Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System.

Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome's cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

The Shigella Type III Secretion System: An Overview from Top to Bottom.

Shigella comprises four species of human-restricted pathogens causing bacillary dysentery. While Shigella possesses multiple genetic loci contributing to virulence, a type III secretion system (T3SS) is its primary virulence factor. The Shigella T3SS nanomachine consists of four major assemblies: the cytoplasmic sorting platform; the envelope-spanning core/basal body; an exposed needle; and a needle-associated tip complex with associated translocon that is inserted into host cell membranes. The initial subversion of host cell activities is carried out by the effector functions of the invasion plasmid antigen (Ipa) translocator proteins, with the cell ultimately being controlled by dedicated effector proteins that are injected into the host cytoplasm though the translocon. Much of the information now available on the T3SS injectisome has been accumulated through collective studies on the T3SS from three systems, those of Shigella flexneri, Salmonella typhimurium and Yersinia enterocolitica/Yersinia pestis. In this review, we will touch upon the important features of the T3SS injectisome that have come to light because of research in the Shigella and closely related systems. We will also briefly highlight some of the strategies being considered to target the Shigella T3SS for disease prevention.

The Structures of SctK and SctD from Pseudomonas aeruginosa Reveal the Interface of the Type III Secretion System Basal Body and Sorting Platform.

Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common architecture in all systems studied thus far, comprising three major components - the cytoplasmic sorting platform, envelope-spanning basal body and external needle with tip complex. The sorting platform consists of an ATPase (SctN) connected to "pods" (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for these residues in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome.

The cytoplasmic domain of MxiG interacts with MxiK and directs assembly of the sorting platform in the Shigella type III secretion system.

Many Gram-negative bacteria use type III secretion systems (T3SSs) to inject virulence effector proteins into eukaryotic cells. The T3SS apparatus (T3SA) is structurally conserved among diverse bacterial pathogens and consists of a cytoplasmic sorting platform, an envelope-spanning basal body, and an extracellular needle with tip complex. The sorting platform is essential for effector recognition and powering secretion. Studies using bacterial "minicells" have revealed an unprecedented level of structural detail of the sorting platform; however, many of the structure-function relationships within this complex remain enigmatic. Here, we report on improved cryo-electron tomographic approaches to enhance the resolution of the Shigella T3SA sorting platform (at ≤2 nm resolution) done in concert with biochemical and genetic methods to define the sorting platform interactome and interactions with the T3SA inner membrane ring (IR). We observed that the sorting platform consists of "pods" with 6-fold symmetry that interact with the Spa47 ATPase via radial extensions comprising MxiN. Most importantly, MxiK maintained an interaction with the IR via specific interactions with the cytoplasmic domain of the IR protein MxiG (MxiGC), which is a noncanonical forkhead-associated domain, and MxiK has an elongated structure that interacts with the IR via MxiGC T4 lysozyme-mediated insertional mutagenesis of MxiK revealed its orientation within the sorting platform and enabled disruption of interactions with its binding partners, which abolished sorting platform assembly. Finally, a comparison with the homologous interactions in the Salmonella T3SS sorting platform revealed clear differences in their IR-sorting platform interfaces that have possible mechanistic implications.

Expression of different ParE toxins results in conserved phenotypes with distinguishable classes of toxicity.

Toxin-antitoxin (TA) systems are found on both chromosomes and plasmids. These systems are unique in that they can confer both fatal and protective effects on bacterial cells-a quality that could potentially be harnessed given further understanding of these TA mechanisms. The current work focuses on the ParE subfamily, which is found throughout proteobacteria and has a sequence identity on average of approximately 12% (similarity at 30%-80%). Our aim is to evaluate the equivalency of chromosomally derived ParE toxin activity depending on its bacterial species of origin. Nine ParE toxins were analyzed, originating from six different bacterial species. Based on the resulting toxicity, three categories can be established: ParE toxins that do not exert toxicity under the experimental conditions, toxins that exert toxicity within the first four hours, and those that exert toxicity only after 10-12 hr of exposure. All tested ParE toxins produce a cellular morphologic change from rods to filaments, consistent with disruption of DNA topology. Analysis of the distribution of filamented cells within a population reveals a correlation between the extent of filamentation and toxicity. No membrane septation is visible along the length of the cell filaments, whereas aberrant lipid blebs are evident. Potent ParE-mediated toxicity is also correlated with a hallmark signature of abortive DNA replication, consistent with the inhibition of DNA gyrase.

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.

The toxin from a ParDE toxin-antitoxin system found in Pseudomonas aeruginosa offers protection to cells challenged with anti-gyrase antibiotics.

Toxin-antitoxin systems are mediators of diverse activities in bacterial physiology. For the ParE-type toxins, their reported role of gyrase inhibition utilized during plasmid-segregation killing indicates they are toxic. However, their location throughout chromosomes leads to questions about function, including potential non-toxic outcomes. The current study has characterized a ParDE system from the opportunistic human pathogen Pseudomonas aeruginosa (Pa). We identified a protective function for this ParE toxin, PaParE, against effects of quinolone and other antibiotics. However, higher concentrations of PaParE are themselves toxic to cells, indicating the phenotypic outcome can vary based on its concentration. Our assays confirmed PaParE inhibition of gyrase-mediated supercoiling of DNA with an IC50 value in the low micromolar range, a species-specificity that resulted in more efficacious inhibition of Escherichia coli derived gyrase versus Pa gyrase, and overexpression in the absence of antitoxin yielded an expected filamentous morphology with multi-foci nucleic acid material. Additional data revealed that the PaParE toxin is monomeric and interacts with dimeric PaParD antitoxin with a KD in the lower picomolar range, yielding a heterotetramer. This work provides novel insights into chromosome-encoded ParE function, whereby its expression can impart partial protection to cultures from selected antibiotics.

Using disruptive insertional mutagenesis to identify the in situ structure-function landscape of the Shigella translocator protein IpaB.

Bacterial type III secretion systems (T3SS) are used to inject proteins into mammalian cells to subvert cellular functions. The Shigella T3SS apparatus (T3SA) is comprised of a basal body, cytoplasmic sorting platform and exposed needle with needle "tip complex" (TC). TC maturation occurs when the translocator protein IpaB is recruited to the needle tip where both IpaD and IpaB control secretion induction. IpaB insertion into the host membrane is the first step of translocon pore formation and secretion induction. We employed disruptive insertional mutagenesis, using bacteriophage T4 lysozyme (T4L), within predicted IpaB loops to show how topological features affect TC functions (secretion control, translocon formation and effector secretion). Insertions within the N-terminal half of IpaB were most likely to result in a loss of steady-state secretion control, however, all but the two that were not recognized by the T3SA retained nearly wild-type hemolysis (translocon formation) and invasiveness levels (effector secretion). In contrast, all but one insertion in the C-terminal half of IpaB maintained secretion control but were impaired for hemolysis and invasion. These nature of the data suggest the latter mutants are defective in a post-secretion event, most likely due to impaired interactions with the second translocator protein IpaC. Intriguingly, only two insertion mutants displayed readily detectable T4L on the bacterial surface. The data create a picture in which the makeup and structure of a functional T3SA TC is highly amenable to physical perturbation, indicating that the tertiary structure of IpaB within the TC is more plastic than previously realized.

Toxin-Antitoxin Modules Are Pliable Switches Activated by Multiple Protease Pathways.

Toxin-antitoxin (TA) modules are bacterial regulatory switches that facilitate conflicting outcomes for cells by promoting a pro-survival phenotypic adaptation and/or by directly mediating cell death, all through the toxin activity upon degradation of antitoxin. Intensive study has revealed specific details of TA module functions, but significant gaps remain about the molecular details of activation via antitoxin degradation used by different bacteria and in different environments. This review summarizes the current state of knowledge about the interaction of antitoxins with cellular proteases Lon and ClpP to mediate TA module activation. An understanding of these processes can answer long-standing questions regarding stochastic versus specific activation of TA modules and provide insight into the potential for manipulation of TA modules to alter bacterial growth.

A GCC-box motif in the promoter of nudix hydrolase 7 (AtNUDT7) gene plays a role in ozone response of Arabidopsis ecotypes.

Arabidopsis nudix hydrolase 7 (AtNudt7) plays an important role in regulating redox homeostasis during stress/defense signaling and seed germination. The early responsiveness of AtNudt7 provides a useful marker especially during oxidative cell death in plants. Nuclear run-on assays demonstrate that AtNudt7 is transcriptionally regulated. AtNUDT7 promoter-GUS transgenic plants show rapid inducibility in response to ozone and pathogens. A 16-bp insertion containing a GCC-box motif was identified in the promoter of a Ws-2 ecotype and was absent in Col-0. The 16-bp sequence was identified in 5% of the Arabidopsis ecotypes used in the 1001 genome sequencing project. The kinetics of expression of Ethylene Response Factor 1 (ERF1), a GCC-box binding factor is in synchrony with expression of AtNudt7 in response to ozone stress. ERF1 protein binds to the GCC-box motif in the AtNUDT7 promoter. In silico analysis of erf1 mutant and overexpressor lines supports a role for this protein in regulating AtNUDT7 expression.

The link between transcript regulation and de novo protein synthesis in the retrograde high light acclimation response of Arabidopsis thaliana.

BACKGROUND: Efficient light acclimation of photosynthetic cells is a basic and important property of plants. The process of acclimation depends on transformation of retrograde signals in gene expression, transcript accumulation and de novo protein synthesis. While signalling cues, transcriptomes and some involved players have been characterized, an integrated view is only slowly emerging, and information on the translational level is missing. Transfer of low (8 µmol quanta.m(-2).s(-1)) or normal light (80 µmol quanta.m(-2).s(-1)) acclimated 30 d old Arabidopsis thaliana plants to high light (800 µmol quanta.m(-2).s(-1)) triggers retrograde signals. Using this established approach, we sought to link transcriptome data with de novo synthesized proteins by in vivo labelling with (35)S methionine and proteome composition. RESULTS: De novo synthesized protein and proteome patterns could reliably be matched with newly annotated master gels. Each molecular level could be quantified for a set of 41 proteins. Among the proteins preferentially synthesized in plants transferred to high light were enzymes including carbonic anhydrase, fructose-1,6-bisphosphate aldolase, O-acetyl serine thiol lyase, and chaperones, while low rates upon transfer to high light were measured for e.g. dehydroascorbate reductase, glyceraldehyde-3-phosphate dehydrogenase and CuZn superoxide dismutase, and opposite responses between 10-fold and 100-fold light increment for e.g. glutamine synthetase and phosphoglycerate kinase. CONCLUSIONS: The results prove the hypothesis that transcript abundance is poorly linked to de novo protein synthesis due to profound regulation at the level of translation. This vertical systems biology approach enables to quantitatively and kinetically link the molecular levels for scrutinizing signal processing and response generation.

The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo.

Hydrogen peroxide (H2O2) evolves during cellular metabolism and accumulates under various stresses causing serious redox imbalances. Many proteomics studies aiming to identify proteins sensitive to H2O2 used concentrations that were above the physiological range. Here the chloroplast proteins were subjected to partial oxidation by exogenous addition of H2O2 equivalent to 10% of available protein thiols which allowed for the identification of the primary targets of oxidation. The chosen redox proteomic approach employed differential labeling of non-oxidized and oxidized thiols using sequential alkylation with N-ethylmaleimide and biotin maleimide. The in vitro identified proteins are involved in carbohydrate metabolism, photosynthesis, redox homeostasis, and nitrogen assimilation. By using methyl viologen that induces oxidative stress in vivo, mostly the same primary targets of oxidation were identified and several oxidation sites were annotated. Ribulose-1,5-bisphosphate (RubisCO) was a primary oxidation target. Due to its high abundance, RubisCO is suggested to act as a chloroplast redox buffer to maintain a suitable redox state, even in the presence of increased reactive oxygen species release. 2-cysteine peroxiredoxins (2-Cys Prx) undergo redox-dependent modifications and play important roles in antioxidant defense and signaling. The identification of 2-Cys Prx was expected based on its high affinity to H2O2 and is considered as a proof of concept for the approach. Targets of Trx, such as phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, transketolase, and sedoheptulose-1,7-bisphosphatase have at least one regulatory disulfide bridge which supports the conclusion that the identified proteins undergo reversible thiol oxidation. In conclusion, the presented approach enabled the identification of early targets of H2O2 oxidation within the cellular proteome under physiological experimental conditions.

Mechanisms and dynamics in the thiol/disulfide redox regulatory network: transmitters, sensors and targets.

Plant cells sense, weigh and integrate various endogenous and exogenous cues in order to optimize acclimation and resource allocation. The thiol/disulfide redox network appears to be in the core of this versatile integration process. In plant cells its complexity exceeds by far that of other organisms. Recent research has elucidated the multiplicity of the diversified input elements, transmitters (thioredoxin, glutaredoxins), targets and sensors (peroxiredoxins and other peroxidases), controlled processes and final acceptors (reactive oxygen species). An additional level of thiol/disulfide regulation is achieved by introducing dynamics in time and subcompartment and complex association.

Arabidopsis chloroplastic glutaredoxin C5 as a model to explore molecular determinants for iron-sulfur cluster binding into glutaredoxins.

Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys(32)) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.

Thiol-disulfide redox proteomics in plant research.

Abiotic stresses often cause metabolic imbalances which affect cellular redox homeostasis and alter the rate of reduction state of functional and regulatory protein thiols and the rate of reactive oxygen species release. Excessive displacement from redox equilibrium causes oxidative damage to cell structures and may elicit cell death. The understanding of the cell response to progressive stress must include knowledge on the thiol redox state of specific proteins. This chapter describes selected gel-based biochemical methods (i) to identify thiol-disulfide redox proteins that undergo major redox-dependent conformational changes by 2D redox SDS-PAGE and (ii) to determine the thiol redox state of proteins by sequential blocking and labeling with N-ethylmaleimide and methoxypolyethylene glycol maleimide-5000 (mPEG-Mal-5000). Both sets of methods provide experimental information that defines the redox proteome of the cell.

Multiple redox and non-redox interactions define 2-Cys peroxiredoxin as a regulatory hub in the chloroplast.

In plants, the highly abundant 2-cysteine peroxiredoxin (2-CysPrx) is associated with the chloroplast and involved in protecting photosynthesis. This work addresses the multiple interactions of the 2-CysPrx in the chloroplast, which depend on its redox state. Transcript co-regulation analysis showed a strong linkage to the peptidyl-prolyl-cis/trans isomerase Cyclophilin 20-3 (Cyp20-3) and other components of the photosynthetic apparatus. Co-expression in protoplasts and quantification of fluorescence resonance energy transfer (FRET) efficiency in vivo confirmed protein interactions of 2-CysPrx with Cyp20-3 as well as NADPH-dependent thioredoxin reductase C (NTRC), while thioredoxin x (Trx-x) did not form complexes that could enable FRET. Likewise, changes in FRET of fluorescently labeled 2-CysPrx in vitro and in vivo proved redox dependent dynamics of 2-CysPrx. Addition of Cyp20-3 to an in vitro peroxidase assay with 2-CysPrx had no significant effect on peroxide reduction. Also, in the presence of NTRC, addition of Cyp20-3 did not further enhance peroxide reduction. In addition, 2-CysPrx functioned as chaperone and inhibited aggregation of citrate synthase during heat treatment. This activity was partly inhibited by Cyp20-3. As a new interaction partner of decameric 2-CysPrx, photosystem II could be identified after chloroplast fractionation and in pull-down assays after reconstitution. In summary, the data indicate a dynamic function of plant 2-CysPrx as redox sensor, chaperone, and regulator in the chloroplast with diverse functions beyond its role as thiol peroxidase.