Substrate recognition by the Yersinia type III protein secretion machinery

Type III secretion is the designation given to those protein secretion pathways, primarily in pathogenic Gram-negative bacteria, whose secretion machinery components share an amino acid sequence homology to components of the flagellar basal body. In Yersinia spp., these secretion machineries inject virulence proteins called Yops into the cytosol of target macrophages in an effort to evade phagocytic killing. To date, a clear mechanism by which Yops are recognized by the type III secretion machinery has not been elucidated. Unlike most, if not all, previously characterized protein sorting pathways, the information that identifies Yops as substrates for secretion seems not to be wholly encoded within the Yop peptide sequence. In fact, it appears that at least some of this information is contained within yop mRNAs. This review summarizes recent observations that have been made in this unusual field and proposes models by which proteins may be initiated into this pathway.     

Substrate specificity classes and the recognition signal for Salmonella type III flagellar export

Most flagellar proteins of Salmonella are exported to their assembly destination via a specialized apparatus. This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria. Extensive studies have been carried out on the export of several of the flagellar proteins, most notably the hook protein (FlgE), the hook-capping protein (FlgD), and the filament protein flagellin (FliC). This has led to the concept of two export specificity classes, the rod/hook type and the filament type. However, little direct experimental evidence has been available on the export properties of the basal-body rod proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction protein (FliE), or the muramidase and putative rod-capping protein (FlgJ). In this study, we have measured the amounts of these proteins exported before and after hook completion. Their amounts in the culture supernatant from a flgE mutant (which is still at the hook-type specificity stage) were much higher than those from a flgK mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the hook-type proteins. Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the hook-capping protein FlgD. We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after protein synthesis had been blocked by spectinomycin or chloramphenicol. We conclude that the amino acid sequence of these proteins suffices to mediate their recognition and export.     

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.     

Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica

The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU^C) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU^C at 2.05 Å and 1.55 Å resolution, respectively. The globular domain is found to consist of a central, mixed β-sheet surrounded by α-helices. The NPTH motif forms a type II β-turn connecting two β-strands. NMR analysis of cleaved and uncleaved YscU^C indicates that the global structure of the protein is retained in cleaved YscU^C. The structure of YscU^C variant N263D reveals that wild type YscU^C is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely α-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.     

The secretion signal of YopN, a regulatory protein of the Yersinia enterocolitica type III secretion pathway

The type III secretion signal of Yersinia enterocolitica YopN was mapped using a gene fusion approach. yopN codons 1 to 12 were identified as critical for signal function. Several synonymous mutations that abolish secretion of hybrid proteins without altering the codon specificity of yopN mRNA were identified.     

Substrate recognition by the bacterial type II secretion system: more than a simple interaction

Type II secretion system (T2SS) is a multiprotein trans‐envelope complex that translocates fully folded proteins through the outer membrane of Gram‐negative bacteria. Although T2SS is extensively studied in several bacteria pathogenic for humans, animals and plants, the molecular basis for exoprotein recruitment by this secretion machine as well as the underlying targeting motifs remain unknown. To address this question, we used bacterial two‐hybrid, surface plasmon resonance, in vivo site‐specific photo‐cross‐linking approaches and functional analyses. We showed that the fibronectin‐like Fn3 domain of exoprotein PelI from Dickeya dadantii interacts with four periplasmic domains of the T2SS components GspD and GspC. The interaction between exoprotein and the GspC PDZ domain is positively modulated by the GspD N1 domain, suggesting that exoprotein secretion is driven by a succession of synergistic interactions. We found that an exposed 9‐residue‐long loop region of PelI interacts with the GspC PDZ domain. This loop acts as a specific secretion signal that controls exoprotein recruitment by the T2SS. Concerted in silico and in vivo approaches reveal the occurrence of equivalent secretion motifs in other exoproteins, suggesting a plausible general mechanism of exoprotein recruitment by the T2SS.     

Yersinia enterocolitica type III secretion: an mRNA signal that couples translation and secretion of YopQ

Pathogenic Yersinia species export Yop proteins via a type III machinery to escape their phagocytic killing during animal infections. Here, we reveal the type III export mechanism of YopQ. In the presence of calcium, when type III secretion was blocked, yopQ mRNA was not translated. The signal of YopQ sufficient for the secretion of translationally fused reporter proteins was contained within the first 10 codons of its open reading frame. Some frameshift mutations that completely altered the peptide sequence specified by this signal did not impair secretion of the reporter protein. Exchanging the upstream untranslated mRNA leader of yopQ for that of E. coli lacZ also did not affect secretion. However, removal of the first 15 codons abolished YopQ export. Pulse-labelled YopE, but not YopQ, could be secreted after the polypeptide had been synthesized within the cytoplasm of Yersinia (post-translational secretion). Thus, YopQ appears to be exported by a mechanism that couples yopQ mRNA translation with the type III secretion of the encoded polypeptide.     

Molecular and functional analysis of the type III secretion signal of the Salmonella enterica InvJ protein

Central to the pathogenicity of Salmonella enterica is the function of a type III secretion system (TTSS) encoded within a pathogenicity island at centisome 63 (SPI-1). An essential component of this system is a supramolecular structure termed the needle complex. Proteins to be delivered into host cells possess specific signals that route them to the type III secretion pathway. In addition, some bacterial proteins have signals that deliver them to the secretion complex to either become their structural components or exert their function at that location. One of these proteins is InvJ, which controls the length of the needle substructure of the needle complex. In this study, we have analysed the signal that targets InvJ to the TTSS. We found that amino acid residues 4 to 7 of InvJ are necessary and sufficient to mediate secretion of InvJ or a reporter protein in a TTSS-dependent manner. InvJ secretion was found to be essential for its function in needle length determination, effector protein secretion and bacterial invasion of epithelial cells. Frameshift mutagenesis analysis indicated that the InvJ type III secretion signal sequence tolerates significant alterations in its amino acid sequence without affecting InvJ secretion. Introduction of silent mutations in the secretion signal coding sequence that result in drastically different predicted mRNA folds had no effect on InvJ secretion or expression.     

Prediction of signal recognition particle RNA genes

We describe a method for prediction of genes that encode the RNA component of the signal recognition particle (SRP). A heuristic search for the strongly conserved helix 8 motif of SRP RNA is combined with covariance models that are based on previously known SRP RNA sequences. By screening available genomic sequences we have identified a large number of novel SRP RNA genes and we can account for at least one gene in every genome that has been completely sequenced. Novel bacterial RNAs include that of Thermotoga maritima, which, unlike all other non-gram-positive eubacteria, is predicted to have an Alu domain. We have also found the RNAs of Lactococcus lactis and Staphylococcus to have an unusual UGAC tetraloop in helix 8 instead of the normal GNRA sequence. An investigation of yeast RNAs reveals conserved sequence elements of the Alu domain that aid in the analysis of these RNAs. Analysis of the human genome reveals only two likely genes, both on chromosome 14. Our method for SRP RNA gene prediction is the first convenient tool for this task and should be useful in genome annotation.     

Revisiting the chlamydial type III protein secretion system: clues to the origin of type III protein secretion

The bacterial type III secretion pathway delivers effector proteins into eukaryotic cells. Analysis of the type III system and flagellar export genes in the obligate parasites of the family Chlamydiales suggests that the type III system arose from the flagellar export system in chlamydiae or related bacteria.     

Yersinia yopQ mRNA encodes a bipartite type III secretion signal in the first 15 codons

The type III machinery of Yersinia transports Yop proteins across the bacterial envelope. The minimal secretion signal of yopQ is located in codons 1-10 that, when fused in frame to the neomycin phosphotransferase gene, is sufficient to promote type III secretion of YopQ(1-10)-Npt. Frame-shift mutations, generated by nucleotide insertions or deletions following the AUG start and suppressed at the fusion site with npt, abrogate signalling of yopQ(1-10) but not of yopQ(1-15). By generating transversions of every single nucleotide in yopQ(1-10), we identified 10 nucleotide positions in codons 2, 3, 5, 7, 9 and 10 that were each required for substrate recognition. One transversion that abolishes secretion, uridyl 9 to adenyl (U9A), is a synonymous codon 3 mutation that retains the original amino acid as confirmed by Edman degradation analysis, suggesting that the mRNA but not the amino acid sequence of yopQ(1-10) is involved in secretion signalling. Although transversion of U8A abrogates signalling of yopQ(1-10), fusion of yopQ codons 11-15 restores secretion. The nucleotides that are required for this suppression by yopQ(11-15) were identified and revealed both synonymous and non-synonymous mutations. Frame-shift mutations introduced into just this suppressor region (codons 11-15) did not abrogate its ability to suppress mutations in the minimal secretion signal (codons 1-10). Thus, elements downstream of the minimal secretion signal of YopQ increase the efficiency of YopQ secretion and suppress mutations elsewhere in the secretion signal.     

The type III secretion injectisome

The type III secretion injectisome is a complex nanomachine that allows bacteria to deliver protein effectors across eukaryotic cellular membranes. In recent years, significant progress has been made in our understanding of its structure, assembly and mode of operation. The principal structural components of the injectisome, from the base located in the bacterial cytosol to the tip of the needle protruding from the cell surface, have been investigated in detail. The structures of several constituent proteins were solved at the atomic level and important insights into the assembly process have been gained. However, despite the ongoing concerted efforts of molecular and structural biologists, the role of many of the constituent components of this nanomachine remain unknown.     

A synonymous mutation in Yersinia enterocolitica yopE affects the function of the YopE type III secretion signal

Yersinia spp. inject virulence proteins called Yops into the cytosol of target eukaryotic cells in an effort to evade phagocytic killing via a dedicated protein-sorting pathway termed type III secretion. Previous studies have proposed that, unlike other protein translocation mechanisms, Yops are not recognized as substrates for secretion via a solely proteinaceous signal. Rather, at least some of this information may be encoded within yop mRNA. Herein, we report that the first seven codons of yopE, when fused to the reporter protein neomycin phosphotransferase (Npt), are sufficient for the secretion of YopE1-7-Npt when type III secretion is induced in vitro. Systematic mutagenesis of yopE codons 1 to 7 reveals that, like yopQ, codons 2, 3, 5, and 7 are sensitive to mutagenesis, thereby defining the first empirical similarity between the secretion signals of two type III secreted substrates. Like that of yopQ, the secretion signal of yopE exhibits a bipartite nature. This is manifested by the ability of codons 8 to 15 to suppress point mutations in the minimal secretion signal that change the amino acid specificities of particular codons or that induce alterations in the reading frame. Further, we have identified a single nucleotide position in codon 3 that, when mutated, conserves the predicted amino acid sequence of the YopE1-7-Npt but abrogates secretion of the reporter protein. When introduced into the context of the full-length yopE gene, the single-nucleotide mutation reduces the type III injection of YopE into HeLa cells, even though the predicted amino acid sequence remains the same. Thus, yopE mRNA appears to encode a property that mediates the type III injection of YopE.     

Conserved features of type III secretion

Type III secretion systems (TTSSs) are essential mediators of the interaction of many Gram-negative bacteria with human, animal or plant hosts. Extensive sequence and functional similarities exist between components of TTSS from bacteria as diverse as animal and plant pathogens. Recent crystal structure determinations of TTSS proteins reveal extensive structural homologies and novel structural motifs and provide a basis on which protein interaction networks start to be drawn within the TTSSs, that are consistent with and help rationalize genetic and biochemical data. Such studies, along with electron microscopy, also established common architectural design and function among the TTSSs of plant and mammalian pathogens, as well as between the TTSS injectisome and the flagellum. Recent comparative genomic analysis, bioinformatic genome mining and genome-wide functional screening have revealed an unsuspected number of newly discovered effectors, especially in plant pathogens and uncovered a wider distribution of TTSS in pathogenic, symbiotic and commensal bacteria. Functional proteomics and analysis further reveals common themes in TTSS effector functions across phylogenetic host and pathogen boundaries. Based on advances in TTSS biology, new diagnostics, crop protection and drug development applications, as well as new cell biology research tools are beginning to emerge.