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.     

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.     

Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation

All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94A resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.     

Recombinant Yersinia enterocolitica YscM1 and YscM2: homodimer formation and susceptibility to thrombin cleavage

Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) make use of a virulence plasmid-encoded type three secretion system (TTSS) to inject effector proteins into host cells. Y. enterocolitica YscM1 (LcrQ in Y. pestis and Y. pseudotuberculosis) and its homologue YscM2 are regulatory components of the TTSS that are also secreted by this transport apparatus. YscM1 and YscM2 share 57% identity and are believed to be functionally equivalent. We have recombinantly expressed and purified YscM1 and YscM2 in Escherichia coli. After expression as glutathione S-transferase (GST) fusions purification to near homogeneity was achieved by glutathione-Sepharose affinity chromatography followed by PreScission protease treatment to cleave off GST and gel filtration on a Superdex 75 column. Such recombinant YscM1 and YscM2 bound efficiently to the specific chaperone SycH, indicating proper folding of the purified proteins. Gel filtration analyses revealed that both YscM1 and YscM2 formed homodimers. The YscM1 and YscM2 homodimers could be dissociated at high ionic strength, indicating that salt bridges essentially contribute to the dimerization. We further demonstrated that YscM1 and YscM2 are susceptible to thrombin cleavage.     

Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation

All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94 Å resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.     

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.     

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.     

A program of Yersinia enterocolitica type III secretion reactions is activated by specific signals

Successful establishment of Yersinia infections requires the type III machinery, a protein transporter that injects virulence factors (Yops) into macrophages. It is reported here that the Yersinia type III pathway responds to environmental signals by transporting proteins to distinct locations. Yersinia enterocolitica cells sense an increase in extracellular amino acids (glutamate, glutamine, aspartate, and asparagine) that results in the activation of the type III pathway. Another signal, provided by serum proteins such as albumin, triggers the secretion of YopD into the extracellular medium. The third signal, a decrease in calcium concentration, appears to be provided by host cells and causes Y. enterocolitica to transport YopE and presumably other virulence factors across the eukaryotic plasma membrane. Mutations in several genes encoding regulatory molecules (lcrG, lcrH, tyeA, yopD, yopN, yscM1, and yscM2) bypass the signal requirement of the type III pathway. Together these results suggest that yersiniae may have evolved distinct secretion reactions in response to environmental signals.     

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.     

Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression.

The yopE gene of Yersinia pseudotuberculosis was recently sequenced, and YopE was identified as an indispensable virulence determinant when tested in a mouse model (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). In the study described here, the DNA sequences of the yopE genes of Yersinia pestis EV76 and Yersinia enterocolitica 8081 were determined and compared with that of the Y. pseudotuberculosis gene. Only two codons were found to differ, both leading to amino acid replacements, when the gene from Y. pestis was compared. These two replacements were also present in the gene from Y. enterocolitica; in addition, 18 other codons were found to differ. Thirteen of these substitutions led to amino acid replacements. Downstream of the yopE gene, the plasmid partition locus par was found to be conserved in all three species. In Y. enterocolitica 8081, the sequence homology was interrupted by a putative insertion sequence element inserted between the yopE gene and the par region at a position only 5 base pairs downstream of the yopE stop codon. Upstream of the yopE gene, 620 base pairs were conserved in the three species. This region contained a 130-amino-acid-long open reading frame reading in the opposite direction to the yopE gene and expressed a 14-kilodalton protein in minicells. An insertion mutation in this region constructed in Y. pseudotuberculosis expressed significantly lower amounts of YopE protein in vitro than did the corresponding wild type. The expression level could be restored by transcomplementation. This new locus was designated yerA, for yopE-regulating gene A. The yerA mutant was avirulent when mice were challenged by oral infection.     

Real-time characterization of virulence factor expression in Yersinia pestis using a GFP reporter system

A real-time reporter system was developed to monitor the thermal induction of virulence factors in Yersinia pestis, the etiological agent of plague. The reporter system consists of a plasmid in Y. pestis in which the expression of green fluorescent protein (GFP) is under the control of the promoters for six virulence factors, yopE, sycE, yopK, yopT, yscN, and lcrE yopN, which are all components of the Type III secretion virulence mechanism of Y. pestis. Induction of the expression of these genes in vivo was determined by the increase in fluorescence intensity of GFP in real time, in 96-well format. Different basal levels of expression at 26 degrees C were observed for the Y. pestis promoters. Expressed as percentages of the level measured for the lac promoter (positive control), the basal expression levels before temperature shift were: yopE (15%), sycE (15%), yopK (13%), yopT (4%), lcrE (3.3%), and yscN (0.8%). Following the shift in temperature from 26 to 37 degrees C, the rates of expression of these genes increased with the yopE reporter showing the strongest degree of induction. The rates of induction of the other virulence factors after the temperature, expressed as percentages of yopE induction, were: yopK (57%), sycE (9%), yscN (3%), lcrE (3%), and yopT (2%). The thermal induction of each of these promoter fusions was repressed by calcium, and the ratios of the initial rates of thermal induction without calcium supplementation compared to the rate with calcium supplementation were: yopE (11-fold), yscN (7-fold), yopK (6-fold), lcrE (3-fold), yopT (2-fold), and sycE (1-fold). This work demonstrates a novel approach to quantify gene induction and provides a method to rapidly determine the effects of external stimuli on expression of Y. pestis virulence factors in real time, in living cells, as a means to characterize virulence determinants.     

Substrate recognition of type III secretion machines--testing the RNA signal hypothesis

Secretion by the type III pathway of Gram-negative microbes transports polypeptides into the extracellular medium or into the cytoplasm of host cells during infection. In pathogenic Yersinia spp., type III machines recognize 14 different Yop protein substrates via discrete signals genetically encoded in 7-15 codons at the 5' portion of yop genes. Although the signals necessary and sufficient for substrate recognition of Yop proteins have been mapped, a clear mechanism on how proteins are recognized by the machinery and then initiated into the transport pathway has not yet emerged. As synonymous substitutions, mutations that alter mRNA sequence but not codon specificity, affect the function of some secretion signals, recent work with several different microbes tested the hypothesis of an RNA-encoded secretion signal for polypeptides that travel the type III pathway. This review summarizes experimental observations and mechanistic models for substrate recognition in this field.