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 Yersinia pestis type III secretion needle plays a role in the regulation of Yop secretion

Activation of bacterial virulence-associated type III secretion systems (T3SSs) requires direct contact between a bacterium and a eukaryotic cell. In Yersinia pestis, the cytosolic LcrG protein and a cytosolic YopN-TyeA complex function to block T3S in the presence of extracellular calcium and prior to contact with a eukaryotic cell. The mechanism by which the bacterium senses extracellular calcium and/or cell contact and transmits these signals to the cytosolic compartment is unknown. We report here that YscF, a small protein that polymerizes to form the external needle of the T3SS, is essential for the calcium-dependent regulation of T3S. Alanine-scanning mutagenesis was used to identify YscF mutants that secrete virulence proteins in the presence and absence of calcium and prior to contact with a eukaryotic cell. Interestingly, one of the YscF mutants that exhibited constitutive T3S was unable to translocate secreted proteins across the eukaryotic plasma membrane. These data indicate that the YscF needle is a multifunctional structure that participates in virulence protein secretion, in translocation of virulence proteins across eukaryotic membranes and in the cell contact- and calcium-dependent regulation of T3S.     

Secretion of YscP from Yersinia enterocolitica is essential to control the length of the injectisome needle but not to change the type III secretion substrate specificity

The length of the needle of the Yersinia Ysc injectisome is determined by a protein called YscP. This protein, which acts both as a molecular ruler and as a substrate-specificity switch for type III secretion is itself secreted by the injectisome. In this report, we address the question why YscP is secreted. By a systematic deletion analysis and by fusing different parts of the molecule to the adenylate cyclase reporter, we identified two independent secretion signals. One of them is encompassed within the 35 N-terminal residues while the second one spans residues 97-137. These two signals are functionally different from Yop secretion signals. When both secretion signals were removed, Yops could still be secreted but the needle length control was lost. YscP possessing only one signal did not control needle length properly but the control was improved when more YscP was produced and secreted. YscP deprived of both signals could not control length, even when overproduced. We conclude from this that YscP needs to be secreted to exert its length control function but not its substrate-specificity switch function.     

Impassable YscP substrates and their impact on the Yersinia enterocolitica type III secretion pathway

Yersinia type III machines secrete protein substrates across the bacterial envelope and, following assembly of their secretion needles, transport effector Yops into host cells. According to their destination during type III secretion, early, middle, and late secretion substrates can be distinguished; however, the signals and mechanisms whereby these proteins are recognized and transported by the secretion machine are not understood. Here, we examine several hybrids between secretion substrates and the impassable reporter protein glutathione S-transferase (GST). YscP-GST and YopR-GST blocked type III secretion; however, YscF-, YopD-, YopN-, and LcrV-GST did not. Unlike YopR-GST, which can block type III machines only during their assembly, expression of YscP-GST led to an immediate and complete block of all secretion. The secretion signal of YscP was mapped to its first 10 codons or amino acids; however, YscP_Δ2-15-GST, lacking this secretion signal, imposed a partial blockade. YscP-GST copurified with the type III ATPase complex (YscN, YscL, and YscQ) and with YscO, suggesting that the association of specific machine components with the impassable substrate may cause the block in type III secretion.     

YopR impacts type III needle polymerization in Yersinia species

A hallmark of Yersinia type III machines is the presence of needles extending from the bacterial surface. Needles perform two functions, serving as the conduits for the transport of effectors into immune cells but also acting as a sensor. The polymerized needle protein, YscF, is thought to perceive threshold levels of environmental calcium ions to trigger secretion. yopR ( yscH ) is a gene downstream of yscEFG , encoding the chaperones and principal building blocks of the needle. Here we investigated the contribution of YopR towards type III secretion and pathogenesis. Yersinia pestis KIM D27 mutants lacking yopR were defective for virulence in a mouse model of septicemic plague. yopR variants of Yersinia enterocolitica W22703 displayed a reduced ability to inject effectors into macrophages and required lower calcium concentrations to activate type III secretion than wild-type yersiniae. Furthermore, yopR mutants failed to assemble YscF into needle complexes and instead secreted YscF into the medium. These results imply that YopR may be involved in controlling the secretion of YscF, thereby impacting the assembly of type III machines. An alternative possibility, which YopR participates directly in the polymerization of YscF, seems less likely as YopR is not associated with purified needles.     

Brefeldin A reversibly blocks early but not late protein transport steps in the yeast secretory pathway.

We have found that brefeldin A (BFA) inhibited the growth of an ise1 mutant of Saccharomyces cerevisiae. Genetic complementation and mapping studies demonstrated that ise1 was allelic to erg6, a gene required for the biosynthesis of the principal membrane sterol of yeast, ergosterol. Treatment of ise1 cells with BFA resulted in an immediate block in protein transport through the secretory pathway. Vacuolar carboxypeptidase Y (CPY) and the secreted pheromone alpha-factor accumulated as both the core glycosylated (ER) and alpha 1,6 mannosylated (early Golgi) forms in drug-treated cells. The modification of alpha-factor with alpha 1,6 mannose in BFA-treated cells did not appear to result from retrograde transport of the alpha 1,6 mannosyl-transferase into the ER. We found that transport of CPY from medial and late Golgi compartments to the vacuole was unaffected by BFA, nor was secretion of alpha 1,3 mannosylated alpha-factor or invertase blocked by BFA. The effects of BFA on the secretory pathway were also reversible after brief exposure (< 40 min) to the drug. We suggest that the primary effect of BFA in S. cerevisiae is restricted to the ER and the alpha 1,6 mannosyltransferase compartment of the Golgi complex.     

Yersinia type III secretion: send in the effectors

Pathogenic Yersinia spp (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) have evolved an exquisite method for delivering powerful effectors into cells of the host immune system where they inhibit signaling cascades and block the cells' response to infection. Understanding the molecular mechanisms of this system has provided insight into the processes of phagocytosis and inflammation.     

Infectious adenovirus type 2 transport through early but not late endosomes

Receptor-mediated endocytosis is a major gate for pathogens into cells. In this study, we analyzed the trafficking of human adenovirus type 2 and 5 (Ad2/5) and the escape-defective temperature-sensitive Ad2-ts1 mutant in epithelial cancer cells. Ad2/5 and Ad2-ts1 uptake into endosomes containing transferrin, major histocompatibility antigen 1 and the Rab5 effector early endosome antigen 1 (EEA1) involved dynamin, amphiphysin, clathrin and Eps15. Cointernalization experiments showed that most of the Ad2/5 and Ad2-ts1 visited the same EEA1-positive endosomes. In contrast to Ad2/5, Ad2-ts1 required functional Rab5 for endocytosis and lysosomal transport and was sensitive to the phosphatidyl-inositol-3 (PI3)-kinase inhibitor wortmannin or the ubiquitin-binding protein Hrs for sorting from early to late endosomes. Endosomal escape of Ad2 was not affected by incubation at 19 degrees C, which blocked membrane sorting in early endosomes and inhibited Ad2-ts1 transport to lysosomes. Unlike Semliki Forest Virus (SFV), sorting of Ad2-ts1 to late endosomes was independent of Rab7 and Ad2/5 infection independent of EEA1. The data indicate that Ad2/5 and Ad2-ts1 use an invariant machinery for clathrin-mediated uptake to early endosomes. We suggest that the infectious Ad2 particles are either directly released from early endosomes to the cytosol or sorted by a temperature-insensitive and PI3-kinase-independent mechanism to an escape compartment different from late endosomes or lysosomes.     

Proteolytic cleavage of the FlhB homologue YscU of Yersinia pseudotuberculosis is essential for bacterial survival but not for type III secretion

Pathogenic Yersinia species employ a type III secretion system (TTSS) to target antihost factors, Yop proteins, into eukaryotic cells. The secretion machinery is constituted of ca. 20 Ysc proteins, nine of which show significant homology to components of the flagellar TTSS. A key event in flagellar assembly is the switch from secreting-assembling hook substrates to filament substrates, a switch regulated by FlhB and FliK. The focus of this study is the FlhB homologue YscU, a bacterial inner membrane protein with a large cytoplasmic C-terminal domain. Our results demonstrate that low levels of YscU were required for functional Yop secretion, whereas higher levels of YscU lowered both Yop secretion and expression. Like FlhB, YscU was cleaved into a 30-kDa N-terminal and a 10-kDa C-terminal part. Expression of the latter in a wild-type strain resulted in elevated Yop secretion. The site of cleavage was at a proline residue, within the strictly conserved amino acid sequence NPTH. A YscU protein with an in-frame deletion of NPTH was cleaved at a different position and was nonfunctional with respect to Yop secretion. Variants of YscU with single substitutions in the conserved NPTH sequence--i.e., N263A, P264A, or T265A--were not cleaved but retained function in Yop secretion. Elevated expression of these YscU variants did, however, result in severe growth inhibition. From this we conclude that YscU cleavage is not a prerequisite for Yop secretion but is rather required to maintain a nontoxic fold.     

Yersinia YopE is targeted for type III secretion by N-terminal, not mRNA, signals

Pathogenic Yersinia species inject virulence proteins, known as Yops, into the cytosol of eukaryotic cells. The injection of Yops is mediated via a type III secretion system. Previous studies have suggested that YopE is targeted for secretion by two signals. One is mediated by its cognate chaperone YerA, whereas the other consists of either the 5' end of yopE mRNA or the N-terminus of YopE. In order to characterize the YopE N-terminal/5' mRNA secretion signal, the first 11 codons of yopE were systematically mutagenized. Frameshift mutations, which completely alter the amino acid sequence of residues 2-11 but leave the mRNA sequence essentially intact, drastically reduce the secretion of YopE in a yerA mutant. In contrast, a mutation that alters the yopE mRNA sequence, while leaving the amino acid sequence of YopE unchanged, does not impair the secretion of YopE. Therefore, the N-terminus of YopE, and not the 5' end of yopE mRNA, serves as a targeting signal for type III secretion. In addition, the chaperone YerA can target YopE for type III secretion in the absence of a functional N-terminal signal. Mutational analysis of the YopE N-terminus revealed that a synthetic amphipathic sequence of eight residues is sufficient to serve as a targeting signal. YopE is also secreted rapidly upon a shift to secretion-permissive conditions. This 'rapid secretion' of YopE does not require de novo protein synthesis and is dependent upon YerA. Furthermore, this burst of YopE secretion can induce a cytotoxic response in infected HeLa cells.     

Crystal structure of Yersinia enterocolitica type III secretion chaperone SycT

Pathogenic Yersinia species use a type III secretion (TTS) system to deliver a number of cytotoxic effector proteins directly into the mammalian host cell. To ensure effective translocation, several such effector proteins transiently bind to specific chaperones in the bacterial cytoplasm. Correspondingly, SycT is the chaperone of YopT, a cysteine protease that cleaves the membrane-anchor of Rho-GTPases in the host. We have analyzed the complex between YopT and SycT and determined the structure of SycT in three crystal forms. Biochemical studies indicate a stoichometric effector/chaperone ratio of 1:2 and the chaperone-binding site contains at least residues 52-103 of YopT. The crystal structures reveal a SycT homodimer with an overall fold similar to that of other TTS effector chaperones. In contrast to the canonical five-stranded anti-parallel beta-sheet flanked by three alpha-helices, SycT lacks the dimerization alpha-helix and has an additional beta-strand capable of undergoing a conformational change. The dimer interface consists of two beta-strands and the connecting loops. Two hydrophobic patches involved in effector binding in other TTS effector chaperones are also found in SycT. The structural similarity of SycT to other chaperones and the spatial conservation of effector-binding sites support the idea that TTS effector chaperones form a single functional and structural group.     

Deciphering the assembly of the Yersinia type III secretion injectisome

The assembly of the Yersinia enterocolitica type III secretion injectisome was investigated by grafting fluorescent proteins onto several components, YscC (outer‐membrane (OM) ring), YscD (forms the inner‐membrane (IM) ring together with YscJ), YscN (ATPase), and YscQ (putative C ring). The recombinant injectisomes were functional and appeared as fluorescent spots at the cell periphery. Epistasis experiments with the hybrid alleles in an array of injectisome mutants revealed a novel outside‐in assembly order: whereas YscC formed spots in the absence of any other structural protein, formation of YscD foci required YscC, but not YscJ. We therefore propose that the assembly starts with YscC and proceeds through the connector YscD to YscJ, which was further corroborated by co‐immunoprecipitation experiments. Completion of the membrane rings allowed the subsequent assembly of cytosolic components. YscN and YscQ attached synchronously, requiring each other, the interacting proteins YscK and YscL, but no further injectisome component for their assembly. These results show that assembly is initiated by the formation of the OM ring and progresses inwards to the IM ring and, finally, to a large cytosolic complex.     

Crystal structure of the Yersinia type III secretion protein YscE

The plague-causing bacterium Yersinia pestis utilizes a contact-dependent (type III) secretion system (T3SS) to transport virulence factors from the bacterial cytosol directly into the interior of mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. The type III secretion apparatus is composed of 20-25 different Yersinia secretion (Ysc) proteins. We report here the structure of YscE, the smallest Ysc protein, which is a dimer in solution. The probable mode of oligomerization is discussed.     

A chromosomally encoded type III secretion pathway in Yersinia enterocolitica is important in virulence

Numerous Gram-negative bacteria use a type III, or contact dependent, secretion system to deliver proteins into the cytosol of host cells. All of these systems identified to date have been shown to have a role in pathogenesis. We have identified 13 genes on the Yersinia enterocolitica chromosome that encode a type III secretion apparatus plus two associated putative regulatory genes. In order to determine the function of this chromosomally-encoded secretion apparatus, we created an in frame deletion of a gene that has homology to the hypothesized inner membrane pore, ysaV. The ysaV mutant strain failed to secrete eight proteins, called Ysps, normally secreted by the parental strain when grown at 28 degrees C in Luria-Bertani (LB) broth supplemented with 0.4 M NaCl. Disruption of the ysaV gene had no effect on motility or phospholipase activity, suggesting this chromosomally encoded type III secretion pathway is distinct from the flagella secretion pathway of Y. enterocolitica. Deletion of the ysaV gene in a virulence plasmid positive strain had no effect on in vitro secretion of Yops by the plasmid-encoded type III secretion apparatus. Secretion of the Ysps was unaffected by the presence or absence of the virulence plasmid, suggesting the chromosomally encoded and plasmid-encoded type III secretion pathways act independently. Y. enterocolitica thus has three type III secretion pathways that appear to act independently. The ysaV mutant strain was somewhat attenuated in virulence compared with the wild type in the mouse oral model of infection (an approximately 0.9 log difference in LD50). The ysaV mutant strain was nearly as virulent as the wild type when inoculated intraperitoneally in the mouse model. A ysaV probe hybridized to sequences in other Yersinia spp. and homologues were found in the incomplete Y. pestis genome sequence, indicating a possible role for this system throughout the genus.     

Yersinia enterocolitica type III secretion: mutational analysis of the yopQ secretion signal

Pathogenic Yersinia spp. secrete Yop proteins via the type III pathway. yopQ codons 1 to 15 were identified as a signal necessary and sufficient for the secretion of a fused reporter protein. Frameshift mutations that alter codons 2 to 15 with little alteration of yopQ mRNA sequence do not abolish type III transport, suggesting a model in which yopQ mRNA may provide a signal for secretion (D. M. Anderson and O. Schneewind, Mol. Microbiol. 31:1139-1148, 2001). In a recent study, the yopE signal was truncated to codons 1 to 12. All frameshift mutations introduced within the first 12 codons of yopE abolished secretion. Also, multiple synonymous mutations that changed the mRNA sequence of yopE codons 1 to 12 without altering the amino acid sequence did not affect secretion. These results favor a model whereby an N-terminal signal peptide initiates YopE into the type III pathway (S. A. Lloyd et al., Mol. Microbiol. 39:520-531, 2001). It is reported here that codons 1 to 10 of yopQ act as a minimal secretion signal. Further truncation of yopQ, either at codon 10 or at codon 2, abolished secretion. Replacement of yopQ AUG with either of two other start codons, UUG or GUG, did not affect secretion. However, replacement of AUG with CUG or AAA and initiating translation at the fusion site with npt did not permit Npt secretion, suggesting that the translation of yopQ codons 1 to 15 is a prerequisite for secretion. Frameshift mutations of yopQ codons 1 to 10, 1 to 11, and 1 to 12 abolished secretion signaling, whereas frameshift mutations of yopQ codons 1 to 13, 1 to 14, and 1 to 15 did not. Codon changes at yopQ positions 2 and 10 affected secretion signaling when placed within the first 10 codons but had no effect when positioned in the larger fusion of yopQ codons 1 to 15. An mRNA mutant of yopQ codons 1 to 10, generated by a combination of nine synonymous mutations, was defective in secretion signaling, suggesting that the YopQ secretion signal is not proteinaceous. A model is discussed whereby the initiation of YopQ polypeptide into the type III pathway is controlled by properties of yopQ mRNA.     

Two independent type III secretion mechanisms for YopE in Yersinia enterocolitica

Pathogenic Yersinia species escape the infected host's defense mechanisms by targeting cytotoxic Yop proteins into the cytoplasm of macrophages via a type III secretion pathway. Two separate secretion signals contained in YopE were identified, each of which were sufficient but not necessary for the secretion of reporter molecules. One signal is located within the coding sequence of the first 15 amino acids and is sufficient for the secretion of fusion proteins but not required for YopE secretion. The second signal is located downstream at residues 15–100 of YopE and is only recognized by the type III machinery when it is bound to SycE. We propose the existence of two independent mechanisms that allow for the secretion of Yop proteins.     

Crystal structure of the Yersinia enterocolitica type III secretion chaperone SycT

Several Gram-negative pathogens deploy type III secretion systems (TTSSs) as molecular syringes to inject effector proteins into host cells. Prior to secretion, some of these effectors are accompanied by specific type III secretion chaperones. The Yersinia enterocolitica TTSS chaperone SycT escorts the effector YopT, a cysteine protease that inactivates the small GTPase RhoA of targeted host cells. We solved the crystal structure of SycT at 2.5 angstroms resolution. Despite limited sequence similarity among TTSS chaperones, the SycT structure revealed a global fold similar to that exhibited by other structurally solved TTSS chaperones. The dimerization domain of SycT, however, differed from that of all other known TTSS chaperone structures. Thus, the dimerization domain of TTSS chaperones does not likely serve as a general recognition pattern for downstream processing of effector/chaperone complexes. Yersinia Yop effectors are bound to their specific Syc chaperones close to the Yop N termini, distinct from their catalytic domains. Here, we showed that the catalytically inactive YopT(C139S) is reduced in its ability to bind SycT, suggesting an ancillary interaction between YopT and SycT. This interaction could maintain the protease inactive prior to secretion or could influence the secretion competence and folding of YopT.     

Murine gammaherpesvirus targets type I IFN receptor but not type III IFN receptor early in infection

The innate immune response represents a primary line of defense against invading viral pathogens. Since epithelial cells are the primary site of gammaherpesvirus replication during infection in vivo and there are no information on activity of IFN-III signaling against gammaherpesviruses in this cell type, in present study, we evaluated the expression profile and virus-host interactions in mouse mammary epithelial cell (NMuMG) infected with three strains of murine gammaherpesvirus, MHV-68, MHV-72 and MHV-4556. Studying three strains of murine gammaherpesvirus, which differ in nucleotide sequence of some structural and non-structural genes, allowed us to compare the strain-dependent interactions with host organism. Our results clearly demonstrate that: (i) MHV-68, MHV-72 and MHV-4556 differentially interact with intracellular signaling and dysregulate IFN signal transduction; (ii) MHV-68, MHV-72 and MHV-4556 degrade type I IFN receptor in very early stages of infection (2–4 hpi), but not type III IFN receptor; (iii) type III IFN signaling might play a key role in antiviral defense of epithelial cells in early stages of murine gammaherpesvirus replication; (iv) NMuMG cells are an appropriate model for study of not only type I IFN signaling, but also type III IFN signaling pathway. These findings are important for better understanding of individual virus-host interactions in lytic as well as in persistent gammaherpesvirus replication and help us to elucidate IFN-III function in early events of virus infection.     

Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD

Many Gram-negative bacteria use a type III secretion (T3S) system to directly inject effector molecules into eucaryotic cells in order to establish a symbiotic or pathogenic relationship with their host. The translocation of many T3S proteins requires specialized chaperones from the bacterial cytosol. SycD belongs to a class of T3S chaperones that assists the secretion of pore-forming translocators and, specifically chaperones the translocators YopB and YopD from enteropathogenic Yersinia enterocolitica. In addition, SycD is involved in the regulation of virulence factor biosynthesis and secretion. In this study, we present two crystal structures of Y. enterocolitica SycD at 1.95 and 2.6 Å resolution, the first experimental structures of a T3S class II chaperone specific for translocators. The fold of SycD is entirely α-helical and reveals three tetratricopeptide repeat-like motifs that had been predicted from amino acid sequence. In both structures, SycD forms dimers utilizing residues from the first tetratricopeptide repeat motif. Using site-directed mutagenesis and size exclusion chromatography, we verified that SycD forms head-to-head homodimers in solution. Although in both structures, dimerization largely depends on the same residues, the two assemblies represent alternative dimers that exhibit different monomer orientations and overall shape. In these two distinct head-to-head dimers, both the concave and the convex surface of each monomer are accessible for interactions with the SycD binding partners YopB and YopD. A SycD variant carrying two point mutations in the dimerization interface is properly folded but defective in dimerization. Expression of this stable SycD monomer in Yersinia does not rescue the phenotype of a sycD null mutant, suggesting a physiological relevance of the dimerization interface.