Modified needle-tip PcrV proteins reveal distinct phenotypes relevant to the control of type III secretion and intoxication by Pseudomonas aeruginosa

The type III secretion system (T3SS) is employed to deliver effector proteins to the cytosol of eukaryotic hosts by multiple species of Gram-negative bacteria, including Pseudomonas aeruginosa. Translocation of effectors is dependent on the proteins encoded by the pcrGVHpopBD operon. These proteins form a T3S translocator complex, composed of a needle-tip complex (PcrV), translocons (PopB and PopD), and chaperones (PcrG and PcrH). PcrV mediates the folding and insertion of PopB/PopD in host plasmic membranes, where assembled translocons form a translocation channel. Assembly of this complex and delivery of effectors through this machinery is tightly controlled by PcrV, yet the multifunctional aspects of this molecule have not been defined. In addition, PcrV is a protective antigen for P. aeruginosa infection as is the ortholog, LcrV, for Yersinia. We constructed PcrV derivatives containing in-frame linker insertions and site-specific mutations. The expression of these derivatives was regulated by a T3S-specific promoter in a pcrV-null mutant of PA103. Nine derivatives disrupted the regulation of effector secretion and constitutively released an effector protein into growth medium. Three of these regulatory mutants, in which the linker was inserted in the N-terminal globular domain, were competent for the translocation of a cytotoxin, ExoU, into eukaryotic host cells. We also isolated strains expressing a delayed-toxicity phenotype, which secrete translocators slowly despite the normal level of effector secretion. Most of the cytotoxic translocation-competent strains retained the protective epitope of PcrV derivatives, and Mab166 was able to protect erythrocytes during infection with these strains. The use of defined PcrV derivatives possessing distinct phenotypes may lead to a better understanding of the functional aspects of T3 needle-tip proteins and the development of therapeutic agents or vaccines targeting T3SS-mediated intoxication.     

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.     

Yersinia enterocolitica evasion of the host innate immune response by V antigen-induced IL-10 production of macrophages is abrogated in IL-10-deficient mice.

The virulence-associated V Ag (LcrV) of pathogenic Yersinia species is part of the translocation apparatus, required to deliver antihost effector proteins (Yersinia outer proteins) into host cells. An orthologous protein (denoted as PcrV) has also been identified in the ExoS regulon of Pseudomonas aeruginosa. Additionally, it is known that LcrV is released by yersiniae into the environment and that LcrV causes an immunosuppressive effect when injected into mice. In this study, we demonstrate for the first time that rLcrV, but not PcrV, is capable of suppressing TNF-alpha production in zymosan A-stimulated mouse macrophages and the human monocytic Mono-Mac-6 cell line. The underlying mechanism of TNF-alpha suppression could be assigned to LcrV-mediated IL (IL)-10 production, because 1) LcrV induces IL-10 release in macrophages, 2) anti-IL-10 Ab treatment completely abrogated TNF-alpha suppression, and 3) TNF-alpha suppression was absent in LcrV-treated macrophages of IL-10-deficient (IL-10-/-) mice. The relevance of LcrV-mediated immunosuppression for the pathogenicity of yersiniae became evident by experimental infection of mice; in contrast to wild-type mice, IL-10-/- mice were highly resistant against Yersinia infection, as shown by lower bacterial load in spleen and liver, absent abscess formation in these organs, and survival.     

Yersinia enterocolitica type III secretion chaperone SycD: recombinant expression, purification and characterization of a homodimer

Yersinia species pathogenic to human benefit from a protein transport machinery, a type three secretion system (T3SS), which enables the bacteria to inject effector proteins into host cells. Several of the transport substrates of the Yersinia T3SS, called Yops (Yersinia outer proteins), are assisted by specific chaperones (Syc for specific Yop chaperone) prior to transport. Yersinia enterocolitica SycD (LcrH in Yersinia pestis and Yersinia pseudotuberculosis) is a chaperone dedicated to the assistance of the translocator proteins YopB and YopD, which are assumed to form a pore in the host cell membrane. In an attempt to make SycD amenable to structural investigations we recombinantly expressed SycD with a hexahistidine tag in Escherichia coli. Combining immobilized nickel affinity chromatography and gel filtration we obtained purified SycD with an exceptional yield of 120mg per liter of culture and homogeneity above 95%. Analytical gel filtration and cross-linking experiments revealed the formation of homodimers in solution. Secondary structure analysis based on circular dichroism suggests that SycD is mainly composed of alpha-helical elements. To prove functionality of purified SycD previously suggested interactions of SycD with Yop secretion protein M2 (YscM2), and low calcium response protein V (LcrV), respectively, were reinvestigated.     

Structure-function analysis of the C-terminal domain of LcrV from Yersinia pestis

LcrV, a multifunctional protein, acts as a positive regulator of effector protein secretion for the type III secretion system (T3SS) in Yersinia pestis by interaction with the negative regulator LcrG. In this study, LcrV was analyzed to identify regions required for LcrG interaction. Random-linker insertion mutagenesis, deletion analysis, and site-directed mutagenesis of hydrophobic amino acids between residues 290 and 311 allowed the isolation of an LcrV mutant (LcrV L291R F308R) defective for LcrG interaction. The new residues identified in LcrG interaction lie in helix 12 of LcrV; residues in helix 7 of LcrV are known to be involved in LcrG interaction. Helix 7 and helix 12 of LcrV interact to form an intramolecular coiled coil; these new results suggest that the intramolecular coiled coil in LcrV is required for LcrG interaction and activation of the T3SS.     

In the Absence of Effector Proteins,the Pseudomonas aeruginosa Type Three Secretion System Needle Tip Complex Contributes to Lung Injury and Systemic Inflammatory Responses

Herein we describe a pathogenic role for the Pseudomonas aeruginosa type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which P. aeruginosa wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (ΔPcrV)] did not. PA103 (ΔU/ΔT) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (ΔPcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (ΔU/ΔT) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (ΔPcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (ΔU/ΔT) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (ΔPcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (ΔU/ΔT) and PA103 (ΔPcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of P. aeruginosa pneumonia-induced lung endothelial barrier disruption.     

pH sensitivity of type III secretion system tip proteins

Many pathogenic gram-negative bacteria employ type III secretion systems to transport proteins into the host cell membrane and cytoplasm to subvert normal cellular functions. The type III secretion apparatus consists of a basal body spanning the inner and outer bacterial membranes and a needle which extends away from the bacterium. Recent work has found that a special class of proteins localizes to the tip of the needle to control secretion of effector proteins. Five of these tip proteins are IpaD (Shigella flexneri), BipD (Burkholderia pseudomallei), SipD (Salmonella spp.), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa). In this study, the conformational stability of these proteins was characterized as a function of pH and temperature. Understanding the stability of the proteins in different pH environments is particularly important since they are expected to encounter different pH environments in their passage through the gastrointestinal tract and are exposed to low pH microenvironments near the surface of target cell membranes. Secondary and tertiary structural changes were monitored using the spectroscopic techniques of far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and ultraviolet absorption spectroscopy. Optical density and right angle scattering measurements were also used to evaluate protein association/dissociation. Empirical phase diagrams were then applied to mathematically combine data from the various spectroscopic techniques to provide a global picture of the proteins' structural behavior in solution. The responses of the proteins to changes in temperature and pH conditions reveal two distinct subfamilies in terms of stability. The first is that of IpaD, BipD, and SipD whose corresponding phase diagrams show conformational differences at pH 5-6. The conserved pH dependence in this subfamily suggests possible common mechanistic function. In the second subfamily (LcrV and PcrV), conformational stability is directly related to pH, also indicating mechanistic similarities.     

Identification and characterization of VopT, a novel ADP-ribosyltransferase effector protein secreted via the Vibrio parahaemolyticus type III secretion system 2

Vibrio parahaemolyticus strain RIMD2210633 has two sets of genes encoding two separate type III secretion systems (T3SSs), called T3SS1 and T3SS2. T3SS2 has a role in enterotoxicity and is present only in Kanagawa phenomenon-positive strains, which are pathogenic to humans. Accordingly, T3SS2 is considered to be closely related to V. parahaemolyticus human pathogenicity. Despite this, the biological actions of T3SS2 and the identity of the effector protein(s) secreted by this system have not been well understood. Here we report that T3SS2 induces a cytotoxic effect in Caco-2 and HCT-8 cells. Moreover, it was revealed that VPA1327 (vopT), a gene encoded within the proximity of T3SS2, is partly responsible for this cytotoxic effect. The VopT shows approximately 45% and 44% identity with the ADP-ribosyltransferase (ADPRT) domain of ExoT and ExoS, respectively, which are two T3SS-secreted effectors of Pseudomonas aeruginosa. T3SS2 was found to be necessary not only for the secretion, but also for the translocation of the VopT into host cells. We also demonstrate that VopT ADP-ribosylates Ras, a member of the low-molecular-weight G (LMWG) proteins both in vivo and in vitro. These results indicate that VopT is a novel ADPRT effector secreted via V. parahaemolyticus T3SS.     

Cross talk between type III secretion and flagellar assembly systems in Pseudomonas aeruginosa

Pseudomonas aeruginosa cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (DeltafliC) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (DeltamotAB) did not. More intriguingly, a strain lacking motCD genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile P. aeruginosa strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a gacA mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types.     

Conserved Salt Bridges Facilitate Assembly of the Helical Core Export Apparatus of a Salmonella enterica Type III Secretion System

Virulence-associated type III secretion systems (T3SS) are utilized by Gram negative bacterial pathogens for injection of effector proteins into eukaryotic host cells. The transmembrane export apparatus at the core of T3SS is composed of a unique helical complex of the hydrophobic proteins SctR, SctS, SctT, and SctU. These components comprise a number of highly conserved charged residues within their hydrophobic domains. The structure of the closed state of the core complex SctR₅S₄T₁ revealed that several of these residues form inter- and intramolecular salt bridges, some of which have to be broken for pore opening. Mutagenesis of individual residues was shown to compromise assembly or secretion of both, the virulence-associated and the related flagellar T3SS. However, the exact role of these conserved charged residues in the assembly and function of T3SS remains elusive. Here we performed an in-depth mutagenesis analysis of these residues in the T3SS of Salmonella Typhimurium, coupled to blue native PAGE, in vivo photocrosslinking and luciferase-based secretion assays. Our data show that these conserved salt bridges are not critical for assembly of the respective protein but rather facilitate the incorporation of the following subunit into the assembling complex. Our data also indicate that these conserved charged residues are critical for type III-dependent secretion and reveal a functional link between SctS_E44 and SctT_R204 and the cytoplasmic domain of SctU in gating the T3SS injectisome. Overall, our analysis provides an unprecedented insight into the delicate requirements for the assembly and function of the machinery at the core of T3SS.     

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.     

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.     

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.     

Multiple lessons from the multiple functions of a regulator of type III secretion system assembly in the plant pathogen Pseudomonas syringae

The assembly of type III secretion systems (T3SSs), which inject bacterial effector proteins into the cytosol of animal and plant hosts, is a highly regulated process. Animal pathogens use a length-control protein to produce T3SS needles of fixed length and then a second regulator, such as YopN in Yersinia spp, to mediate host contact-dependent effector delivery. For Pseudomonas syringae and other plant pathogens, regulation of the assembly process differs because the T3SS pilus must grow through variably thick plant cell walls before contacting the host plasma membrane. In this issue of Molecular Microbiology, Crabill et al. (2012) report evidence that the YopN homologue HrpJ is a multifunctional regulator of T3SS assembly in DC3000. A hrpJ mutant hyper-secretes pilus protein and no longer secretes four translocator proteins in culture, and it fails to inject effectors in planta. As with other proteins in this class, HrpJ is itself a T3SS substrate, but secretion-incompetent forms retain regulatory function. However, HrpJ is unusual in suppressing innate immune responses within host cells, as demonstrated with transgenic plants. The multiple capabilities of HrpJ appear to couple host contact sensing with pilus length control and translocator secretion while also contributing to immunity suppression early in the interaction.     

New structural insights into the bacterial type III secretion system

The virulence-associated type III secretion system (T3SS) enables many Gram-negative bacterial pathogens to translocate proteins into the eukaryotic host cells that they infect. This unique protein transport process is mediated by the type III secretion apparatus (T3SA), a multisubunit membrane-spanning macromolecular assembly comprising >20 different proteins. Recent studies have identified biochemical and structural properties of the core T3SA, in addition to several components constituting this complex, with important implications for both the assembly process and the overall function of the T3SA.     

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.     

铜绿假单胞菌中Ⅲ型分泌系统调控机制及针对性治疗的研究进展

铜绿假单胞菌是临床上重要的条件致病菌,具有多种毒力因子且极易产生耐药性。Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)是铜绿假单胞菌中重要的毒性因子分泌系统,该菌通过Ⅲ型分泌系统将多种毒力因子注入到真核宿主细胞内并逃逸宿主细胞免疫系统的清除,引起宿主细胞相应的病理变化。对Ⅲ型分泌系统的研究,不仅有助于明确铜绿假单胞菌的致病机理,更可为临床治疗和药物研发提供理论基础。本文主要对铜绿假单胞菌中Ⅲ型分泌系统的结构、功能、调控机制以及针对性治疗策略等方面的研究进行了综述。     

Advances on Vibrio parahaemolyticus research in the postgenomic era

Vibrio parahaemolyticus is a leading cause of seafood-borne bacterial gastroenteritis in humans. Since its discovery in 1950, this bacterium has been isolated in widespread outbreaks and in sporadic cases of gastroenteritis worldwide. Although the exotoxin, thermostable direct hemolysin, had been the focus of extensive research on the pathogenicity of V. parahaemolyticus, the whole-genome sequencing of a clinical isolate, RIMD2210633 strain, was a breakthrough in this field. The possession of two sets of gene clusters for type III secretion systems (T3SS1 and T3SS2) was unveiled by that genome project. T3SS is a protein export apparatus that delivers bacterial proteins, called effectors, directly into the host's cytosol, to disrupt host cell function. The subsequent studies have established that T3SS2, which is encoded in an 80 kb pathogenicity island called V. parahaemolyticus pathogenicity island (Vp-PAI), is closely related to enteropathogenicity. Recent functional analyses of Vp-PAI-encoded genes revealed the sophisticated mechanisms in V. parahaemolyticus for sensing the intestinal environment and host cell contact, and a dozen T3SS2-exported proteins encoded in Vp-PAI. In this review, we summarize recent advances in V. parahaemolyticus research regarding the control of the expression of Vp-PAI-encoded genes, structural components and the secretory regulation of T3SS2, and the biological activities of T3SS2-exported effectors. Thus, Vp-PAI-encoded T3SS2 becomes an important key in the postgenomic era to shed light on the enteropathogenic mechanism of V. parahaemolyticus.