Proteins Yops Research Articles

Targeting Rac1 by the Yersinia Effector Protein YopE Inhibits Caspase-1-mediated Maturation and Release of Interleukin-1β

Yersinia bacteria can take control of the host cell by injecting so-called Yop effector proteins into the cytosol of the cells to which they adhere. Using Yersinia enterocolitica strains that are deficient for one or more Yops, we could show that YopE and, to a lesser extent, YopT interfere with the caspase-1-mediated maturation of prointerleukin-1beta in macrophages. In addition, overexpression of YopE and YopT was shown to prevent the autoproteolytic activation of caspase-1 in a way that is dependent on their inhibitory effect on Rho GTPases. Expression of constitutive-active or dominant-negative Rho GTPase mutants or treatment with Rho GTPase inhibitors confirmed the role of Rho GTPases and, in particular, Rac1 in the autoactivation of caspase-1. Rac1-induced caspase-1 activation was mediated by its effect on LIM kinase-1, which is targeting the actin cytoskeleton. Rac-1 and LIM kinase-1 dominant-negative mutants were shown to inhibit caspase-1 activation induced by overexpression of Asc, which is a caspase-1-activating adaptor protein. Moreover, Rac1 as well as YopE and YopT significantly modulated caspase-1 oligomerization. These results highlight a previously unknown function of Rho GTPases in the activation of caspase-1 and give new insight on the role of YopE in immune-escape mechanisms of Yersinia.

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 osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain’s Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.

MP037 Virulence of Yersinia enterocolitica Yop mutants in the oral mouse infection model

Polymorphonuclear leukocytes, in addition to their direct bactericidal activities, produce cytokines involved in the activation and regulation of the innate and adaptive immune response to infection. In this study we evaluated the cytokine response of human PMNs following incubation with the pathogenic Yersinia species. Yersinia pestis strains with the pCD1 virulence plasmid, which encodes cytotoxic Yop proteins that are translocated into host cells, stimulated little or no cytokine production compared to pCD1-negative strains. In particular, PMNs incubated with pCD1-negative Y. pestis secreted 1000-fold higher levels of interleukin-8 (IL-8 or CXCL8), a proinflammatory chemokine important for PMN recruitment and activation. Deletion of yopE, -H, -T, -M or ypkA had no effect on pCD1-dependent inhibition, whereas deletion of yopJ resulted in significantly increased IL-8 production. Like Y. pestis, the enteropathogenic Yersinia species inhibited IL-8 secretion by PMNs, and strains lacking the virulence plasmid induced high levels of IL-8. Our results show that virulence plasmid-encoded effector Yops, particularly YopJ, prevent IL-8 secretion by human PMNs. Suppression of the chemotactic IL-8 response by Y. pestis may contribute to the delayed PMN recruitment to the infected lymph node that typifies bubonic plague.

Requirement of the Yersinia pseudotuberculosis effectors YopH and YopE in colonization and persistence in intestinal and lymph tissues.

The gram-negative enteric pathogen Yersinia pseudotuberculosis employs a type III secretion system and effector Yop proteins that are required for virulence. Mutations in the type III secretion-translocation apparatus have been shown to cause defects in colonization of the murine cecum, suggesting roles for one or more effector Yops in the intestinal tract. To investigate this possibility, isogenic yop mutant strains were tested for their ability to colonize and persist in intestinal and associated lymph tissues of the mouse following orogastric inoculation. In single-strain infections, a yopHEMOJ mutant strain was unable to colonize, replicate, or persist in intestinal and lymph tissues. A yopH mutant strain specifically fails to colonize the mesenteric lymph nodes, but yopE and yopO mutant strains showed only minor defects in persistence in intestinal and lymph tissues. While no single Yop was found to be essential for colonization or persistence in intestinal tissues in single-strain infections, the absence of both YopH and YopE together almost eliminated colonization of all tissues, indicating either that these two Yops have some redundant functions or that Y. pseudotuberculosis employs multiple strategies for colonization. In competition infections with wild-type Y. pseudotuberculosis, the presence of wild-type bacteria severely hindered the ability of the yopH, yopE, and yopO mutants to persist in many tissues, suggesting that the wild-type bacteria either fills colonization niches or elicits host responses that the yop mutants are unable to withstand.

TyeA of Yersinia pseudotuberculosis is involved in regulation of Yop expression and is required for polarized translocation of Yop effectors.

Type III secretion-dependent translocation of Yop (Yersinia outer proteins) effector proteins into host cells is an essential virulence mechanism common to the pathogenic Yersinia species. One unique feature of this mechanism is the polarized secretion of Yops, i.e. Yops are only secreted at the site of contact with the host cell and not to the surrounding medium. In vitro, secretion occurs in Ca2+-depleted media, a condition believed to somehow mimic cell contact. Three proteins, YopN, LcrG and TyeA have been suggested to control secretion and mutating any of these genes results in constitutive secretion. In addition, in Y. enterocolitica TyeA has been implied to be specifically required for delivery of a subset of Yop effectors into infected cells. In this work we have investigated the role of TyeA in secretion and translocation of Yop effectors by Y. pseudotuberculosis. An in frame deletion mutant of tyeA was found to be temperature-sensitive for growth and this phenotype correlated to a lowered expression of the negative regulatory element LcrQ. In medium containing Ca2+, Yop expression was somewhat elevated compared to the wild-type strain and low levels of Yop secretion was also seen. Somewhat surprisingly, expression and secretion of Yops was lower than for the wild-type strain when the tyeA mutant was grown in Ca2+-depleted medium. Translocation of YopE, YopH, YopJ and YopM into infected HeLa cells was significantly lower in comparison with the isogenic wild-type strain and Yop proteins could also be recovered in the tissue culture medium. This indicated that the tyeA mutant had lost the ability to translocate Yop proteins by a polarized mechanism. In order to exclude that the defect in translocation seen in the tyeA mutant was a result of lowered expression/secretion of Yops, a double lcrQ/tyeA mutant was constructed. This strain was de-repressed for Yop expression and secretion but was still impaired for translocation of both YopE and YopM. In addition, the low level of YopE translocation in the tyeA mutant was independent of the YopE chaperone YerA/SycE. TyeA was found to localize to the cytoplasm of the bacterium and we were unable to find any evidence that TyeA was secreted or surface located. From our studies in Y. pseudotuberculosis we conclude that TyeA is involved in regulation of Yop expression and required for polarized delivery of Yop effectors in general and is not as suggested in Y. enterocolitica directly required for translocation of a subset of Yop effectors.

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.

Yop fusions to tightly folded protein domains and their effects on Yersinia enterocolitica type III secretion.

Yersinia enterocolitica organisms secrete Yop proteins via the type III pathway. Translational fusion of yop genes to ubiquitin or dihydrofolate reductase results in hybrid proteins that cannot be secreted. The folding of hybrids prevents their own transport, but it does not hinder the type III secretion of other Yops.

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.

YopD and LcrH regulate expression of Yersinia enterocolitica YopQ by a posttranscriptional mechanism and bind to yopQ RNA.

Pathogenic yersiniae secrete 14 Yop proteins via the type III pathway. Synthesis of YopQ occurs when the type III machinery is activated by a low-calcium signal, but not when the calcium concentration is above 100 microM. To characterize the mechanism that regulates the expression of yopQ, mutants that permit synthesis of YopQ in the presence of calcium were isolated. Yersiniae bearing deletion mutations in yopN, tyeA, sycN, or yscB synthesized and secreted YopQ in both the presence and the absence of calcium. In contrast, yersiniae with a deletion in yopD or lcrH synthesized YopQ in the presence of calcium but did not secrete the polypeptide. These variants displayed no defect in YopQ secretion under low-calcium conditions, revealing that yopD and lcrH are required for the regulation of yopQ expression. Experiments with transcriptional and translational fusions to the npt reporter gene suggest that yopD and lcrH regulate yopQ expression at a posttranscriptional step. YopD and LcrH form a complex in the bacterial cytosol and bind yopQ mRNA. Models that can account for posttranscriptional regulatory mechanisms of yop expression are discussed.

Role of fraction 1 antigen of Yersinia pestis in inhibition of phagocytosis.

Yersinia pestis, the causative agent of plague, expresses a capsule-like antigen, fraction 1 (F1), at 37 degrees C. F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is unique to Y. pestis. F1 is a surface polymer composed of a protein subunit, Caf1, with a molecular mass of 15.5 kDa. The secretion and assembly of F1 require the caf1M and caf1A genes, which are homologous to the chaperone and usher protein families required for biogenesis of pili. F1 has been implicated to be involved in the ability of Y. pestis to prevent uptake by macrophages. In this study we addressed the role of F1 antigen in inhibition of phagocytosis by the macrophage-like cell line J774. The Y. pestis strain EV76 was found to be highly resistant to uptake by J774 cells. An in-frame deletion of the caf1M gene of the Y. pestis strain EV76 was constructed and found to be unable to express F1 polymer on the bacterial surface. This strain had a somewhat lowered ability to prevent uptake by J774 cells. Strain EV76C, which is cured for the virulence plasmid common to the pathogenic Yersinia species, was, as expected, much reduced in its ability to resist uptake. A strain lacking both the virulence plasmid and caf1M was even further hampered in the ability to prevent uptake and, in this case, essentially all bacteria (95%) were phagocytosed. Thus, F1 and the virulence plasmid-encoded type III system act in concert to make Y. pestis highly resistant to uptake by phagocytes. In contrast to the type III effector proteins YopE and YopH, F1 did not have any influence on the general phagocytic ability of J774 cells. Expression of F1 also reduced the number of bacteria that interacted with the macrophages. This suggests that F1 prevents uptake by interfering at the level of receptor interaction in the phagocytosis process.

Molecular characterization of type III secretion signals via analysis of synthetic N-terminal amino acid sequences.

Yersinia species utilize a type III secretion system to inject toxins, called Yops (Yersinia outer proteins), into eukaryotic cells. The N-termini of the Yops serve as type III secretion signals, but they do not share a consensus sequence. To simplify the analysis of type III secretion signals, we replaced amino acids 2-8 of the secreted protein YopE with all permutations (27 or 128) of synthetic serine/isoleucine sequences. The results demonstrate that amphipathic N-terminal sequences, containing four or five serine residues, have a much greater probability than hydrophobic or hydrophilic sequences to target YopE for secretion. Multiple linear regression analysis of the synthetic sequences was used to obtain a model for N-terminal secretion signals. The model accurately classifies the N-terminal sequences of native type III substrates as efficient secretion signals.

The level of Yop proteins secreted by Yersinia enterocolitica is changed in maltose mutants.

Enteropathogenic Yersinia enterocolitica strains express a set of plasmid-encoded proteins called Yops, involved in pathogenicity. We studied the influence of the maltose system on the production of Yop proteins and found that the level of Yop proteins of Y. enterocolitica O:9 was reduced in the presence of maltose. Transposon insertion mutants impaired with the maltose transport activity showed a decreased level in the production of Yop proteins. The transcription of the yopH gene for YopH phosphatase in these maltose mutants was unchanged and revealed a maltose mutation impaired in the secretion of Yop proteins instead of their expression.

The level of Yop proteins secreted by Yersinia enterocolitica is changed in maltose mutants

Enteropathogenic Yersinia enterocolitica strains express a set of plasmid-encoded proteins called Yops, involved in pathogenicity. We studied the influence of the maltose system on the production of Yop proteins and found that the level of Yop proteins of Y. enterocolitica O:9 was reduced in the presence of maltose. Transposon insertion mutants impaired with the maltose transport activity showed a decreased level in the production of Yop proteins. The transcription of the yopH gene for YopH phosphatase in these maltose mutants was unchanged and revealed a maltose mutation impaired in the secretion of Yop proteins instead of their expression.

Regulated secretion of YopN by the type III machinery of Yersinia enterocolitica.

During infection, Yersinia enterocolitica exports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 microM (low-calcium response). Yersiniae lacking yopN (lcrE), yscB, sycN, or tyeA do not inactivate the type III pathway even when the concentration of calcium is above 100 microM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of yopN gene sequences to the 5' end of the npt reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of yopN. Expression of plasmid-borne yopN, but not of yopN(1-294)-npt, complements the calcium-blind phenotype of yopN mutants. Surprisingly, yopN mutants respond to environmental changes in calcium concentration and secrete YopN(1-294)-Npt in the absence but not in the presence of calcium. tyeA is required for the low-calcium regulation of YopN(1-294)-Npt secretion, whereas sycN and yscB mutants fail to secrete YopN(1-294)-Npt in the presence of calcium. Experiments with yopN-npt fusions identified two other signals that regulate the secretion of YopN. yopN codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of yscB and sycN. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional tyeA. These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.

Roles of LcrG and LcrV during type III targeting of effector Yops by Yersinia enterocolitica.

Yersinia enterocolitica target effector Yop proteins into the cytosol of eukaryotic cells by a mechanism requiring the type III machinery. LcrG and LcrV have been suggested to fulfill essential functions during the type III targeting of effector Yops. It is reported here that knockout mutations of lcrG caused mutant yersiniae to prematurely secrete Yops into the extracellular medium without abolishing the type III targeting mechanism (Los phenotype [loss of type III targeting specificity]). Knockout mutations in lcrV reduced type III targeting of mutant yersiniae but did not promote secretion into the extracellular medium (Not [no type III targeting]). However, knockout mutations in both genes caused DeltalcrGV yersiniae to display a Los phenotype similar to that of strains carrying knockout mutations in lcrG alone. LcrG binding to LcrV resulted in the formation of soluble LcrGV complexes in the bacterial cytoplasm. Membrane-associated, bacterial-surface-displayed or -secreted LcrG could not be detected. Most of LcrV was located in the bacterial cytoplasm; however, small amounts were secreted into the extracellular medium. These data support a model whereby LcrG may act as a negative regulator of type III targeting in the bacterial cytoplasm, an activity that is modulated by LcrG binding to LcrV. No support could be gathered for the hypothesis whereby LcrG and LcrV may act as a bacterial surface receptor for host cells, allowing effector Yop translocation across the eukaryotic plasma membrane.

Identification of attenuated Yersinia pseudotuberculosis strains and characterization of an orogastric infection in BALB/c mice on day 5 postinfection by signature-tagged mutagenesis.

Yersinia pseudotuberculosis localizes to the distal ileum, cecum, and proximal colon of the gastrointestinal tract after oral infection. Using signature-tagged mutagenesis, we isolated 13 Y. pseudotuberculosis mutants that failed to survive in the cecum of mice after orogastric inoculation. Twelve of these mutants were also attenuated for replication in the spleen after intraperitoneal infection, whereas one strain, mutated the gene encoding invasin, replicated as well as wild-type bacteria in the spleen. Several mutations were in operons encoding components of the type III secretion system, including components involved in translocating Yop proteins into host cells. This indicates that one or more Yops may be necessary for survival in the gastrointestinal tract. Three mutants were defective in O-antigen biosynthesis; these mutants were also unable to invade epithelial cells as efficiently as wild-type Y. pseudotuberculosis. Several other mutations were in genes that had not previously been associated with growth in a host, including cls, ksgA, and sufl. In addition, using Y. pseudotuberculosis strains marked with signature tags, we counted the number of different bacterial clones that were present in the cecum, mesenteric lymph nodes, and spleen 5 days postinfection. We find barriers in the host animal that limit the number of bacteria that succeed in reaching and/or replicating in the mesenteric lymph nodes and spleen after breaching the gut mucosa.

LcrV, a Substrate for Yersinia enterocolitica Type III Secretion, Is Required for Toxin Targeting into the Cytosol of HeLa Cells

Pathogenic Yersinia species employ type III machines to transport virulence factors across the bacterial envelope. Some substrates for the type III machinery are secreted into the extracellular medium, whereas others are targeted into the cytosol of host cells. We found that during infection of tissue culture cells, yersiniae secrete small amounts of LcrV into the extracellular medium. Knockout mutations of lcrV abolish Yersinia targeting and reduce expression of the lcrGVHyopBD operon. In contrast, a block in LcrV secretion does not affect targeting, but results in premature expression and secretion of Yop proteins into the extracellular medium. LcrV-mediated activation of the type III pathway is thought to occur by sequestration of the regulatory factor LcrG, presumably via the formation of LcrV.LcrG complexes. These results suggest that intrabacterial LcrV regulates the expression and targeting of Yop proteins during Yersinia infection, whereas secreted LcrV is required to ensure specificity of Yop injection into eukaryotic cells.

Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: Development of a delivery system for heterologous proteins

Y. enterocolitica translocates virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Here we investigated whether Y. enterocolitica could translocate Yops into a range of eukaryotic cells including neurons and insect cells. Y. enterocolitica translocated the hybrid reporter protein YopE-Cya into each of the eukaryotic cell types tested. In addition, Y. enterocolitica was cytotoxic for each of the adherent cell types. Thus we detected no limit to the range of eukaryotic cells into which Y. enterocolitica can translocate Yops. The Yop effectors YopE, YopH and YopT were each cytotoxic for the adherent cell types tested, showing that not only is Y. enterocolitica not selective in its translocation of particular Yop effectors into each cell type, but also that the action of these Yop effectors is not cell type specific. Invasin and/or YadA, two powerful adhesins were required for translocation of Yop into non-phagocytic cells but not for translocation into macrophages. To use the Yersinia translocation system for broad applications, a Y. enterocolitica translocation strain and vector for the delivery of heterologous proteins into eukaryotic cells was constructed. This strain + vector combination lacks the translocated Yop effectors and allows delivery into eukaryotic cells of heterologous proteins fused to the minimal N-terminal secretion/translocation signal of YopE. Using this strategy translocation of a YopE-Diphtheria toxin subunit A hybrid protein into several cell types has been shown.

YscP, a Yersinia protein required for Yop secretion that is surface exposed, and released in low Ca2+.

The Yersinia Ysc apparatus is made of more than 20 proteins, 11 of which have homologues in many type III systems. Here, we characterize YscP from Yersinia enterocolitica. This 515-residue protein has a high proline content, a large tandem repetition and a slow migration in SDS-PAGE. Unlike the products of neighbouring genes, it has a counterpart only in Pseudomonas aeruginosa and it varies even between Yersinia Ysc machineries. An yscPDelta97-465 mutant was unable to secrete any Yop, even under conditions overcoming feedback inhibition of Yop synthesis. Interestingly, a cloned yscPDelta57-324 from Yersinia pestis introduced in the yscPDelta97-465 mutant can sustain a significant Yop secretion and thus partially complemented the mutation. This explains the leaky phenotype observed with the yscP mutant of Y. pestis. In accordance with this secretion deficiency, YscP is required for the delivery of Yop effectors into macrophages. Mechanical shearing, immunolabelling and electron microscopy experiments showed that YscP is exposed at the bacterial surface when bacteria are incubated at 37 degrees C in the presence of Ca2+ and thus do not secrete Yops. At 37 degrees C, when Ca2+ ions are chelated, YscP is released like a Yop protein. We conclude that YscP is a part of the Ysc injectisome which is localized at the bacterial surface and is destabilized by Ca2+ chelation.