Inject Effector Proteins Research Articles

Unraveling neutrophil- Yersinia interactions during tissue infection.

The human and animal pathogens Yersinia pestis, which causes bubonic and pneumonic plague, and Yersinia pseudotuberculosis and Yersinia enterocolitica, which cause gastroenteritis, share a type 3 secretion system which injects effector proteins, Yops, into host cells. This system is critical for virulence of all three pathogens in tissue infection. Neutrophils are rapidly recruited to infected sites and all three pathogens frequently interact with and inject Yops into these cells during tissue infection. Host receptors, serum factors, and bacterial adhesins appear to collaborate to promote neutrophil– Yersinia interactions in tissues. The ability of neutrophils to control infection is mixed depending on the stage of infection and points to the efficiency of Yops and other bacterial factors to mitigate bactericidal effects of neutrophils. Yersinia in close proximity to neutrophils has higher levels of expression from yop promoters, and neutrophils in close proximity to Yersinia express higher levels of pro-survival genes than migrating neutrophils. In infected tissues, YopM increases neutrophil survival and YopH targets a SKAP2/SLP-76 signal transduction pathway. Yet the full impact of these and other Yops and other Yersinia factors on neutrophils in infected tissues has yet to be understood.

Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation.

Like many Gram-negative pathogens, Shigella rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo-hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supporting Spa47 oligomerization and enzymatic activation. Mutational analysis and a series of high-resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation of Shigella virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulates Shigella virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross-species therapeutic that broadly targets T3SS ATPase oligomerization and activation.

The cytoplasmic portion of the T3SS inner membrane ring components sort into distinct families based on biophysical properties

Type III secretion systems are used by many Gram-negative bacteria to inject effector proteins into eukaryotic cells to subvert their normal activities. Structurally conserved portions of the type III secretion apparatus include a: basal body located within the bacterial envelope; an exposed needle with tip complex that delivers effectors across the target cell membrane; and cytoplasmic sorting platform that selects cargo and powers secretion. While structurally conserved, the individual proteins that make up this nanomachine are typically not interchangeable though they do tend to fall into families. Here we selected a single domain from the inner membrane ring of the basal body from six different type III secretion systems (called SctD using a proposed unifying nomenclature). The selected domain creates an integral interface between the basal body and the sorting platform. Therefore, it represents a pivotal point between two distinct assemblies. All six protein domains possess a structural motif called a forkhead-associated-like (FHA-like) domain but differ greatly in their sequences and solution behaviors. These differences are used here to define family boundaries for these FHA-like domains. The data parallel, though not precisely, family boundaries defined by other proteins within the apparatus and by phylogenetic analysis. Ultimately, differences in the families are likely to reflect differences in the activities of these type III secretion systems or the host niches in which these pathogens are found.

A bacterial secreted translocator hijacks riboregulators to control type III secretion in response to host cell contact.

Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact.

Identification and characterization of putative Aeromonas spp. T3SS effectors.

The genetic determinants of bacterial pathogenicity are highly variable between species and strains. However, a factor that is commonly associated with virulent Gram-negative bacteria, including many Aeromonas spp., is the type 3 secretion system (T3SS), which is used to inject effector proteins into target eukaryotic cells. In this study, we developed a bioinformatics pipeline to identify T3SS effector proteins, applied this approach to the genomes of 105 Aeromonas strains isolated from environmental, mutualistic, or pathogenic contexts and evaluated the cytotoxicity of the identified effectors through their heterologous expression in yeast. The developed pipeline uses a two-step approach, where candidate Aeromonas gene families are initially selected using Hidden Markov Model (HMM) profile searches against the Virulence Factors DataBase (VFDB), followed by strict comparisons against positive and negative control datasets, greatly reducing the number of false positives. This approach identified 21 Aeromonas T3SS likely effector families, of which 8 represent known or characterized effectors, while the remaining 13 have not previously been described in Aeromonas. We experimentally validated our in silico findings by assessing the cytotoxicity of representative effectors in Saccharomyces cerevisiae BY4741, with 15 out of 21 assayed proteins eliciting a cytotoxic effect in yeast. The results of this study demonstrate the utility of our approach, combining a novel in silico search method with in vivo experimental validation, and will be useful in future research aimed at identifying and authenticating bacterial effector proteins from other genera.

A Pathogen Effector Subverts Translational Regulation to Boost Host Polyamine Levels

Pathogenic bacteria inject effector proteins into host cells to manipulate cellular processes promoting infection. Transcription-activator-like effectors (TALEs), an effector class found in some plant-pathogenic bacteria, transcriptionally activate host genes to promote disease. We have identified arginine decarboxylase (ADC) genes as the sole host targets of Brg11, a TALE-like effector from the bacterial broad-host-range plant pathogen Ralstonia solanacearum. Brg11 targets the ADC-box, a 50-bp motif upstream of ADC genes broadly conserved across land plants. The transcribed ADC-box attenuates translation from native ADC mRNAs. Yet, Brg11 induces transcription of truncated ADC mRNA lacking the 5’ ADC-box, thus bypassing translational control. ADCs are rate-limiting enzymes of polyamine biosynthesis, and we show that the Brg11-induced elevation of polyamine levels triggers a plant defense reaction that likely inhibits bacterial niche-competitors, but not R. solanacearum. Our findings illustrate a novel concept in effector biology whereby bacterial effectors act as mediators of tripartite microbe-host-microbe interactions.

Solution NMR Structure of the Gtpase Activating (GAP) Domain of Vope, A Vibrio Cholerae T3Ss Effector Protein

Vibrio cholerae uses a type III secretion system to inject effector proteins into a host cell. Recently, a putative GTPase Activating Protein (GAP) called VopE was identified as a pathogenic protein that affected host mitochondrial dynamics and immune response. However, biophysical and structural characterization has been missing. We describe solution NMR structure of the putative GAP domain (73-204) of VopE. Using SEC-SAXS data, we improved the quality of the output calculated structures. Structural comparison with other ToxGAP's revealed a conserved overall fold with several unique features to VopE. By examining the conservation of residues at putative binding interface, we again find features conserved to all ToxGAP's and some only observed in VopE. Based on NMR data with its putative GTPase target, we hypothesize on other potential target mitochondrial GTPases.

Structures of Type III Secretion System Needle Filaments.

Among the Gram-negative bacterial secretion systems, type III secretion systems (T3SS) possess a unique extracellular molecular apparatus called the needle. This macromolecular protein assembly is a nanometre-size filament formed by the helical arrangement of hundreds of copies of a single, small protein, which is highly conserved between T3SSs from animal to plant bacterial pathogens. The needle filament forms a hollow tube with a channel ~20Å in diameter that serves as a conduit for proteins secreted into the targeted host cell. In the past ten years, technical breakthroughs in biophysical techniques such as cryo-electron microscopy (cryo-EM) and solid-state NMR (SSNMR) spectroscopy have uncovered atomic resolution details about the T3SS needle assembly. Several high-resolution structures of Salmonella typhimurium and Shigella flexneri T3SS needles have been reported demonstrating a common structural fold. These structural models have been used to explain the active role of the needle in transmitting the host-cell contact signal from the tip to the base of the T3SS through conformational changes as well as during the injection of effector proteins. In this chapter, we summarize the current knowledge about the structure and the role of the T3SS needle during T3SS assembly and effector secretion.

The Structure of the Type III Secretion System Needle Complex.

The type III secretion system (T3SS) is an essential virulence factor of many pathogenic bacterial species including Salmonella, Yersinia, Shigella and enteropathogenic Escherichia coli (EPEC). It is an intricate molecular machine that spans the bacterial membranes and injects effector proteins into target host cells, enabling bacterial infection. The T3SS needle complex comprises of proteinaceous rings supporting a needle filament which extends out into the extracellular environment. It serves as the central conduit for translocating effector proteins. Multiple laboratories have dedicated a remarkable effort to decipher the structure and function of the needle complex. A combination of structural biology techniques such as cryo-electron microscopy (cryoEM), X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and computer modelling have been utilized to study different structural components at progressively higher resolutions. This chapter will provide an overview of the structural details of the T3SS needle complex, shedding light on this essential component of this fascinating bacterial system.

The Tip Complex: From Host Cell Sensing to Translocon Formation.

Type III secretion systems are used by some Gram-negative bacteria to inject effector proteins into targeted eukaryotic cells for the benefit of the bacterium. The type III secretion injectisome is a complex nanomachine comprised of four main substructures including a cytoplasmic sorting platform, an envelope-spanning basal body, an extracellular needle and an exposed needle tip complex. Upon contact with a host cell, secretion is induced, resulting in the formation of a translocon pore in the host membrane. Translocon formation completes the conduit needed for effector secretion into the host cell. Control of type III secretion occurs in response to environmental signals, with the final signal being host cell contact. Secretion control occurs primarily at two sites-the cytoplasmic sorting platform, which determines secretion hierarchy, and the needle tip complex, which is critical for sensing and responding to environmental signals. The best-characterized injectisomes are those from Yersinia, Shigella and Salmonella species where there is a wealth of information on the tip complex and the two translocator proteins. Of these systems, the best characterized from a secretion regulation standpoint is Shigella. In the Shigella system, the tip complex and the first secreted translocon both contribute to secretion control and, thus, both are considered components of the tip complex. In this review, all three of these type III secretion systems are described with discussion focused on the structure and formation of the injectisome tip complex and what is known of the transition from nascent tip complex to assembled translocon pore.

Hsp90 Interacts with the Bacterial Effector NleH1.

Enterohemorrhagic Escherichia coli (EHEC) utilizes a type III secretion system (T3SS) to inject effector proteins into host cells. The EHEC NleH1 effector inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway by reducing the nuclear translocation of the ribosomal protein S3 (RPS3). NleH1 prevents RPS3 phosphorylation by the IκB kinase-β (IKKβ). IKKβ is a central kinase in the NF-κB pathway, yet NleH1 only restricts the phosphorylation of a subset of the IKKβ substrates. We hypothesized that a protein cofactor might dictate this inhibitory specificity. We determined that heat shock protein 90 (Hsp90) interacts with both IKKβ and NleH1 and that inhibiting Hsp90 activity reduces RPS3 nuclear translocation.

Hpa1 is a type III translocator in Xanthomonas oryzae pv. oryzae

BackgroundPathogenic Gram-negative bacteria interact with their eukaryotic hosts by deploying the type III translocon to inject effector proteins into the cytosol of eukaryotic cells. The translocon compositions, the number and biochemical characteristics of type III translocators in animal-pathogenic bacteria have been well elucidated, but information is lacking for plant-pathogenic bacteria. With extensive studies on biological functions of the Hpa1 protein secreted by the type III secretion system in Xanthomonas oryzae pv. oryzae (Xoo), we show here that Hpa1 is a type III translocator based on measurements of two proteins categorized as transcription activator-like (TAL) effector.ResultsHpa1 was functionally associated with the TAL effector PthXo1 or AvrXa10 by genetic analysis of the wild-type Xoo strain and related mutants or recombinant strains. Inoculation experiments suggested that Hpa1 is required not only for the virulent role of PthXo1 in the susceptible rice variety Nipponbare, but also for the avirulent function of AvrXa10 on the resistant rice variety IRBB10. Hpa1 is unrelated to the secretion of PthXo1 and AvrXa10 out of bacterial cells. However, Hpa1 is critical for both TAL effectors to be translocated from bacterial cells into the cytosol of rice cells based on replicate experiments performed on the susceptible and resistant varieties, respectively. Hpa1-mediated translocation of PthXo1 is coincident with induced expression of rice SWEET11 gene, which is the regulatory target of PthXo1, resulting in the occurrence of the bacterial blight disease in the susceptible rice variety. By contrast, the immune hypersensitive response is induced in agreement with induced expression of rice Xa10 gene, which is the target of AvrXa10, only when AvrXa10 is translocated from bacteria into cells of the resistant rice variety. All the virulent or avirulent performances of the TAL effectors are nullified by directed mutation that removes the α-helix motif from the Hpa1 sequence.ConclusionsThe genetic and biochemical data demonstrate that Hap1 is a type III translocator at least for TAL effectors PthXo1 and AvrXa10. The effect of the directed mutation suggests that Hpa1 depends on its α-helical motif to fulfil the translocator function.

Characterization of the two conformations adopted by the T3SS inner-membrane protein PrgK.

The pathogenic bacterium Salmonella enterica serovar Typhimurium utilizes two type III secretion systems (T3SS) to inject effector proteins into target cells upon infection. The T3SS secretion apparatus (the injectisome) is a large macromolecular assembly composed of over twenty proteins, many in highly oligomeric states. A sub-structure of the injectisome, termed the basal body, spans both membranes and the periplasmic space of the bacterium. It is primarily composed of three integral membranes proteins, InvG, PrgH, and PrgK, that form ring structures through which components are secreted. In particular, PrgK possesses a periplasmic region consisting of two globular domains joined by a linker polypeptide. We showed previously that in isolation, this region adopts two distinct conformations, of with only one is observed in the assembled basal body complex. Here, using NMR spectroscopy, we further characterize these two conformations. In particular, we demonstrate that the interaction of the linker region with the first globular domain, as found in the intact basal body, is dependent upon the cis conformation of the Leu77-Pro78 peptide. Furthermore, this interaction is pH-dependent due to coupling with hydrogen bond formation between Tyr75 and His42 in its neutral Nδ1 H tautomeric form. This pH-dependent interaction may play a role in the regulation of the secretion apparatus disassembly in the context of bacterial infection.

Role of Plasmids in Plant-Bacteria Interactions.

Plants are colonized by diverse microorganisms, which may positively or negatively influence the plant fitness. The positive impact includes nutrient acquisition, enhancement of resistance to biotic and abiotic stresses, both important factors for plant growth and survival, while plant pathogenic bacteria can cause diseases. Plant pathogens are adapted to negate or evade plant defense mechanisms, e.g. by the injection of effector proteins into the host cells or by avoiding the recognition by the host. Plasmids play an important role in the rapid bacterial adaptation to stresses and changing environmental conditions. In the plant environment, plasmids can further provide a selective advantage for the host bacteria, e.g. by carrying genes encoding metabolic pathways, metal and antibiotic resistances, or pathogenicity-related genes. However, we are only beginning to understand the role of mobile genetic elements and horizontal gene transfer for plant-associated bacteria. In this review, we aim to provide a short update on what is known about plasmids and horizontal gene transfer of plant associated bacteria and their role in plant-bacteria interactions. Furthermore, we discuss tools available to study the plant-associated mobilome, its transferability, and its bacterial hosts.

Structural Insight Into Conformational Changes Induced by ATP Binding in a Type III Secretion-Associated ATPase From Shigella flexneri.

Gram-negative bacteria utilize the type III secretion system (T3SS) to inject effector proteins into the host cell cytoplasm, where they subvert cellular functions and assist pathogen invasion. The conserved type III-associated ATPase is critical for the separation of chaperones from effector proteins, the unfolding of effector proteins and translocating them through the narrow channel of the secretion apparatus. However, how ATP hydrolysis is coupled to the mechanical work of the enzyme remains elusive. Herein, we present a complete description of nucleoside triphosphate binding by surface presentation antigens 47 (Spa47) from Shigella flexneri, based on crystal structures containing ATPγS, a catalytic magnesium ion and an ordered water molecule. Combining the crystal structures of Spa47-ATPγS and unliganded Spa47, we propose conformational changes in Spa47 associated with ATP binding, the binding of ATP induces a conformational change of a highly conserved luminal loop, facilitating ATP hydrolysis by the Spa47 ATPase. Additionally, we identified a specific hydrogen bond critical for ATP recognition and demonstrated that, while ATPγS is an ideal analog for probing ATP binding, AMPPNP is a poor ATP mimic. Our findings provide structural insight pertinent for inhibitor design.

Spa47 Oligomerization Plays a Key Role in ATPase Activation and Function in the Shigella Type III Secretion System

Several Gram‐negative pathogens, including Shigella, utilize a conserved type three secretion system (T3SS) for infection. The type three secretion apparatus (T3SA) provides a unidirectional conduit for injection of effector proteins from the bacteria to the host cell cytoplasm. The conserved T3SS ATPase found at the base of the T3SA is believed to provide the energy for apparatus formation and effector secretion, however, the mechanisms of ATP hydrolysis, effector secretion, and apparatus formation by the T3SS ATPase remain unclear. We have recently shown that Spa47, the T3SS ATPase of Shigella, forms a unique activated oligomeric species during the course of purification. Using our recently published crystal structure of Spa47 as a guide, we modeled an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. To explore the dynamics of Spa47 protomer interaction in vitro and in vivo, we performed a series of ATP hydrolysis, fluorescence microscopy and Shigella invasion experiments. We observe a positive Hill coefficient with the monomeric Spa47 species that is not present with the oligomeric Spa47 species in vitro. Interestingly, we continue to see a positive Hill coefficient even with the addition of engineered ATPase inactive Spa47 point mutants to WT Spa47 monomer species. We have utilized live cell fluorescence microscopy to show that these ATPase inactive Spa47 point mutants localize to the T3SA though they do not support secretion. Furthermore, each of the ATPase inactive Spa47 point mutants as well as an oligomerization deficient Spa47 construct exhibits a dominant negative phenotype. Together, these results suggest that while ATP hydrolysis is not the major driving factor for Spa47 localization to the apparatus base, the Spa47 oligomer may undergo a series of concerted ATP hydrolysis events that are critical for apparatus function, similar to ATP synthase. As we better understand the nuances of T3SS ATPase mechanism and function, the results of this work will provide a strong platform for future work to perhaps target the T3SS ATPase for novel therapeutics.Support or Funding InformationNational Science Foundation Graduate Research Fellowship and National Institute of Health (1R15AI124108‐01A1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins.

The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

Multitalented EspB of enteropathogenic Escherichia coli (EPEC) enters cells autonomously and induces programmed cell death in human monocytic THP-1 cells

Enteropathogenic Escherichia coli (EPEC) subvert host cell signaling pathways by injecting effector proteins via a Type 3 Secretion System (T3SS). The T3SS-dependent EspB protein is a multi-functional effector protein, which contributes to adherence and translocator pore formation and after injection exhibits several intracellular activities. In addition, EspB is also secreted into the environment. Effects of secreted EspB have not been reported thus far. As a surrogate for secreted EspB we employed recombinant EspB (rEspB) derived from the prototype EPEC strain E2348/69 and investigated the interactions of the purified protein with different human epithelial and immune cells including monocytic THP-1 cells, macrophages, dendritic cells, U-937, epithelial T84, Caco-2, and HeLa cells. To assess whether these proteins might exert a cytotoxic effect we monitored the release of lactate dehydrogenase (LDH) as well as propidium iodide (PI) uptake. For comparison, we also investigated several homologs of EspB such as IpaD of Shigella, and SipC, SipD, SseB, and SseD of Salmonella as purified recombinant proteins. Interestingly, cytotoxicity was only observed in THP-1 cells and macrophages, whereas epithelial cells remained unaffected. Cell fractionation and immune fluorescence experiments showed that rEspB enters cells autonomously, which suggests that EspB might qualify as a novel cell-penetrating effector protein (CPE). Using specific organelle tracers and inhibitors of signaling pathways we found that rEspB destroys the mitochondrial membrane potential – an indication of programmed cell death induction in THP-1 cells. Here we show that EspB not only constitutes an essential part of the T3SS-nanomachine and contributes to the arsenal of injected effector proteins but, furthermore, that secreted (recombinant) EspB autonomously enters host cells and selectively induces cell death in immune cells.

Functional Analysis of a Putative Type III Secretion System in Stress Adaption by Mesorhizobium alhagi CCNWXJ12-2T.

Mesorhizobium alhagi CCNWXJ12-2T, isolated from root nodules of the desert plant Alhagi sparsifolia, contains two type III secretion systems (T3SSs). T3SSs are specialized machinery with wide distribution in bacteria that inject effector proteins into target cells. Our previous study showed that the expression of M. alhagi T3SS1 is upregulated in high-salt conditions. Here, phylogenetic analysis of T3SS1 using the core protein RhcU suggested that T3SS1 belongs to the α-Rhc II subgroup of the Rhc T3SS family. To elaborate the function of M. alhagi CCNWXJ12-2T T3SS1 in stress adaption, two T3SS1 mutants (ΔrhcQ and ΔMA29250) were constructed and analyzed. β-galactosidase transcriptional fusion assays showed that activity of the promoter of T3SS1 was induced by salts. Mutant ΔrhcQ was more sensitive to NaCl and LiCl than the wild-type, but ΔMA29250 was not. Both mutants were more sensitive to KCl than the wild-type. The intracellular Na+ concentration in ΔrhcQ in high-NaCl conditions (0.4 M) increased by 37% compared to that of the wild-type strain, while the Na+ concentration in ΔMA29250 increased by 13%. The K+ concentration in both mutants increased by 16% compared to the wild-type in high-KCl conditions (0.3 M). Strain ΔrhcQ showed decreased survival compared to the wild-type after treatment with H2O2, while the survival rate of ΔMA29250 was almost the same as that of the wild-type. Antioxidant enzyme activities in ΔrhcQ were lower than those in the wild-type strain, but this was not the case for ΔMA29250. Our data elucidate the beneficial effects of T3SS1 in the adaption of M. alhagi CCNWXJ12-2T to stress.

Novel insights into the mechanism of SepL-mediated control of effector secretion in enteropathogenic Escherichia coli.

Type three secretion systems (T3SSs) are virulence determinants employed by several pathogenic bacteria as molecular syringes to inject effector proteins into host cells. Diarrhea‐producing enteropathogenic Escherichia coli (EPEC) uses a T3SS to colonize the intestinal tract. T3S is a highly coordinated process that ensures hierarchical delivery of three classes of substrates: early (inner rod and needle subunits), middle (translocators), and late (effectors). Translocation of effectors is triggered upon host‐cell contact in response to different environmental cues, such as calcium levels. The T3S substrate specificity switch from middle to late substrates in EPEC is regulated by the SepL and SepD proteins, which interact with each other and form a trimeric complex with the chaperone CesL. In this study, we investigated the link between calcium concentration and secretion regulation by the gatekeeper SepL. We found that calcium depletion promotes late substrate secretion in a translocon‐independent manner. Furthermore, the stability, formation, and subcellular localization of the SepL/SepD/CesL regulatory complex were not affected by the absence of calcium. In addition, we demonstrate that SepL interacts in a calcium‐independent manner with the major export gate component EscV, which in turn interacts with both middle and late secretion substrates, providing a docking site for T3S. These results suggest that EscV serves as a binding platform for both the SepL regulatory protein and secreted substrates during the ordered assembly of the T3SS.