ExoS controls the cell contact-mediated switch to effector secretion in Pseudomonas aeruginosa

Type III secretion is used by many gram-negative bacterial pathogens to directly deliver protein toxins (effectors) into targeted host cells. In all cases, secretion of effectors is triggered by host cell contact, although the mechanism is unclear. In Pseudomonas aeruginosa, expression of all type III secretion-related genes is up-regulated when secretion is triggered. We were able to visualize this process using a green fluorescent protein reporter system and to use it to monitor the ability of bacteria to trigger effector secretion on cell contact. Surprisingly, the action of one of the major type III secreted effectors, ExoS, prevented triggering of type III secretion by bacteria that subsequently attached to cells, suggesting that triggering of secretion is feedback regulated. Evidence is presented that translocation (secretion of effectors across the host cell plasma membrane) of ExoS is indeed self-regulated and that this inhibition of translocation can be achieved by either of its two enzymatic activities. The translocator proteins PopB, PopD, and PcrV are secreted via the type III secretion system and are required for pore formation and translocation of effectors across the host cell plasma membrane. Here we present data that secretion of translocators is in fact not controlled by calcium, implying that triggering of effector secretion on cell contact represents a switch in secretion specificity, rather than a triggering of secretion per se. The requirement for a host cell cofactor to control effector secretion may help explain the recently observed phenomenon of target cell specificity in both the Yersinia and P. aeruginosa type III secretion systems.     

Type III secretion system translocator has a molten globule conformation both in its free and chaperone-bound forms

Type III secretion systems of Gram-negative pathogenic bacteria allow the injection of effector proteins into the cytosol of host eukaryotic cells. Crossing of the eukaryotic plasma membrane is facilitated by a translocon, an oligomeric structure made up of two bacterial proteins inserted into the host membrane during infection. In Pseudomonas aeruginosa, a major human opportunistic pathogen, these proteins are PopB and PopD. Their interactions with their common chaperone PcrH in the cytosol of the bacteria are essential for the proper function of the injection system. The interaction region between PopD and PcrH was identified using limited proteolysis, revealing that the putative PopD transmembrane fragment is buried within the PopD/PcrH complex. In addition, structural features of PopD and PcrH, either individually or within the binary complex, were characterized using spectroscopic methods and 1D NMR. Whereas PcrH possesses the characteristics of a folded protein, PopD is in a molten globule state either alone or in the PopD/PcrH complex. The molten globule state is known to enable the membrane insertion of translocation/pore-forming domains of bacterial toxins. Therefore, within the bacterial cytoplasm, PopD is preserved in a state that is favorable to secretion and insertion into cell membranes.     

Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis

Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa , on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia , YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa . Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants ( yopB / yopD ) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.     

The Type III Secretion Translocation Pore Senses Host Cell Contact

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.     

Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion

The type III secretion system (T3SS) is a specialized apparatus evolved by Gram-negative bacteria to deliver effector proteins into host cells, thus facilitating the establishment of an infection. Effector translocation across the target cell plasma membrane is believed to occur via pores formed by at least two secreted translocator proteins, the functions of which are dependent upon customized class II T3SS chaperones. Recently, three internal tetratricopeptide repeats (TPRs) were identified in this class of chaperones. Here, defined mutagenesis of the class II chaperone PcrH of Pseudomonas aeruginosa revealed these TPRs to be essential for chaperone activity towards the translocator proteins PopB and PopD and subsequently for the translocation of exoenzymes into host cells.     

Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages

The Pseudomonas aeruginosa cystic fibrosis isolate CHA induces type III secretion system-dependent but ExoU-independent oncosis of neutrophils and macrophages. Time-lapse microscopy of the infection process revealed the rapid accumulation of motile bacteria around infected cells undergoing the process of oncosis, a phenomenon we termed pack swarming. Characterization of the non-chemotactic CHAcheZ mutant showed that pack swarming is a bacterial chemotactic response to infected macrophages. A non-cytotoxic mutant, lacking the type III-secreted proteins PcrV, PopB and PopD, was able to pack swarm only in the presence of the parental strain CHA or when macrophages were pretreated with the pore-forming toxin streptolysin O. Interaction of P. aeruginosa with red blood cells (RBCs) showed that the contact-dependent haemolysis provoked by CHA requires secretion via the type III system and the PcrV, PopB/PopD proteins. The pore inserted into RBC membrane was estimated from osmoprotection experiments to be between 2.8 and 3.5 nm. CHA-infected macrophages could be protected from cell lysis with PEG3350, indicating that the pore introduced into RBC and macrophage membranes is of similar size. The time course uptake of the vital fluorescent dye, Yo-Pro-1, into infected macrophages confirmed that the formation of transmembrane pores by CHA precedes cellular oncosis. Therefore, CHA-induced macrophage death results from a pore-forming activity that is dependent on the intact pcrGVHpopBD operon.     

Oligomerization of PcrV and LcrV, protective antigens of Pseudomonas aeruginosa and Yersinia pestis

Protective antigens of Pseudomonas aeruginosa (PcrV) and Yersinia pestis (LcrV) are key elements of specialized machinery, the type III secretion system (T3SS), which enables the injection of effector molecules into eukaryotic cells. Being positioned at the injectisome extremity, V proteins participate in the translocation process across the host cell plasma membrane. In this study, we demonstrate the assembly of V proteins into oligomeric doughnut-like complexes upon controlled refolding of the proteins in vitro. The oligomeric nature of refolded PcrV was revealed by size exclusion chromatography, native gel electrophoresis, and native mass spectrometry, which ascertain the capacity of the protein to multimerize into higher-order species. Furthermore, transmission electron microscopy performed on oligomers of both PcrV and LcrV revealed the presence of distinct structures with approximate internal and external diameters of 3-4 and 8-10 nm, respectively. The C-terminal helix, alpha12, of PcrV and notably the hydrophobic residues Val(255), Leu(262), and Leu(276) located within this helix, were shown to be crucial for oligomerization. Moreover, the corresponding mutant proteins produced in P. aeruginosa were found to be non-functional in in vivo type III-dependent cytotoxicity assays by directly affecting the correct assembly of PopB/D translocon within the host cell membranes. The detailed understanding of structure-function relationships of T3SS needle tip proteins will be of value in further developments of new vaccines and antimicrobials.     

Injection of Pseudomonas aeruginosa Exo toxins into host cells can be modulated by host factors at the level of translocon assembly and/or activity

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ?-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-?-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation.     

Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas

Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon'). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.     

The Pseudomonas aeruginosa Type III Secretion System Has an Exotoxin S/T/Y Independent Pathogenic Role during Acute Lung Infection

The type III secretion system (T3SS) is a complex nanomachine of many pathogenic Gram-negative bacteria. It forms a proteinaceous channel that is inserted into the host eukaryotic cell membrane for injection of bacterial proteins that manipulate host cell signaling. However, few studies have focused on the effector-independent functions of the T3SS. Using a murine model of acute lung infection with Pseudomonas aeruginosa, an important human opportunistic pathogen, we compared the pathogenicity of mutant bacteria that lack all of the known effector toxins ( ΔSTY), with mutant bacteria that also lack the major translocator protein PopB (ΔSTY/ΔPopB) and so cannot form a functional T3SS channel in the host cell membrane. Mortality was higher among mice challenged with ΔSTY compared to mice challenged with ΔSTY/ΔPopB mutant bacteria. In addition, mice infected with ΔSTY showed decreased bacterial clearance from the lungs compared to those infected with ΔSTY/ΔPopB. Infection was in both cases associated with substantial killing of lung infiltrating macrophages. However, macrophages from ΔSTY-infected mice died by pro-inflammatory necrosis characterized by membrane permeabilization and caspase-1 mediated IL-1β production, whereas macrophages from ΔSTY/ΔPopB infected mice died by apoptosis, which is characterized by annexin V positive staining of the cell membrane and caspase-3 activation. This was confirmed in macrophages infected in vitro. These results demonstrate a T3SS effector toxin independent role for the T3SS, in particular the T3SS translocator protein PopB, in the pathogenicity of P. aeruginosa during acute lung infection.     

The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation

Many gram-negative pathogens use a type IV secretion system (T4SS) to deliver effector proteins into eukaryotic host cells. The fidelity of protein translocation depends on the efficient recognition of effector proteins by the T4SS. Legionella pneumophila delivers a large number of effector proteins into eukaryotic cells using the Dot/Icm T4SS. How the Dot/Icm system is able to recognize and control the delivery of effectors is poorly understood. Recent studies suggest that the IcmS and IcmW proteins interact to form a stable complex that facilitates translocation of effector proteins by the Dot/Icm system by an unknown mechanism. Here we demonstrate that the IcmSW complex is necessary for the productive translocation of multiple Dot/Icm effector proteins. Effector proteins that were able to bind IcmSW in vitro required icmS and icmW for efficient translocation into eukaryotic cells during L. pneumophila infection. We identified regions in the effector protein SidG involved in icmSW-dependent translocation. Although the full-length SidG protein was translocated by an icmSW-dependent mechanism, deletion of amino terminal regions in the SidG protein resulted in icmSW-independent translocation, indicating that the IcmSW complex is not contributing directly to recognition of effector proteins by the Dot/Icm system. Biochemical and genetic studies showed that the IcmSW complex interacts with a central region of the SidG protein. The IcmSW interaction resulted in a conformational change in the SidG protein as determined by differences in protease sensitivity in vitro. These data suggest that IcmSW binding to effectors could enhance effector protein delivery by mediating a conformational change that facilitates T4SS recognition of a translocation domain located in the carboxyl region of the effector protein.     

Synergistic pore formation by type III toxin translocators of Pseudomonas aeruginosa

Type III secretion/translocation systems are essential actors in the pathogenicity of Gram-negative bacteria. The injection of bacterial toxins across the host cell plasma membranes is presumably accomplished by a proteinaceous structure, the translocon. In vitro, Pseudomonas aeruginosa translocators PopB and PopD form ringlike structures observed by electron microscopy. We demonstrate here that PopB and PopD are functionally active and sufficient to form pores in lipid vesicles. Furthermore, the two translocators act in synergy to promote membrane permeabilization. The size-based selectivity observed for the passage of solutes indicates that the membrane permeabilization is due to the formation of size-defined pores. Our results provide also new insights into the mechanism of translocon pore formation that may occur during the passage of toxins from the bacterium into the cell. While proteins bind to lipid vesicles equally at any pH, the kinetics of membrane permeabilization accelerate progressively with decreasing pH values. Electrostatic interactions and the presence of anionic lipids were found to be crucial for pore formation whereas cholesterol did not appear to play a significant role in functional translocon formation.     

Structural Basis of Chaperone Recognition of Type III Secretion System Minor Translocator Proteins

The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the “translocators”) are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.     

Control of effector export by the Pseudomonas aeruginosa type III secretion proteins PcrG and PcrV

Pseudomonas aeruginosa uses a type III secretion system to inject protein effectors into a targeted host cell. Effector secretion is triggered by host cell contact. How effector secretion is prevented prior to cell contact is not well understood. In all secretion systems studied to date, the needle tip protein is required for controlling effector secretion, but the mechanism by which needle tip proteins control effector secretion is unclear. Here we present data that the P. aeruginosa needle tip protein, PcrV, controls effector secretion by assembling into a functional needle tip complex. PcrV likely does not simply obstruct the secretion channel because the pore‐forming translocator proteins can still be secreted while effector secretion is repressed. This finding suggests that PcrV controls effector secretion by affecting the conformation of the apparatus, shifting it from the default, effector secretion ‘on’ conformation, to the effector secretion ‘off’ conformation. We also present evidence that PcrG, which can bind to PcrV and is also involved in controlling effector export, is cytoplasmic and that the interaction between PcrG and PcrV is not required for effector secretion control by either protein. Taken together, these data allow us to propose a working model for control of effector secretion by PcrG and PcrV.     

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly.     

Protective effects of affinity-purified antibody and truncated vaccines against Pseudomonas aeruginosa V-antigen in neutropenic mice

Virulent P. aeruginosa strains express PcrV, one of the translocational components of the type III secretion system. PcrV has been reported to be a protective antigen against lethal P. aeruginosa infection. The PcrV region, which contributes to protective immunity against P. aeruginosa infection, was investigated by using genetically engineered, truncated PcrV proteins and affinity-purified anti-PcrV antibodies against the truncated PcrV proteins. The efficacy of active and passive immunization against PcrV was tested in mice with cyclophosphamide-induced immunosuppression by intraabdominal challenge of P. aeruginosa . Active immunization with either full-length PcrV1-294 or PcrV139-294 significantly improved the survival of mice infected with P. aeruginosa , while PcrV139-258, PcrV139-234, PcrV197-294, and PcrV261-294 were not protective. These results suggest that an effective PcrV vaccine needs to contain not only the Mab166 epitope (PcrV144-257) but also the carboxyl terminal tail of PcrV. In the case of passive immunization, administration of affinity-purified anti-PcrV IgG against either PcrV1-294 or PcrV139-258 showed significantly higher efficacy against lethal P. aeruginosa infection than did original anti-PcrV IgG and Mab166. The increased efficacy of affinity-purified anti-PcrV IgG implies that more potent anti-PcrV strategies are possible. The results of this study are crucial to the development of an effective PcrV vaccine for active immunization and to an appropriate blocking anti-PcrV antibody against P. aeruginosa infection in humans.     

Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Damage to the lung epithelium is associated with the expression of toxins that are directly injected into eukaryotic cells through a type Ill-mediated secretion and translocation mechanism. Here we show that the P. aeruginosa homolog of the Yersinia V antigen, PcrV, is involved in the translocation of type III toxins. Vaccination against PcrV ensured the survival of challenged mice and decreased lung inflammation and injury. Antibodies to PcrV inhibited the translocation of type III toxins.     

Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems

Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are alpha-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins.     

LcrV is a channel size-determining component of the Yop effector translocon of Yersinia

Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact-dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127-195 of LcrV and to amino acids 106-173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size-determining structural component of the Yop translocon.     

A translocator‐specific export signal establishes the translocator–effector secretion hierarchy that is important for type III secretion system function

Type III secretion systems are used by many Gram‐negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host‐cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore‐forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the Pseudomonas aeruginosa translocator protein PopD as a model to identify its export signals. The N‐terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone binding site and one at the very C‐terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator–effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells.