HrpB7 from Xanthomonas campestris pv. vesicatoria is an essential component of the type III secretion system and shares features of HrpO/FliJ/YscO family members

The Gram‐negative bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a type III secretion system (T3SS) into eukaryotic cells. The T3SS spans both bacterial membranes and consists of more than 20 proteins, 9 of which are conserved in plant and animal pathogens and constitute the core subunits of the secretion apparatus. T3S in X. campestris pv. vesicatoria also depends on nonconserved proteins with yet unknown function including HrpB7, which contains predicted N‐ and C‐terminal coiled‐coil regions. In the present study, we provide experimental evidence that HrpB7 forms stable oligomeric complexes. Interaction and localisation studies suggest that HrpB7 interacts with inner membrane and predicted cytoplasmic (C) ring components of the T3SS but is dispensable for the assembly of the C ring. Additional interaction partners of HrpB7 include the cytoplasmic adenosinetriphosphatase HrcN and the T3S chaperone HpaB. The interaction of HrpB7 with T3SS components as well as complex formation by HrpB7 depends on the presence of leucine heptad motifs, which are part of the predicted N‐ and C‐terminal coiled‐coil structures. Our data suggest that HrpB7 forms multimeric complexes that associate with the T3SS and might serve as a docking site for the general T3S chaperone HpaB.     

HpaC controls substrate specificity of the Xanthomonas type III secretion system

The Gram-negative bacterial plant pathogen Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject bacterial effector proteins into the host cell cytoplasm. One essential pathogenicity factor is HrpB2, which is secreted by the T3S system. We show that secretion of HrpB2 is suppressed by HpaC, which was previously identified as a T3S control protein. Since HpaC promotes secretion of translocon and effector proteins but inhibits secretion of HrpB2, HpaC presumably acts as a T3S substrate specificity switch protein. Protein-protein interaction studies revealed that HpaC interacts with HrpB2 and the C-terminal domain of HrcU, a conserved inner membrane component of the T3S system. However, no interaction was observed between HpaC and the full-length HrcU protein. Analysis of HpaC deletion derivatives revealed that the binding site for the C-terminal domain of HrcU is essential for HpaC function. This suggests that HpaC binding to the HrcU C terminus is key for the control of T3S. The C terminus of HrcU also provides a binding site for HrpB2; however, no interaction was observed with other T3S substrates including pilus, translocon and effector proteins. This is in contrast to HrcU homologs from animal pathogenic bacteria suggesting evolution of distinct mechanisms in plant and animal pathogenic bacteria for T3S substrate recognition.     

Identification of Novel Type III Secretion Chaperone-Substrate Complexes of Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of C. trachomatis we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipation after protein expression in Yersinia enterocolitica and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a C. trachomatis T3S substrate using Y. enterocolitica as a heterologous system. Further T3S assays in Yersinia indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in C. trachomatis Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082.     

A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in Shigella flexneri

The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.     

Type III-dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell

Many plant pathogenic bacteria utilize a conserved type III secretion system (TTSS) to deliver effector proteins into the host tissue. Indirect evidence has suggested that at least some effector proteins are translocated from the bacterial cytoplasm into the plant cell. Using an immunocytochemical approach, we demonstrate that the type III effector AvrBs3 from Xanthomonas campestris pv. vesicatoria localizes to nuclei of infected pepper leaves. Importantly, AvrBs3 translocation was observed in situ in native tissues of susceptible and resistant plants. AvrBs3 was detected in the nucleus as soon as 4 h post infection, which was dependent on a functional TTSS and the putative translocator HrpF. N-terminal AvrBs3 deletion derivatives are no longer secreted by the TTSS in vitro and could not be detected inside the host cells, suggesting that the N-terminus of AvrBs3 is important for secretion. Deletion of the nuclear localization signals in the AvrBs3 C-terminus, which are required for the AvrBs3-mediated induction of the hypersensitive reaction in resistant pepper plants, abolished AvrBs3 localization to the nucleus. This is the first report on direct evidence for translocation of a native type III effector protein from a plant pathogenic bacterium into the host cell.     

Structures of the Shigella flexneri type 3 secretion system protein MxiC reveal conformational variability amongst homologues

Many Gram-negative pathogenic bacteria use a complex macromolecular machine, known as the type 3 secretion system (T3SS), to transfer virulence proteins into host cells. The T3SS is composed of a cytoplasmic bulb, a basal body spanning the inner and outer bacterial membranes, and an extracellular needle. Secretion is regulated by both cytoplasmic and inner membrane proteins that must respond to specific signals in order to ensure that virulence proteins are not secreted before contact with a eukaryotic cell. This negative regulation is mediated, in part, by a family of proteins that are thought to physically block the entrance to the secretion apparatus until an appropriate signal is received following host cell contact. Despite weak sequence homology between proteins of this family, the crystal structures of Shigella flexneri MxiC we present here confirm the conservation of domain topology with the homologue from Yersinia sp. Interestingly, comparison of the Shigella and Yersinia structures reveals a significant structural change that results in substantial domain re-arrangement and opening of one face of the molecule. The conservation of a negatively charged patch on this face suggests it may have a role in binding other components of the T3SS.     

HpaA from Xanthomonas is a regulator of type III secretion

The Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject effector proteins into the host cell cytoplasm. Efficient secretion of several effector proteins depends on the cytoplasmic global T3S chaperone HpaB. In this study, we show that HpaB interacts with the virulence factor HpaA, which is secreted by the T3S system and translocated into the plant cell. HpaA promotes secretion of pilus, translocon and effector proteins and therefore appears to be an important control protein of the T3S system. Protein-protein interaction studies and the analysis of HpaA deletion derivatives revealed that the C-terminal protein region, which contains a HpaB binding site, is crucial for the contribution of HpaA to T3S. Secretion of pilus and translocon proteins is not affected when HpaA is expressed as an N-terminal deletion derivative that lacks the secretion and translocation signal. Our data suggest that binding of HpaA to HpaB within the bacterial cell favours secretion of extracellular components of the secretion apparatus. Secretion of HpaA presumably liberates HpaB and thus promotes effector protein secretion after assembly of the T3S apparatus.     

Functionally Essential Interaction between Yersinia YscO and the T3S4 Domain of YscP

The type III secretion (T3S) system is essential to the virulence of a large number of Gram-negative bacterial pathogens, including Yersinia. YscO is required for T3S in Yersinia and is known to interact with several other T3S proteins, including the chaperone SycD and the needle length regulator YscP. To define which interactions of YscO are required for T3S, we pursued model-guided mutagenesis: three conserved and surface-exposed regions of modeled YscO were targeted for multiple alanine substitutions. Most of the mutations abrogated T3S and did so in a recessive manner, consistent with a loss of function. Both functional and nonfunctional YscO mutant proteins interacted with SycD, indicating that the mutations had not affected protein stability. Likewise, both functional and nonfunctional versions of YscO were exclusively intrabacterial. Functional and nonfunctional versions of YscO were, however, distinguishable with respect to interaction with YscP. This interaction was observed only for wild-type YscO and a T3S-proficient mutant of YscO but not for the several T3S-deficient mutants of YscO. Evidence is presented that the YscO-YscP interaction is direct and that the type III secretion substrate specificity switch (T3S4) domain of YscP is sufficient for this interaction. These results provide evidence that the interaction of YscO with YscP, and in particular the T3S4 domain of YscP, is essential to type III secretion.     

SecA Localization and SecA-Dependent Secretion Occurs at New Division Septa in Group B Streptococcus

Exported proteins of Streptococcus agalactiae (GBS), which include proteins localized to the bacterial surface or secreted into the extracellular environment, are key players for commensal and pathogenic interactions in the mammalian host. These proteins are transported across the cytoplasmic membrane via the general SecA secretory pathway and those containing the so-called LPXTG sorting motif are covalently attached to the peptidoglycan by sortase A. How SecA, sortase A, and LPXTG proteins are spatially distributed in GBS is not known. In the close relative Streptococcus pyogenes, it was shown that presence of the YSIRKG/S motif (literally YSIRK_X3Gx₂S) in the signal peptide (SP) constitutes the targeting information for secretion at the septum. Here, using conventional and deconvolution immunofluorescence analyses, we have studied in GBS strain NEM316 the localization of SecA, SrtA, and the secreted protein Bsp whose signal peptide contains a canonical YSIRKG/S motif (YSLRKykfGlaS). Replacing the SP of Bsp with four other SPs containing or not the YSIRKG/S motif did not alter the localized secretion of Bsp at the equatorial ring. Our results indicate that secretion and cell wall-anchoring machineries are localized at the division septum. Cell wall- anchored proteins displayed polar (PilB, Gbs0791), punctuate (CspA) or uniform distribution (Alp2) on the bacterial surface. De novo secretion of Gbs0791 following trypsin treatment indicates that it is secreted at the septum, then redistributed along the lateral sides, and finally accumulated to the poles. We conclude that the ±YSIRK SP rule driving compartimentalized secretion is not true in S. agalactiae.     

Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates

A remarkable feature of the flagellar‐specific type III secretion system (T3SS) is the selective recognition of a few substrate proteins among the many thousand cytoplasmic proteins. Secretion substrates are divided into two specificity classes: early substrates secreted for hook‐basal body (HBB) construction and late substrates secreted after HBB completion. Secretion was reported to require a disordered N‐terminal secretion signal, mRNA secretion signals within the 5′‐untranslated region (5′‐UTR) and for late substrates, piloting proteins known as the T3S chaperones. Here, we utilized translational β‐lactamase fusions to probe the secretion efficacy of the N‐terminal secretion signal of fourteen secreted flagellar substrates in Salmonella enterica. We observed a surprising variety in secretion capability between flagellar proteins of the same secretory class. The peptide secretion signals of the early‐type substrates FlgD, FlgF, FlgE and the late‐type substrate FlgL were analysed in detail. Analysing the role of the 5′‐UTR in secretion of flgB and flgE revealed that the native 5′‐UTR substantially enhanced protein translation and secretion. Based on our data, we propose a multicomponent signal that drives secretion via the flagellar T3SS. Both mRNA and peptide signals are recognized by the export apparatus and together with substrate‐specific chaperones allowing for targeted secretion of flagellar substrates.     

The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate bacterial effector proteins into eukaryotic host cells. The membrane-spanning secretion apparatus consists of 11 core components and several associated proteins with yet unknown functions. In this study, we analyzed the role of HrpB1, which was previously shown to be essential for T3S and the formation of the extracellular T3S pilus. We provide experimental evidence that HrpB1 localizes to the bacterial periplasm and binds to peptidoglycan, which is in agreement with its predicted structural similarity to the putative peptidoglycan-binding domain of the lytic transglycosylase Slt70 from Escherichia coli. Interaction studies revealed that HrpB1 forms protein complexes and binds to T3S system components, including the inner membrane protein HrcD, the secretin HrcC, the pilus protein HrpE, and the putative inner rod protein HrpB2. The analysis of deletion and point mutant derivatives of HrpB1 led to the identification of amino acid residues that contribute to the interaction of HrpB1 with itself and HrcD and/or to protein function. The finding that HrpB1 and HrpB2 colocalize to the periplasm and both interact with HrcD suggests that they are part of a periplasmic substructure of the T3S system.     

Reassessing the Role of DotF in the Legionella pneumophila Type IV Secretion System

Legionella pneumophila, the causative agent of a severe pneumonia termed Legionnaires’ Disease, survives and replicates within both protozoan hosts and human alveolar macrophages. Intracellular survival is dependent upon secretion of a plethora of protein effectors that function to form a replicative vacuole, evade the endocytic pathway and subvert host immune defenses. Export of these factors requires a type IV secretion system (T4SS) called Dot/Icm that is composed of twenty-seven proteins. This report focuses on the DotF protein, which was previously postulated to have several different functions, one of which centered on binding Dot/Icm substrates. In this report, we examined if DotF functions as the T4SS inner membrane receptor for Dot/Icm substrates. Although we were able to recapitulate the previously published bacterial two-hybrid interaction between DotF and several substrates, the interaction was not dependent on the Dot/Icm substrates’ signal sequences as predicted for a substrate:receptor interaction. In addition, binding did not require the cytoplasmic domain of DotF, which was anticipated to be involved in recognizing substrates in the cytoplasm. Finally, inactivation of dotF did not abolish intracellular growth of L. pneumophila or translocation of substrates, two phenotypes dependent on the T4SS receptor. These data strongly suggest that DotF does not act as the major receptor for Dot/Icm substrates and therefore likely performs an accessory function within the core-transmembrane subcomplex of the L. pneumophila Dot/Icm type IV secretion system.     

Suppression of XopQ–XopX‐induced immune responses of rice by the type III effector XopG

Effectors that suppress effector‐triggered immunity (ETI) are an essential part of the arms race in the co‐evolution of bacterial pathogens and their host plants. Xanthomonas oryzae pv. oryzae uses multiple type III secretion system (T3SS) secreted effectors such as XopU, XopV, XopP, XopG, and AvrBs2 to suppress rice immune responses that are induced by the interaction of two other effectors, XopQ and XopX. Here we show that each of these five suppressors can interact individually with both XopQ and XopX. One of the suppressors, XopG, is a predicted metallopeptidase that appears to have been introduced into X. oryzae pv. oryzae by horizontal gene transfer. XopQ and XopX interact with each other in the nucleus while interaction with XopG sequesters them in the cytoplasm. The XopG E76A and XopG E85A mutants are defective in interaction with XopQ and XopX, and are also defective in suppression of XopQ–XopX‐mediated immune responses. Both mutations individually affect the virulence‐promoting ability of XopG. These results indicate that XopG is important for X. oryzae pv. oryzae virulence and provide insights into the mechanisms by which this protein suppresses ETI in rice.     

Tandem translation generates a chaperone for the Salmonella type III secretion system protein SsaQ

Type III secretion systems (T3SSs) of bacterial pathogens involve the assembly of a surface-localized needle complex, through which translocon proteins are secreted to form a pore in the eukaryotic cell membrane. This enables the transfer of effector proteins from the bacterial cytoplasm to the host cell. A structure known as the C-ring is thought to have a crucial role in secretion by acting as a cytoplasmic sorting platform at the base of the T3SS. Here, we studied SsaQ, an FliN-like putative C-ring protein of the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS. ssaQ produces two proteins by tandem translation: a long form (SsaQ(L)) composed of 322 amino acids and a shorter protein (SsaQ(S)) comprising the C-terminal 106 residues of SsaQ(L). SsaQ(L) is essential for SPI-2 T3SS function. Loss of SsaQ(S) impairs the function of the T3SS both ex vivo and in vivo. SsaQ(S) binds to its corresponding region within SsaQ(L) and stabilizes the larger protein. Therefore, SsaQ(L) function is optimized by a novel chaperone-like protein, produced by tandem translation from its own mRNA species.     

EscI: a crucial component of the type III secretion system forms the inner rod structure in enteropathogenic Escherichia coli

The T3SS (type III secretion system) is a multi-protein complex that plays a central role in the virulence of many gram-negative bacterial pathogens. This apparatus spans both bacterial membranes and transports virulence factors from the bacterial cytoplasm into eukaryotic host cells. The T3SS exports substrates in a hierarchical and temporal manner. The first secreted substrates are the rod/needle proteins which are incorporated into the T3SS apparatus and are required for the secretion of later substrates, the translocators and effectors. In the present study, we provide evidence that rOrf8/EscI, a poorly characterized locus of enterocyte effacement-encoded protein, functions as the inner rod protein of the T3SS of EPEC (enteropathogenic Escherichia coli). We demonstrate that EscI is essential for type III secretion and is also secreted as an early substrate of the T3SS. We found that EscI interacts with EscU, the integral membrane protein that is linked to substrate specificity switching, implicating EscI in the substrate-switching event. Furthermore, we showed that EscI self-associates and interacts with the outer membrane secretin EscC, further supporting its function as an inner rod protein. Overall, the results of the present study suggest that EscI is the YscI/PrgJ/MxiI homologue in the T3SS of attaching and effacing pathogens.     

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA. T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1‐11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1‐11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein–protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.     

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.     

HpaB from Xanthomonas campestris pv. vesicatoria acts as an exit control protein in type III-dependent protein secretion

The hrp (hypersensitive response and pathogenicity) gene cluster of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria encodes a type III secretion (TTS) system, which injects bacterial effector proteins into the plant cell. Here, we characterized hpaB (hpa, hrp-associated), which encodes a pathogenicity factor with typical features of a TTS chaperone. We show that HpaB is important for the efficient secretion of at least five effector proteins but is dispensable for the secretion of non-effectors such as XopA and the TTS translocon protein HrpF. GST pull-down assays revealed that HpaB interacts with two unrelated effector proteins, AvrBs1 and AvrBs3, but not with XopA. The HpaB-binding site is located within the first 50 amino acids of AvrBs3. This region also contains the targeting signal for HpaB-dependent secretion, which is missing in HrpF and XopA. Intriguingly, the N-termini of HrpF and XopA target the AvrBs3Delta2 reporter for translocation in a DeltahpaB mutant but not in the wild-type strain. This indicates that HpaB plays an essential role in the exit control of the TTS system. Our data suggest that HpaB promotes the secretion of a large set of effector proteins and prevents the delivery of non-effectors into the plant cell.