Type III secretion systems (T3SS) are nano-syringes used by a wide range of Gram-negative pathogens to promote infection by directly injecting effector proteins into targeted host cells. Translocation of effectors is triggered by host-cell contact and requires assembly of a pore in the host-cell plasma membrane, which consists of two translocator proteins. Our understanding of the translocation pore, how it is assembled in the host cell membrane and its precise role in effector translocation, is extremely limited. Here we use a genetic technique to identify protein-protein contacts between pore-forming translocator proteins, as well as the T3SS needle-tip, that are critical for translocon function. The data help establish the orientation of the translocator proteins in the host cell membrane. Analysis of translocon function in mutants that break these contacts demonstrates that an interaction between the pore-forming translocator PopD and the needle-tip is required for sensing host cell contact. Moreover, tethering PopD at a dimer interface also specifically prevents host-cell sensing, arguing that the translocation pore is actively involved in detecting host cell contact. The work presented here therefore establishes a signal transduction pathway for sensing host cell contact that is initiated by a conformational change in the translocation pore, and is subsequently transmitted to the base of the apparatus via a specific contact between the pore and the T3SS needle-tip.
Type III secretion systems (T3SSs) are molecular syringes employed by a wide range of Gram-negative pathogens to directly inject effector proteins into targeted host cells, and thereby promote disease [1,2]. A hallmark of these virulence-associated T3SSs is that effector secretion is triggered by host cell contact, and that injection of effector proteins into the cell is vectorial [3]. Only T3SS that engage the host cell trigger effector secretion, and effectors are injected into the host cell, but not into the surrounding milieu. The structure that facilitates this directed injection of effector proteins is the translocon [4–8]. Upon cell contact, the T3SS needle is brought into close proximity of the host cell plasma membrane. The specialized tip structure at the distal end of the T3SS needle facilitates the insertion of the pore forming translocator proteins into the targeted host cell membrane [9–11]. Animal pathogens produce two hydrophobic translocators that assemble to form the translocation pore [7,12,13]. The pore likely docks to the needle tip, forming the translocon [14,15], through which effectors are subsequently injected into the host cell. Atomic force imaging of pores assembled by enteropathogenic E. coli into red blood cell membranes suggests that the pores have a 6–8 fold symmetry [16]. Triggering of effector secretion depends on the translocon, and does not occur if the translocon is not assembled [17]. Our understanding of the translocon is limited. While pore forming translocator proteins can insert into membranes in vitro [18–20] the organization of the translocation pore, including the stoichiometry of the pore forming translocators, and in some instances, their orientation in the membrane are unknown. Moreover, the behavior of translocator proteins in vitro does not perfectly mimic observations made in the context of bacterial infection. For example, while the Pseudomonas aeruginosa pore-forming translocator proteins, PopB and PopD, can form pores individually in lipid vesicles in vitro [18], pore formation by translocator proteins delivered by the bacterium, requires both PopB and PopD [9]. Similarly, while PcrV facilitates translocator insertion [9,10], it does not interact with PopB or PopD in vitro [18,21]. In a similar vein, potential triggers for effector secretion have been proposed for a number of T3SS [22,23], however, it is not known what component of the T3SS senses host cell contact to initiate injection of effector proteins. Here we use a genetic approach to map interactions between translocator proteins that are critical for the delivery of effectors into host cells. The approach is based on the fact that translocator proteins between closely related T3SS are often unable to cross-complement [24–26]. We exploited the incompatibility of the translocator proteins of P. aeruginosa and Yersinia pseudotuberculosis, to map critical interactions by generating hybrid proteins that restore translocon function. In this manner we mapped two contacts, one between the needle tip protein PcrV and the pore-forming translocator protein PopD, as well as a second region of contact between PopB and PopD. We also determined that both PopB and PopD form dimers. Importantly, we demonstrate that tethering the PopD dimer by forming a disulfide bond specifically interferes with triggering of effector secretion. Similarly, breaking the PopD-PcrV contact prevents transmission of the host-cell contact signal to commence effector secretion. Our data support a model in which the translocation pore is actively involved in sensing host cell contact, a signal that is transmitted to the base of the apparatus via contacts between the pore-forming translocator proteins and the needle tip.
Given the difficulty of studying translocon function in vitro, we employed a genetic approach to map translocator interactions that are critical for function. To this end we exploited the incompatibility between related translocator proteins. Previous work by others had shown that the translocator proteins of P. aeruginosa cannot substitute for the corresponding proteins in Yersinia sp. , despite the overall similarity between these T3SSs. Specifically popD was unable to complement a yopD mutant of Y. pseudotuberculosis [12], and pcrV could not restore translocation in an lcrV mutant of Y. eneterocolitica. Interestingly, in the case of PcrV, function could be restored by exchanging the N-terminal globular domain of PcrV with that of LcrV, generating an LcrV-PcrV hybrid [27]. We recapitulated these data by producing the Y. pseudotuberculosis pore-forming translocator proteins, YopB and YopD, in P. aeruginosa and assaying their ability to restore translocation of effectors into A549 epithelial cells in a strain lacking the corresponding P. aeruginosa translocator proteins, PopB and PopD. Consistent with the data from Y. enterocolitica, YopB and YopD were unable to restore effector translocation unless the needle-tip protein PcrV was replaced by a hybrid protein in which the N-terminal domain of PcrV was replaced with the corresponding domain of LcrV (LcrV-PcrV hybrid “V1”) (Fig 1A). We next expanded on this observation by combining P. aeruginosa and Y. pseudotuberculosis translocators. Combining YopD with PopB, resulted in a translocon that was functional in the context of the V1 needle tip, but not wild-type PcrV (Fig 1A). These data indicate that PopB and YopD can form functional pores, but that YopD makes a critical contact with the N-terminal domain of LcrV that is broken when YopD is produced in the context of the P. aeruginosa needle-tip protein PcrV. The reciprocal combination, YopB and PopD, was non-functional regardless of the needle-tip, arguing that YopB and PopD are unable to form a functional translocon (Fig 1A). We conclude that combining Yersinia translocator proteins with P. aeruginosa translocator proteins breaks two interactions that are required for T3SS function: one between PopD and PcrV, the other between PopB and PopD. We initially focused on the PopD-PcrV interaction since the mismatch incompatibility between YopD and PcrV resulted in the stronger defect in T3SS function. To map the region of PopD that interacts with PcrV, we generated YopD-PopD hybrid proteins in which portions of the C-terminal half of YopD were replaced by the corresponding region of PopD (S1A Fig). Strains producing these YopD-PopD hybrid proteins (D1-D3) in the context of the wild-type, PcrV, needle-tip were assayed for their ability to intoxicate epithelial cells (Fig 1B). YopD-PopD fusion proteins in which as little as the C-terminal 27 amino acids of YopD were replaced with the corresponding residues of PopD were able to fully substitute for PopD in this assay, arguing that it is the very C-terminus of PopD that has to interact with PcrV to allow intoxication of host cells. Indeed, substituting individual residues that differ between YopD and PopD in this region demonstrates that two of these differing residues contribute significantly to the loss of function when YopD is paired with PcrV (Fig 1C). Notably, the phenylalanine at position 303 of YopD is particularly deleterious for the interaction with PcrV, and a significant amount of T3SS function can be recovered by simply changing this residue to the corresponding alanine residue of PopD (Fig 1C). We similarly mapped the PopD-PcrV interaction by systematically replacing smaller portions of the N-terminal domain of PcrV with the corresponding regions of LcrV (Figs 2A, 2B and S2). The ability of these hybrid needle tips to support injection of host cells by bacteria producing YopD was assayed by monitoring intoxication of A549 epithelial cells (Fig 2C). YopD (F303A), which can function with PcrV and LcrV served as a control. These data indicate that the C-terminus of PopD has to contact a region of PcrV formed by α-helices 4,5, and 6. This region was modeled by threading PcrV into the known structure of LcrV (Fig 2B). In order to demonstrate this contact directly, we substituted alanine 292 of PopD (corresponding to residue 303 of YopD) and residue Q87 of PcrV (Fig 2B) with cysteines to determine if these two residues can form a disulfide bond, linking PopD and PcrV. PopB, PopD, and PcrV are naturally devoid of cysteines. Infection of cells in the presence of copper as an oxidant resulted in formation of a PcrV-PopD disulfide bond which depended on the presence of both cysteines, demonstrating that the C-terminus of PopD indeed binds to PcrV at the positions indicated by our genetic mapping (Fig 2D). Notably, PopD (R243C), in which the cysteine lies outside of the C-terminal 27 amino acids that are responsible for the YopD-PcrV incompatibility, could also be crosslinked to PcrV (Q87C). While the latter result is consistent with the overall orientation of the translocator proteins and our assertion that the C-terminus of PopD interacts with the amino-terminal globular domain of PcrV, we cannot infer the exact manner in which the C-terminus of PopD binds to PcrV from these data. Instead, this result would argue that there is some mobility of the C-terminus of PopD relative to PcrV. Consistent with a function in translocation, the interactions could only be trapped in the context of the host cell, since the heterodimer could not be visualized in protein secreted into the culture supernatant. Given that we can specifically break the PopD-PcrV contact by substituting the C-terminus of PopD with the corresponding region of YopD, we next sought to assign a specific function to this interaction. In principle, the interaction between PopD and PcrV could be involved in one of three aspects of translocon function: insertion of PopD into the host cell membrane, triggering of effector secretion, and delivery of effectors into the host cell (e. g. by forming a conduit between the bacterium and the host-cell). In order to distinguish between these three possibilities, we first examined the effect of breaking the PopD-PcrV contact on translocator insertion. Insertion of translocator proteins into red blood cells, and subsequent pore formation, results in hemolysis, which can be assayed spectrophotometrically [7]. To examine the effect of breaking the PopD-PcrV interaction on translocator insertion, we generated a strain that produces a hybrid PopD-YopD fusion protein (D4) in which the C-terminal 27 amino acids of PopD have been replaced with the corresponding residues of YopD. This strain is severely defective for delivery of effectors into host cells, unless produced in the context of the V1 needle tip (see below). Hemolysis in this assay is strictly T3SS-dependent, as illustrated by the fact that deletion of pcrV abrogates hemolysis entirely (Fig 3A). The strain producing the D4 hybrid translocator, on the other hand, promoted hemolysis to nearly the same level as cells producing PopD. Moreover, there was no statistical difference in hemolysis between a strain producing the D4 hybrid in the context of the wild-type PcrV needle tip (no interaction), as opposed to the hybrid V1 needle tip (restores interaction), arguing that translocon insertion is not significantly impaired. Indeed, assaying translocator insertion directly by isolating the RBC plasma membranes on sucrose density gradients illustrated that translocator insertion was not impaired (Fig 3B). Consistent with previously published data on PopB and PopD [9], the D4 hybrid was resistant to extraction of the membranes with high salt or high pH solutions, arguing that it is inserted into the membrane and not associated peripherally. Based on these results, the defect incurred by breaking the PopD-PcrV interaction could therefore be either at the level of triggering of effector secretion, or delivery of effectors into the host cell (docking/conduit formation). We distinguished between these two possibilities by making use of a mutation that abrogates the need for a specific trigger for effector secretion [30,31]. Deleting pcr1, encoding a component of the PopN complex, which prevents effector secretion before cell contact, results in secretion of effector proteins in the absence of host cell contact [32,33]. Notably, a pcr1 mutant is still able to inject effectors into host cells, an activity that relies on a functional translocon (Fig 4A). Therefore, if breaking the PopD-PcrV contact blocks triggering, removing pcr1 should restore effector translocation, since the system no longer needs a specific trigger to commence secreting effectors. Conversely, if the PopD-PcrV contact is need for conduit formation, then removing pcr1 should not correct the defect incurred by breaking this interaction (Fig 4B). To determine which of these two possibilities is the case we assayed injection of ExoS into epithelial cells directly. Here we relied on a strain that produces a mutant version of ExoS in which both active sites have been inactivated by point mutation. This strain increases the signal in the assay, since ExoS feedback inhibits its own translocation thereby severely limiting the amount of the wild-type protein injected [17]. As can be seen in Fig 4C, breaking the PopD-PcrV contact severely impairs translocation of ExoS into host cells. Restoring the contact by also producing the V1 needle tip restores translocation, indicating that the defect in translocation in strains producing the D4 PopD-YopD hybrid is due to the lost PopD-PcrV interaction. Importantly, in a strain lacking pcr1 translocation of ExoS by the strain producing the D4 hybrid is restored to near wild type levels, arguing that the PopD-PcrV contact is specifically required for triggering of effector secretion (Figs 4C and S3). We next set out to map the critical contact between PopB and PopD that is disrupted by pairing PopD with YopB. We again generated strains producing PopD-YopD and PopB-YopB hybrid proteins (S1 Fig), paired with YopB and PopD, respectively. Restoration of T3SS function was assayed by monitoring the ability to intoxicate epithelial cells (Fig 5). These experiments allowed us to narrow down the region of interaction to residues 228–245 of PopD and 274–297 of PopB. However, we were unable to assign the bulk of the defect in the YopB-PopD interaction to a specific residue, as had been the case for the PopD-PcrV interaction. Instead, the defect appears to be due to numerous small incompatibilities that are difficult to map genetically. In the course of attempting to introduce cysteines into PopB and PopD to crosslink the two proteins, we found that both PopB and PopD form dimers, which can be trapped by an intermolecular disulfide bond (Figs 6A and S4A, lane 4). In order to demonstrate that these higher molecular weight species represent dimers of PopB and PopD, respectively, we co-expressed functional, size-tagged versions of either translocator, in which four copies of the VSV-G-epitope had been inserted at a neutral site. Insertion of the VSV-G tags allowed us to distinguish the tagged version of the translocator from that expressed by the untagged chromosomal gene (Figs 6A and S4A, lanes 5–9). Indeed, if PopD (R243C) and the size-tagged version, PopD (R243C) -4VG, were co-expressed in the presence of copper, we detected three higher molecular weight complexes, indicating the formation of homodimers of either protein, as well as a heterodimer of intermediate molecular weight (Fig 6A, lane 8). The latter species would not be expected if PopD were disulfide bonding to a different protein. Reduction of the sample through the addition of DTT dissolved the higher molecular weight complexes (Fig 6A, lane 9), demonstrating that they are tethered through a disulfide bond. The same was true for PopB (A280C) (S4 Fig). Since we can use the PopD (R243C) disulfide bond to tether the PopD dimer, we next asked whether linking the two PopD molecules interferes with translocon function. Indeed, tethering the PopD dimer at position 243 dramatically reduced the amount of ExoS translocated into epithelial cells (Fig 6B). Importantly, this defect was not evident when the experiment was performed in the presence of the membrane-impermeant reductant TCEP, demonstrating that PopD (R243C) is not defective per se, rather formation of the disulfide bond prevents effector translocation. Tethering PopB at position A280, on the other hand, only had a minor effect on translocation (S4 Fig), demonstrating that the defect in translocation is not an artifact of the growth conditions. We next removed pcr1 to test whether the defect in translocation is due to a defect in triggering of effector secretion, or due to malformation of the translocon, preventing effector delivery. As for the PopD-PcrV interaction above, removing pcr1 restored effector translocation in the presence of copper by the strain producing PopD (R243C). Deleting pcr1 does not interfere with dimer formation (Fig 6A), arguing that tethering the PopD-dimer at position 243 specifically interferes with triggering of effector secretion. By extension, these data demonstrate that the translocon is actively involved in sensing host-cell contact. Tethering the PopD dimer presumably blocks a conformational change that is induced by host-cell contact and is transmitted to the base of the apparatus by an interaction with the needle-tip.
We exploited the incompatibility between the related translocator proteins of P. aeruginosa and Y. pseudotuberculosis to map protein-protein contacts among translocator proteins that are critical for translocon function. Our analysis indicated that expressing Y. pseudotuberculosis translocator proteins in P. aeruginosa disrupts two interactions: one between PopD and PcrV, the second between PopB and PopD. The PopD-PcrV interaction is specifically required for triggering of effector secretion. Moreover, our analysis indicates that triggering of effector secretion likely involves a conformational change in the translocation pore, which is then transmitted to the needle tip. Our data also lend insight into the overall organization of the translocon. Both pore-forming translocator proteins form dimers, and the location of the contacts we mapped allow us to orient PopB, PopD, and PcrV relative to one another. The organization of the translocation pore has been difficult to study. In part, this is due to the fact that it has not been possible to isolate translocation pores assembled in the context of an infection. Export of translocators before cell contact [17], and presumably non-specific binding to cell surfaces, has further muddled the analysis of the topology of inserted translocators. For example, three orientations have been proposed for the translocator equivalent to PopD in the P. aeruginosa system: N-terminus in/C-terminus out, N-terminus out/C-terminus in and both N and C-terminus out [34–36]. By mapping contacts that are required for translocon function, we can sidestep these difficulties since any protein-protein interaction we identify has to occur either during assembly of, or in the context of the fully formed translocon. Accordingly, our data indicate that the C-terminus of PopD faces the extracellular milieu, where it contacts the PcrV needle tip (Fig 7A). Our data are also consistent with the recently published topology of PopB, whereby PopB is proposed to insert into the host cell plasma membrane with both the N- and C-terminus facing the extracellular milieu [37]. It seems likely that PopD is inserted into the host cell plasma membrane with the N-terminus facing the cytoplasm rather than being attached peripherally since PopD is resistant to extraction by high salt or high pH solutions [9] (Fig 3B). This has been disputed due to the difficulty of inserting PopD into artificial membranes in vitro, leading to the proposal that only PopB inserts to form the pore, while PopD serves to connect PopB to the needle-tip. [38]. A possible explanation for this discrepancy is the fact that insertion of the pore-forming translocator proteins, when delivered by the bacterium, is facilitated by the needle-tip [9,11,27], which prompted Cornelis and co-workers to propose that the needle-tip functions as a scaffold for the assembly of the translocation pore [8,27]. Triggering of effector secretion on cell contact is a hallmark of T3SSs, however the mechanism of triggering remains enigmatic. Up to this point, the role of the translocon in triggering effector secretion has been unclear. In the case of the Shigella flexneri T3SS it was proposed that insertion of one of the translocator proteins, IpaB, results in recruitment of the second pore-forming translocator protein, IpaC, which in turn allows formation of the translocation pore and subsequent effector export [39]. While several triggers have been proposed, for example exposure of the needle to the low-calcium environment of the host cell, or changes in pH [22,23], it is unclear from these data whether the translocon has a passive role in triggering of effector secretion (e. g. by establishing the pore which then allows another part of the secretion needle to respond to a chemical property of the connected compartment), or whether the translocon actively senses host cell contact. Our data indicate that the latter is the case. Tethering the dimer of PopD at position 243 by introducing a disulfide bond specifically interfered with triggering of effector secretion, but not pore-formation or injection of effectors. These data argue that the translocation pore has to undergo a conformational change to trigger effector export, which is prevented by the disulfide bond at position 243. Our data indicate that the C-terminus of PopD has to interact with PcrV in order to transmit the signal to commence effector secretion. Early data using the Y. pseudotuberculosis system indicated that the C-terminus of YopD is important for translocation. Small deletions of residues 278–292, or 293–305, blocked effector translocation [40,41], however, these deletions also impacted assays of pore-formation, such as translocon-mediated hemolysis, arguing that the deletion mutants had lost the ability to form a functional translocon altogether [41]. Subsequent analysis of point mutations in a putative amphipathic α-helix located near the C-terminus of YopD (residues 278–292) revealed that a subset of these mutations had a reduced ability to bind to the needle-tip protein LcrV in a pull-down assay [42]. Several of these mutants had also lost the ability to deliver effectors into HeLa cells, which led the authors to propose that the YopD-LcrV interaction is needed for function. Our analysis is consistent with- and significantly extends these findings. First, we were able to map the point of contact of PopD on PcrV. Moreover, we demonstrated through direct crosslinking, that this interaction occurs specifically at the cell surface upon translocon assembly (as noted in the introduction, PcrV fails to bind to PopD in vitro, [18,21]). Finally, our data indicate that the interaction of the extreme C-terminus of PopD with PcrV (which differs slightly from the region analyzed by Costa et al.) is specifically required for triggering of effector secretion. The proposed role of the N-terminal domain of PcrV in signal transduction is further bolstered by a previously published linker insertion mutagenesis analysis of PcrV function. Here, three insertions in the N-terminal domain (after amino acids 44,52, and 64 of PcrV) resulted in proteins that still supported translocation of effectors into host cells, but resulted in deregulated effector secretion [43]. PcrV prevents effector secretion by constraining the T3SS in an effector secretion off conformation [44]. These insertion mutants have specifically lost the ability to constrain the apparatus in the effector secretion “off” conformation. By extension, we would propose that the triggering conformational change in PopD similarly relieves the constraint PcrV imposes on effector secretion through its interaction with the N-terminal domain of PcrV. A similar constraint on effector secretion by the needle-tip complex has been proposed in S. flexneri. Here the tip complex is composed of two proteins, IpaD and IpaB [39]. A short, three amino acid C-terminal truncation of IpaB results in a protein that still associates with the needle-tip, but has lost the ability to prevent effector secretion before cell contact [45]. The model is also compatible with known needle mutations that trigger effector secretion in the absence of cell contact, as well as mutations throughout the basal body that similarly result in premature secretion of effectors [46–49]. Our data identifies a signal transduction pathway (Fig 7B), whereby an as yet unknown trigger effects a conformational change in the translocation pore. This structural change is then transmitted to the needle tip via an interaction between PopD and PcrV, and from there, through a conformational change in the needle, to the base of the apparatus.
Bacterial strains and plasmids used in this study are listed in S1 Table. E. coli strains were grown in LB medium (10g tryptone, 5g yeast extract, 10g NaCl per liter), supplemented with 15μg/ml gentamicin when necessary. P. aeruginosa strains were grown on “high salt” LB (10g/L tryptone, 5g/L yeast extract, 200mM NaCl, 5mM MgCl2,0. 5mM CaCl2), supplemented with 30μg/ml of gentamicin when necessary. Assembly of the T3SS is controlled by the osmolarity of the medium. Use of “high salt” LB allows for consistent production of the T3SS under laboratory conditions [50]. Production of translocator proteins was induced through the addition of IPTG to the growth medium. A549 cells (American Type Culture Collection, Cat. #CCL-185) were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (RP10) at 37°C in a 5% CO2 atmosphere. Cells were maintained in the presence of penicillin and streptomycin. Before experiments, cells to be infected were washed 1x with Dulbecco’s phosphate buffered saline (DPBS), and the medium was exchanged with RP10 lacking antibiotics. Mutations were introduced into the chromosome of strains through allelic exchange as described previously [51]. Plasmids were constructed using standard molecular biological techniques. The indicated translocator (or portion of a translocator) was amplified by polymerase chain reaction using the primers listed in S1 Table. Y. pseudotuberculosis YPIII DNA was used as template for the amplification of amplification of yopD, yopB, and lcrV. Hybrid translocators were generated using splicing by overlap extension (SOE) PCR. PCR products were cut with the appropriate restriction enzymes (listed in S2 Table) and ligating into plasmids pEXG2 (allelic exchange vector) or pPSV37 (plasmid that can replicate in P. aeruginosa with lacUV5 promoter and lacIq). P. aeruginosa strains were grown to mid-logarithmic phase in “High salt” LB, pelleted and resuspended in PBS-MC [Dulbeccos’s phosphate buffered saline, Invitrogen Cat# 14190–144, supplemented with 5mM MgCl2 and 0. 5mM CaCl2 (final concentrations) ]. A549 epithelial cells were infected at an MOI of 10 for 2–4 hours. Where necessary, 100μM IPTG was added to the media to induce translocator gene expression from plasmid constructs. Following the infection period, the media was removed, and the cells were fixed using 3. 7% formaldehyde for 15 min. Rounded versus flat cells were counted by low-power phase contrast microscopy. Data are reported as mean of three biological replicates with standard deviation. P. aeruginosa strains were grown to mid-logarithmic phase in “High salt” LB, pelleted and resuspended in PBS-MC. A549 epithelial cells were infected with P. aeruginosa strains at an MOI of 25 for 2 hours. The cells were then washed three times with PBS-MC, and rinsed with 1ml of 250 μg/mL proteinase K in PBS-MC. The protease solution was then removed, and the cells were incubated at room temperature for 15 minutes to digest extracellular protein. The protease-treated cells were resuspended in 1ml of PBS-MC with 2mM phenylmethylsulfonyl fluoride (PMSF), and pelleted (3 minutes, 5000 rpm). The cells were resuspended in 300μL of PBS-MC with 0. 5% Triton X-100 and incubated on ice for 15 minutes. 150μL of the cell suspension were removed and mixed with 50μl of 4x SDS sample buffer (SDS sample). The remaining cells were pelleted, and 150μL of supernatant were removed and combined with 50μL of 4x SDS sample buffer (Triton solubilized fraction). The samples were separated by SDS-PAGE and analyzed by Western blot. Membranes were probed with antibodies directed against ExoS, RpoA (Neoclone) and either human glucose-6-phoshate isomerase (G6PI, Santa Cruz Biotechnology Inc.), actin (Developmental Studies Hybridoma bank), or tubulin (Santa Cruz Biotechnology Inc.). Antibodies were detected using a horseradish peroxidase labeled secondary antibody, and WesternBright Quantum reagent and imaged using a GE ImageQuant LAS4000 imaging system. Protein levels were quantitated using ImageJ, and ExoS levels were normalized to G6PI (or actin, or tubulin) -levels. Pseudomonas strains producing cysteine mutants of PopD, PopB, or PcrV (as indicated) were grown to mid-logarithmic phase in “High salt” LB with 200μM IPTG, pelleted and resuspended in PBS-MC. A549 epithelial cells were infected for 2 hours in the presence of 200μM IPTG and either 25 μM copper phenathroline or 1mM TCEP. After infection cells were washed with 25 mM iodoacetamide for 3 min, to alkylate free cysteine residues. Cells were then washed once with high salt PBS (PBS-MC + 1 M KCl) before the cells were scraped up in 1ml of PBS-MC with 2mM phenylmethylsulfonyl fluoride (PMSF), and pelleted (3 minutes, 5000 rpm). The cells were resuspended in 45μL of PBS-MC with 0. 5% Triton X-100 and incubated on ice for 15 minutes. The cells were pelleted, and 45μL of supernatant were removed and combined with 15μL of 4x SDS sample buffer without DTT. Samples were analyzed by Western blot. Membranes were probed for the presence of PcrV, PopB, or PopD using affinity-purified antibodies. The protocol we used was based on the assay published by Blocker et al. [7]. Sheep red blood cells (Quadfive) were washed three times with PBS-MC, and resuspended in RPMI without phenol red or FBS to a concentration of 5 x10^8 cells/mL. Bacterial cultures were diluted 1: 250 from overnight cultures into high salt LB and allowed to grow to mid-log phase. Bacterial cultures were spun down and resuspended in PBS MC, the OD600 was measured and bacteria was resuspended to a concentration of 2. 5 x 10^9 cells/mL. Bacteria and red blood cells were mixed 1: 1 (MOI of 5) in a v-bottomed 96-well plate. Samples were centrifuged at 2000 g for 10 minutes and then incubated for 1 hour at 37°C. The samples were then resuspended and centrifuged at 2000 g for 10 minutes before collecting 100 μL of supernatant. Supernatants were placed in a clean flat-bottomed 96-well plate and then hemoglobin release was calculated by measuring absorbance at 415 nm. Base line lysis was determined by red blood cells mixed and incubated with PBS MC. Percent lysis was determined by comparing samples to 100% lysis using 0. 1% SDS. Red blood cells and bacterial samples were prepared as noted for the hemolysis assay. Samples were prepared by mixing 1. 25 mL of a 8x10^8 cells/mL red blood cell suspension with 250 μL of a 2x10^10 cells/mL bacterial suspension in the presence of 1X cOmplete protease inhibitor. They were then centrifuged at 2000 g for 10 minutes, and incubated at 37°C for 1 hour. Samples were resuspended and centrifuged at 2000g for 10 min before lysing with distilled water on ice for 10 minutes. Intact cells were centrifuged out at 5000 rpm for 10 minutes. The supernatant containing the lysed membranes were removed. A 30μl aliquot was removed and mixed with 10μL of 4xSDS sample buffer to serve as input control. A sucrose gradient was formed by layering 0. 5 mL 50%, 1 mL 41%, and 1 mL 20% sucrose in 20 mM HEPES pH7. 6 1mM EDTA. Red blood cell membranes were isolated by separating the supernatant on a sucrose gradient (Beckman Coulter Optima Max-XP ultracentrifuge; MLS50 rotor; 34,407rpm (95,000 rcf) for 2 hours), and collecting the membranes at the interface between the 41% and 20% sucrose phases. Samples were split into three parts. One part was diluted with ~10x volume of 10 mM HEPES pH7. 6 1mM EDTA buffer, one with 1M KCl, and the last part with 1 M Na2CO3. The membranes were pelleted by ultracentrifugation (TLA100. 3 rotor, 40,000 RPM for 1h), resuspended in 50μL 1X SDS sample buffer, and boiled at 95°C for 10 min before. Samples were desalted using Zebra spin desalting columns (Thermo) and mixed with 4xSDS sample buffer (80μl final volume). Samples were analyzed by Western blot (input samples were diluted 1: 2 and 10μl were loaded on the gels, 20μl of the membrane preps were loaded). Membranes were probed with antibodies directed against PopB and PopD.
Type III secretion systems (T3SSs) are molecular syringes used by a wide variety of Gram-negative pathogens to directly deliver proteins (effectors) into host cells, allowing the bacteria to cause disease. Injection of proteins is triggered by host-cell contact, but how the machinery to deliver effectors is assembled (the translocon), or indeed, how cell contact is perceived, is unclear. Here we identify protein-protein contacts that are critical for translocon function. Our analysis sheds light on the organization of the translocon, and reveals that host cell contact is perceived by a change in the structure of the translocation pore. This signal is then transmitted to the tip of the T3SS needle, and down to the base of the apparatus.
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The Type III Secretion Translocation Pore Senses Host Cell Contact