Quantification of the physiochemical constraints on the export of spider silk proteins by <Emphasis Type="Italic">Salmonella</Emphasis> type III secretion

Background  

The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein.     

Genetically distinct pathways guide effector export through the type VI secretion system

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co‐regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone‐like quality of Hcp. Application of this approach to the Hcp secretion island I‐encoded T6SS (H1‐T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (t ype VI s ecretion e xported 4), subsequently shown to act as a potent intra‐specific H1‐T6SS‐delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1‐T6SS effectors, Tse5 and Tse6, which differ from Hcp‐stabilized substrates by the presence of toxin‐associated PAAR‐repeat motifs and genetic linkage to members of the valine‐glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp‐stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1‐T6SS‐exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.     

Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP

Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion‐competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well‐characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two‐hybrid, co‐precipitation, cross‐linking and size exclusion chromatography we show that Slc1 (SycE‐like chaperone 1; CT043) specifically interacts with a 200‐amino‐acid residue N‐terminal region of TARP (TARP^1–200). Slc1 formed homodimers in vitro, as shown in cross‐linking and gel filtration experiments. Biochemical analysis of an isolated Slc1–TARP^1–200 complex was consistent with a characteristic 2:1 chaperone–effector stoichiometry. Furthermore, Slc1 was co‐immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP.     

Regulation of Vibrio parahaemolyticus T3SS2 gene expression and function of T3SS2 effectors that modulate actin cytoskeleton

Vibrio parahaemolyticus is a leading causative agent of seafood‐borne gastroenteritis worldwide. Most clinical isolates from patients with diarrhoea possess two sets of genes for the type III secretion system (T3SS) on each chromosome (T3SS1 and T3SS2). T3SS is a protein secretion system that delivers effector proteins directly into eukaryotic cells. The injected effectors modify the normal cell functions by altering or disrupting the normal cell signalling pathways. Of the two sets of T3SS genes present in V. parahaemolyticus, T3SS2 is essential for enterotoxicity in several animal models. Recent studies have elucidated the biological activities of several T3SS2 effectors and their roles in virulence. This review focuses on the regulation of T3SS2 gene expression and T3SS2 effectors that specifically target the actin cytoskeleton.     

Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.     

The ESX/type VII secretion system modulates development,but not virulence,of the plant pathogen Streptomyces scabies

Streptomyces scabies is a model organism for the investigation of plant–microbe interactions in Gram‐positive bacteria. Here, we investigate the type VII protein secretion system (T7SS) in S. scabies; the T7SS is required for the virulence of other Gram‐positive bacteria, including Mycobacterium tuberculosis and Staphylococcus aureus. The hallmarks of a functional T7SS are an EccC protein that forms an essential component of the secretion apparatus and two small, sequence‐related substrate proteins, EsxA and EsxB. A putative transmembrane protein, EccD, may also be associated with T7S in Actinobacteria. In this study, we constructed strains of the plant pathogen S. scabies carrying marked mutations in genes coding for EccC, EccD, EsxA and EsxB. Unexpectedly, we showed that all four mutant strains retain full virulence towards several plant hosts. However, disruption of the esxA or esxB, but not eccC or eccD, genes affects S. scabies development, including a delay in sporulation, abnormal spore chains and resistance to lysis by the Streptomyces‐specific phage ?C31. We further showed that these phenotypes are specific to the loss of the T7SS substrate proteins EsxA and EsxB, and are not observed when components of the T7SS secretion machinery are lacking. Taken together, these results imply an unexpected intracellular role for EsxA and EsxB.     

Pseudomonas syringae pv. tomato DC3000 polymutants deploying coronatine and two type III effectors produce quantifiable chlorotic spots from individual bacterial colonies in Nicotiana benthamiana leaves

Primary virulence factors of Pseudomonas syringae pv. tomato DC3000 include the phytotoxin coronatine (COR) and a repertoire of 29 effector proteins injected into plant cells by the type III secretion system (T3SS). DC3000 derivatives differentially producing COR, the T3SS machinery and subsets of key effectors were constructed and assayed in leaves of Nicotiana benthamiana. Bacteria were inoculated by the dipping of whole plants and assayed for population growth and the production of chlorotic spots on leaves. The strains fell into three classes. Class I strains are T3SS⁺ but functionally effectorless, grow poorly in planta and produce faint chlorotic spots only if COR⁺. Class II strains are T3SS^– or, if T3SS⁺, also produce effectors AvrPtoB and HopM1. Class II strains grow better than class I strains in planta and, if COR⁺, produce robust chlorotic spots. Class III strains are T3SS⁺ and minimally produce AvrPtoB, HopM1 and three other effectors encoded in the P. syringae conserved effector locus. These strains differ from class II strains in growing better in planta, and produce chlorotic spots without COR if the precursor coronafacic acid is produced. Assays for chlorotic spot formation, in conjunction with pressure infiltration of low‐level inoculum and confocal microscopy of fluorescent protein‐labelled bacteria, revealed that single bacteria in the apoplast are capable of producing colonies and associated leaf spots in a 1 : 1 : 1 manner. However, COR makes no significant contribution to the bacterial colonization of the apoplast, but, instead, enables a gratuitous, semi‐quantitative, surface indicator of bacterial growth, which is determined by the strain's effector composition.     

Crystal structure of the C‐terminal domain of the Salmonella type III secretion system export apparatus protein InvA

InvA is a prominent inner‐membrane component of the Salmonella type III secretion system (T3SS) apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria. InvA is made up of an N‐terminal integral membrane domain and a C‐terminal cytoplasmic domain that is proposed to form part of a docking platform for the soluble export apparatus proteins notably the T3SS ATPase InvC. Here, we report the novel crystal structure of the C‐terminal domain of Salmonella InvA which shows a compact structure composed of four subdomains. The overall structure is unique although the first and second subdomains exhibit structural similarity to the peripheral stalk of the A/V‐type ATPase and a ring building motif found in other T3SS proteins respectively.     

EseD, a Putative T3SS Translocon Component of Edwardsiella tarda, Contributes to Virulence in Fish and is a Candidate for Vaccine Development

Edwardsiella tarda has a type III secretion system (T3SS) essential for pathogenesis. EseD, together with EseB and EseC, has been suggested to form a putative T3SS translocon complex, although its further function is unclear. To investigate the physiological role of EseD, a mutant strain of E. tarda was constructed with an in-frame deletion of the entire eseD gene. One finding was that the ?eseD mutant decreased the secretion levels of EseC and EseB proteins. Additionally, the ?eseD mutant showed attenuated swarming and contact-hemolysis abilities. However, the ?eseD mutant showed increased biofilm formation. Complementation of the mutant strain with eseD restored these phenotypes to those similar to the wild-type strain. Furthermore, infection experiments in fish showed that the ?eseD mutant exhibited slower proliferation and a tenfold decrease in virulence in fish. These results indicate a specific role of EseD in the pathogenesis of E. tarda. Finally, recombinant EseD protein elicited high antibody titers in immunized fish and various levels of protection against lethal challenge with the wild-type strain. These results indicate that EseD protein may be a candidate antigen for development of a subunit vaccine against Edwardsiellosis.     

Stretching the arms of the type VI secretion sheath protein

The bacterial type VI secretion system (T6SS) is a multicomponent complex responsible for the translocation of effector proteins into the external milieu. The T6SS consists of an external sheath, an internal rigid tube, a baseplate, and a T6SS‐specific membrane complex. Secretion is accomplished by the contraction of the sheath, which expels the effector‐loaded tube. In this issue of EMBO reports, Brackmann et al 1 show how modifications of the sheath subunits can lock the T6SS assembly in the extended state. These findings allowed Wang et al 2 and Nazarov et al 3 to purify the T6SS sheath–tube–baseplate complex in the extended pre‐secretion state and to analyze its structure using cryo‐electron microscopy (cryoEM).     

Distribution of type III secretion systems in Vibrio parahaemolyticus from the northern Gulf of Mexico

Aims: Two well‐characterized Vibrio parahaemolyticus pathogenicity factors – thermostable direct haemolysin (TDH) and TDH‐related haemolysin – are produced by strains containing the tdh and trh genes, respectively. Most strains of V. parahaemolyticus contain two nonredundant type III secretion systems (T3SS), T3SS1 and T3SS2, both of which contribute to pathogenicity. Furthermore, a recent study has revealed two distinct lineages of the V. parahaemolyticus T3SS2: T3SS2α and T3SS2β. The aim of this study was to determine the incidence of these pathogenicity factors in environmental isolates of V. parahaemolyticus. Methods and Results: We collected 130 V. parahaemolyticus isolates (TCBS agar) containing tdh and/or trh (determined by colony hybridization) from sediment, oyster and water in the northern Gulf of Mexico and screened them and 12 clinical isolates (PCR and agarose gel electrophoresis) for pathogenicity factors tdh, trh, T3SS1, T3SS2α and T3SS2β. The majority of potential pathogens were detected in the sediment, including all tdh^?/trh⁺ isolates. T3SS2α components were detected in all tdh⁺/trh ^? isolates and zero of 109 trh⁺ isolates. One T3SS2α gene, vopB2, was found in all tdh⁺/trh^? clinical strains but not in any of the 130 environmental strains. Fluorescence in situ hybridization adapted for individual gene recognition (RING‐FISH) was used to confirm the presence/absence of vopB2. T3SS2β was found in all tdh^?/trh⁺ isolates and in no tdh⁺/trh^? isolates. Conclusions: The combination of haemolysins found in each isolate consistently corresponded to the presence and type of T3SS detected. The vopB2 gene may represent a novel marker for identifying increased virulence among strains. Significance and Impact of the Study: This is the first study to confirm the presence of T3SS2β genes in V. parahaemolyticus strains isolated from the Gulf of Mexico and one of the few that examines the distribution and co‐existence of tdh, trh, T3SS1, T3SS2α and T3SS2β in a large collection of environmental strains.     

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.     

Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae

Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.     

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence,gliding motility and the degradation of complex biopolymers

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres‐Chlorobi‐Bacteroidetes superphylum. Proteins secreted by the T9SS have an N‐terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C‐terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C‐terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components.     

Steps Toward High‐Performance PLA: Economical Production of d‐Lactate Enabled by a Newly Isolated Sporolactobacillus terrae Strain

Optically pure d ‐lactate production has received much attention for its critical role in high‐performance polylactic acid production. However, the current technology can hardly meet the comprehensive demand of industrialization on final titer, productivity, optical purity, and raw material costs. Here, an efficient d ‐lactate producer strain, Sporolactobacillus terrae (S. terrae) HKM‐1, is isolated for d ‐lactate production. The strain HKM‐1 shows extremely high d ‐lactate fermentative capability by using peanut meal, soybean meal, or corn steep liquor powder as a sole nitrogen source; the final titers (205.7 g L^?1, 218.9 g L^?1, and 193.9 g L^?1, respectively) and productivities (5.56 g L^?1 h^?1, 5.34 g L^?1 h^?1, and 3.73 g L^?1 h^?1, respectively) of d ‐lactate reached the highest level ever reported. A comparative genomic analysis between S. terrae HKM‐1 and previously reported d ‐lactate high‐producing Sporolactobacillus inulinus (S. inulinus) CASD is conducted. The results show that many unrelated genetic features may contribute to the superior performance in d ‐lactate production of S. terrae HKM‐1. This d ‐lactate producer HKM‐1, along with its fermentation process, is promising for sustainable d ‐lactate production by using agro‐industrial wastes.     

New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens

Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS‐secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self‐resistance (‘immunity’) against T6SS‐dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS‐secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS‐secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches.     

Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential

The enterohepatic Epsilonproteobacterium Helicobacter hepaticus persistently colonizes the intestine of mice and causes chronic inflammatory symptoms in susceptible mouse strains. The bacterial factors causing intestinal inflammation are poorly characterized. A large genomic pathogenicity island, HHGI1, which encodes components of a type VI secretion system (T6SS), was previously shown to contribute to the colitogenic potential of H. hepaticus. We have now characterized the T6SS components Hcp, VgrG1, VgrG2 and VgrG3, encoded on HHGI1, including the potential impact of the T6SS on intestinal inflammation in a mouse T‐cell transfer model. The H. hepaticus T6SS components were expressed during the infection and secreted in a T6SS‐dependent manner, when the bacteria were cultured either in the presence or in the absence of mouse intestinal epithelial cells. Mutants deficient in VgrG1 displayed a significantly lower colitogenic potential in T‐cell‐transferred C57BL/6 Rag2^?/? mice, despite an unaltered ability to colonize mice persistently. Intestinal microbiota analyses demonstrated only minor changes in mice infected with wild‐typeH. hepaticus as compared with mice infected with VgrG1‐deficient isogenic bacteria. In addition, competitive assays between both wild‐type and T6SS‐deficient H. hepaticus, and between wild‐type H. hepaticus and Campylobacter jejuni or Enterobacteriaceae species did not show an effect of the T6SS on interbacterial competitiveness. Therefore, we suggest that microbiota alterations did not play a major role in the changes of pro‐inflammatory potential mediated by the T6SS. Cellular innate pro‐inflammatory responses were increased by the secreted T6SS proteins VgrG1 and VgrG2. We therefore concluded that the type VI secretion component VgrG1 can modulate and specifically exacerbate the innate pro‐inflammatory effect of the chronic H. hepaticus infection.     

Crystal structure of YwpF from Staphylococcus aureus reveals its architecture comprised of a β‐barrel core domain resembling type VI secretion system proteins and a two‐helix pair

The ywpF gene (SAV2097) of the Staphylococcus aureus strain Mu50 encodes the YwpF protein, which may play a role in antibiotic resistance. Here, we report the first crystal structure of the YwpF superfamily from S. aureus at 2.5‐Å resolution. The YwpF structure consists of two regions: an N‐terminal core β‐barrel domain that shows structural similarity to type VI secretion system (T6SS) proteins (e.g., Hcp1, Hcp3, and EvpC) and a C‐terminal two‐helix pair. Although the monomer structure of S. aureus YwpF resembles those of T6SS proteins, the dimer/tetramer model of S. aureus YwpF is distinct from the functionally important hexameric ring of T6SS proteins. We therefore suggest that the S. aureus YwpF may have a different function compared to T6SS proteins. Proteins 2015; 83:781–788. © 2015 Wiley Periodicals, Inc.