, although numerous reports have focused on translocons, no earlier study has investigated whether IpaB and SipB have any specific regions required for bacterial surface localization. Here, we used deletion analysis to identify regions essential to the surface localization of SipB. Our results revealed that the N-terminal 160 aa of SipB (SipB160) were sufficient to direct outer-membrane localization. More detailed analysis identified the region between amino acids 100 and 140 as being essential for the outer-membrane localization of SipB160. Construction of plasmids encoding alanine-substituted full-length SipB and their expression in the sipB deletion strain revealed that construct pSA7, encoding SipBAla135-138, exhibited reduced outer-membrane localization. These findings collectively indicated that the N-terminal amino acids 100–140 of SipB are essential to its surface localization, and further suggested that SipB160 and wild-type SipB are localized to the same region of the Salmonella cell surface.
Many proteins secreted through T3SS components have been predicted to share a common coiled-coil structure (Pallen et al., 1997; Delahay & Frankel, 2002). The coiled-coil proteins are reported to interact with themselves and/or each other (Delahay & Frankel, 2002). Therefore, we hypothesized that residues 111–118 of SipB are required for an interaction with the bacterial cell surface. However, our results revealed that N-terminal residues 135–138 were indispensable for the surface localization of SipB. To further characterize residues 135–138, we aligned the amino acid sequences of BipB, IpaB and SipB. Interestingly, the amino acid 135 (E, Glu) in SipB (corresponding to residues 152 in BipB and 129 in IpaB) was identical in all three proteins, and a high sequence homology was found among residues 135–138 (data not shown). Glutamic acid is a negatively charged amino acid typically found on the surface of proteins. Thus, residue 135 may play a role in the binding of SipB to the outer membrane by interacting with proteins or other molecules.
This study revealed that a recombinant SipB protein (pSSV2-30) lacking a secretion signal could not be localized to the outer membrane, and invA mutant cells failing to secrete SipB also failed to show its localization to the outer membrane, suggesting that SipB is localized to the outer membrane after its secretion outside the cell. Our findings of the secretion-dependent localization of SipB to the outer membrane are consistent with an earlier report that showed that an invJ mutant, which was defective in secretion of SipB protein to the culture supernatant, also failed to show SipB localization to the bacterial cell surface (Collazo & Galan, 1996). Although the SPI-2-encoded translocon (SseB, SseC and SseD) is structurally different from that involving SPI-1, the former is also known to be localized to the outer surface of the bacterial cell after secretion in vitro (Beuzon et al., 1999; Klein & Jones, 2001; Nikolaus et al., 2001). Therefore, it seems as though the secretion of translocons such as SipB is indispensable for their localization to the bacterial cell surface.
Although future work will be required to elucidate the mechanism(s) underlying the specific targeting of SipB to the bacterial cell surface, our novel finding that the N-terminal region of SipB is required for its localization to the surface of Salmonella may provide new insights into the presence of translocons on the bacterial cell surface.
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ACKNOWLEDGEMENTS |
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Edited by: P. H. Everest
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Blocker, A., Jouihri, N., Larquet, E., Gounon, P., Ebel, F., Parsot, C., Sansonetti, P. & Allaoui, A. (2001). Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton. Mol Microbiol 39, 652–663.[CrossRef][Medline]
Bullas, L. R. & Ryu, J. I. (1983). Salmonella typhimurium LT2 strains which are r– m+ for all three chromosomally located systems of DNA restriction and modification. J Bacteriol 156, 471–474.
Collazo, C. M. & Galan, J. E. (1996). Requirement for exported proteins in secretion through the invasion-associated type III system of Salmonella typhimurium. Infect Immun 64, 3524–3531.[Abstract]
Collazo, C. M. & Galan, J. E. (1997). The invasion-associated type III system of Salmonella typhimurium directs the translocation of Sip proteins into the host cell. Mol Microbiol 24, 747–756.[CrossRef][Medline]
Cornelis, G. R. & Van Gijsegem, F. (2000). Assembly and function of type III secretory systems. Annu Rev Microbiol 54, 735–774.[CrossRef][Medline]
Curtiss, R., III, Porter, S. B., Munson, M., Tinge, S. A., Hassan, J. O., Gentry-Weeks, C. & Kelly, S. M. (1991). Nonrecombinant and recombinant avirulent salmonella vaccines for poultry. In Colonization Control of Human Bacterial Enteropathogens in Poultry, pp. 169–198. Edited by L. C. Blankenship, J. H. S. Bailey, N. A. Cox, N. J. Stern & R. J. Meinersmann. New York: Academic Press.
Daniell, S. J., Delahay, R. M., Shaw, R. K., Hartland, E. A., Pallen, M. J., Booy, F., Ebel, F., Knutton, S. & Frankel, G. (2001). Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis. Infect Immun 69, 4055–4064.
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.
Davis, R. W., Bolstein, D. & Roth, J. R. (1980). Advanced Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Davis, R., Marquart, M. E., Lucius, D. & Picking, W. D. (1998). Protein–protein interactions in the assembly of Shigella flexneri invasion plasmid antigens IpaB and IpaC into protein complexes. Biochim Biophys Acta 1429, 45–56.[CrossRef][Medline]
Delahay, R. M. & Frankel, G. (2002). Coiled-coil proteins associated with type III secretion systems: a versatile domain revisited. Mol Microbiol 45, 905–916.[CrossRef][Medline]
Faudry, E., Vernier, G., Neumann, E., Forge, V. & Attree, I. (2006). Synergistic pore formation by type III toxin translocators of Pseudomonas aeruginosa. Biochemistry 45, 8117–8123.[CrossRef][Medline]
Galán, J. E. & Collmer, A. (1999). Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284, 1322–1328.
Galan, J. E. & Curtiss, R., III (1991). Distribution of the invA, -B, -C, and -D genes of Salmonella typhimurium among other Salmonella serovars: invA mutants of Salmonella typhi are deficient for entry into mammalian cells. Infect Immun 59, 2901–2908.
Hayward, R. D. & Koronakis, V. (1999). Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella. EMBO J 18, 4926–4934.[CrossRef][Medline]
Hayward, R. D., McGhie, E. J. & Koronakis, V. (2000). Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion. Mol Microbiol 37, 727–739.[CrossRef][Medline]
Hersh, D., Monack, D. M., Smith, M. R., Ghori, N., Falkow, S. & Zychlinsky, A. (1999). The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc Natl Acad Sci U S A 96, 2396–2401.
Hueck, C. J. (1998). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62, 379–433.
Ide, T., Laarmann, S., Greune, L., Schillers, H., Oberleithner, H. & Schmidt, M. A. (2001). Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli. Cell Microbiol 3, 669–679.[CrossRef][Medline]
Johnson, S., Roversi, P., Espina, M., Olive, A., Deane, J. E., Birket, S., Field, T., Picking, W. D., Blocker, A. J. & other authors (2006). Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD. J Biol Chem 282, 4035–4044.[CrossRef][Medline]
Kaniga, K., Tucker, S., Trollinger, D. & Galán, J. E. (1995). Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells. J Bacteriol 177, 3965–3971.
Kim, B. H., Kim, H. G., Kim, J. S., Jang, J. I. & Park, Y. K. (2007). Analysis of functional domains present in the N-terminus of the SipB protein. Microbiology 153, 2998–3008.
Kimbrough, T. G. & Miller, S. I. (2002). Assembly of the type III secretion needle complex of Salmonella typhimurium. Microbes Infect 4, 75–82.[CrossRef][Medline]
Klein, J. R. & Jones, B. D. (2001). Salmonella pathogenicity island 2-encoded proteins SseC and SseD are essential for virulence and are substrates of the type III secretion system. Infect Immun 69, 737–743.
Kubori, T. & Galan, J. E. (2002). Salmonella type III secretion-associated protein InvE controls translocation of effector proteins into host cells. J Bacteriol 184, 4699–4708.
Kuwae, A., Ohishi, M., Watanabe, M., Nagai, M. & Abe, A. (2003). BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cells. Cell Microbiol 5, 973–983.[CrossRef][Medline]
McGhie, E. J., Hume, P. J., Hayward, R. D., Torres, J. & Koronakis, V. (2002). Topology of the Salmonella invasion protein SipB in a model bilayer. Mol Microbiol 44, 1309–1321.[CrossRef][Medline]
Menard, R., Sansonetti, P. & Parsot, C. (1994). The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. EMBO J 13, 5293–5302.[Medline]
Mills, J. A., Buysse, J. M. & Oaks, E. V. (1988). Shigella flexneri invasion plasmid antigens B and C: epitope location and characterization with monoclonal antibodies. Infect Immun 56, 2933–2941.
Neyt, C. & Cornelis, G. R. (1999). Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG. Mol Microbiol 33, 971–981.[CrossRef][Medline]
Nikolaus, T., Deiwick, J., Rappl, C., Freeman, J. A., Schröder, W., Miller, S. I. & Hensel, M. (2001). SseBCD proteins are secreted by the type III secretion system of Salmonella pathogenicity island 2 and function as a translocon. J Bacteriol 183, 6036–6045.
Nogawa, H., Kuwae, A., Matsuzawa, T. & Abe, A. (2004). The type III secreted protein BopD in Bordetella bronchiseptica is complexed with BopB for pore formation on the host plasma membrane. J Bacteriol 186, 3806–3813.
Olive, A. J., Kenjale, R., Espina, M., Moore, D. S., Picking, W. L. & Picking, W. D. (2007). Bile salts stimulate recruitment of IpaB to the Shigella flexneri surface, where it colocalizes with IpaD at the tip of the type III secretion needle. Infect Immun 75, 2626–2629.
Osborn, M. J. & Munson, R. (1974). Separation of the inner (cytoplasmic) and outer membranes of Gram-negative bacteria. Methods Enzymol 31, 642–653.[Medline]
Pallen, M. J., Dougan, G. & Frankel, G. (1997). Coiled-coil domains in proteins secreted by type III secretion systems. Mol Microbiol 25, 423–425.[CrossRef][Medline]
Ramamurthi, K. S. & Schneewind, O. (2005). A synonymous mutation in Yersinia enterocolitica yopE affects the function of the YopE type III secretion signal. J Bacteriol 187, 707–715.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Scherer, C. A., Cooper, E. & Miller, S. I. (2000). The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection. Mol Microbiol 37, 1133–1145.[CrossRef][Medline]
Suparak, S., Kespichayawattana, W., Haque, A., Easton, A., Damnin, S., Lertmemongkolchai, G., Bancroft, G. J. & Korbsrisate, S. (2005). Multinucleated giant cell formation and apoptosis in infected host cells is mediated by Burkholderia pseudomallei type III secretion protein BipB. J Bacteriol 187, 6556–6560.
Veenendaal, A. K., Hodgkinson, J. L., Schwarzer, L., Stabat, D., Zenk, S. F. & Blocker, A. J. (2007). The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Mol Microbiol 63, 1719–1730.[CrossRef][Medline]
Walberg, M., Gaustad, P. & Steen, H. B. (1999). Uptake kinetics of nucleic acid targeting dyes in S. aureus, E. faecalis and B. cereus: a flow cytometric study. J Microbiol Methods 35, 167–176.[CrossRef][Medline]
Watarai, M., Tobe, T., Yoshikawa, M. & Sasakawa, C. (1995). Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness. EMBO J 14, 2461–2470.[Medline]
Received 11 July 2007;
revised 22 September 2007;
accepted 10 October 2007.
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