Yop Secretion Research Articles

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.

Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion

Secretion of Yop effector proteins by the Yersinia pestis plasmid pCD1-encoded type III secretion system (T3SS) is regulated in response to specific environmental signals. Yop secretion is activated by contact with a eukaryotic cell or by growth at 37 degrees C in the absence of calcium. The secreted YopN protein, the SycN/YscB chaperone and TyeA form a cytosolic YopN/SycN/YscB/TyeA complex that is required to prevent Yop secretion in the presence of calcium and prior to contact with a eukaryotic cell. The mechanism by which these proteins prevent secretion and the subcellular location where the block in secretion occurs are not known. To further investigate both the mechanism and location of the YopN-dependent block, we isolated and characterized several YopN mutants that constitutively block Yop secretion. All the identified amino-acid substitutions that resulted in a constitutive block in Yop secretion mapped to a central domain of YopN that is not directly involved in the interaction with the SycN/YscB chaperone or TyeA. The YopN mutants required an intact TyeA-binding domain and TyeA to block secretion, but did not require an N-terminal secretion signal, an intact chaperone-binding domain or the SycN/YscB chaperone. These results suggest that a C-terminal domain of YopN complexed with TyeA blocks Yop secretion from a cytosolic, not an extracellular, location. A hypothetical model for how the YopN/SycN/YscB/TyeA complex regulates Yop secretion is presented.

Mechanisms of Protein Export across the Bacterial Outer Membrane

Gram-negative bacteria secrete a wide range of proteins whose functions include biogenesis of organelles, such as pili and flagella; nutrient acquisition; virulence; and efflux of drugs and other toxins. Export of these proteins to the bacterial surface involves transport across the inner membrane (IM), periplasm, and outer membrane (OM) of the cell envelope. Several pathways have evolved to fulfill the task of secretion (Table ​(Table1).1). These pathways can be divided into two main groups: (i) Sec dependent and (ii) Sec independent (Fig. ​(Fig.1)1) (64). FIG. 1. Schematic presentation of Sec-dependent and Sec-independent secretion pathways in gram-negative bacteria. The type I pathway is exemplified by the hemolysin A (HlyA) secretion in pathogenic E. coli, the type II pathway by YopE secretion in Yersinia pestis ... TABLE 1. Representative members of the eight secretion systems Proteins secreted via the Sec-dependent pathways utilize a common machinery, the Sec translocase, for transport across the IM and are mainly differentiated based on their mechanisms of secretion across the OM (64). Sec-dependent pathways include the type II secretion (T2S); the autotransporter (AT), or type V secretion; the two-partner secretion (TPS); and the chaperone/usher (CU) secretion systems (34, 46, 58, 70, 73). The type IV secretion (T4S), or adapted-conjugation, pathway can be Sec dependent but is mostly considered Sec independent (21). Sec-independent pathways tend to allow direct export from the cytoplasm to the extracellular environment in one step and do not involve periplasmic intermediates. These pathways also include the type I secretion (T1S), or ABC (ATP-binding cassette), exporters (5) and the type III secretion (T3S) systems (31). An alternative Sec-independent pathway known as twin-arginine translocation (Tat) is employed for transport of already-folded proteins across the IM. Generally, Tat substrates are targeted to the periplasm of the cell (4) but can also be transported across the OM via the type II pathway, such as the phospholipases of Pseudomonas aeruginosa (77). The Sec and Tat machineries for transport across the IM will not be discussed further. In this review, we summarize the current knowledge of OM protein secretion in terms of the structure of the exporters and the mechanism of secretion and examine similarities and differences among the different export pathways (Table ​(Table2).2). Since many virulence factors are secreted proteins, a more complete understanding of the mechanisms underlying bacterial protein secretion is crucial to the development of new vaccines and treatments for a wide range of human pathogens. TABLE 2. Structural features of the OM components of protein secretion systems

Small-molecule inhibitors specifically targeting type III secretion.

The type III secretion (TTS) system is used by several animal and plant pathogens to deliver effector proteins into the cytosol of the eukaryotic target cell as a strategy to evade the defense reactions elicited by the infected organism. The fact that these systems are highly homologous implies that novel antibacterial agents that chemically attenuate the pathogens via a specific interaction with the type III secretion mechanism can be identified. A number of small organic molecules having this potential have recently been identified (A. M. Kauppi, R. Nordfelth, H. Uvell, H. Wolf-Watz, and M. Elofsson, Chem. Biol. 10:241-249, 2003). Using different reporter gene constructs, we showed that compounds that belong to a class of acylated hydrazones of different salicylaldehydes target the TTS system of Yersinia pseudotuberculosis. One of these compounds, compound 1, was studied in detail and was found to specifically block Yop effector secretion under in vitro conditions by targeting the TTS system. In this respect the drug mimics the well-known effect of calcium on Yop secretion. In addition, compound 1 inhibits Yop effector translocation after infection of HeLa cells without affecting the eukaryotic cells or the bacteria. A HeLa cell model that mimics in vivo conditions showed that compound 1 chemically attenuates the pathogen to the advantage of the eukaryotic cell. Thus, our results show proof of concept, i.e., that small compounds targeting the TTS system can be identified, and they point to the possible use of TTS inhibitors as a novel class of antibacterial agents.

Yersinia pestis Yop secretion protein F: Purification, characterization, and protective efficacy against bubonic plague

Yersinia pestis is a gram-negative human pathogen that uses a type III secretion system to deliver virulence factors into human hosts. The delivery is contact-dependent and it has been proposed that polymerization of Yop secretion protein F (YscF) is used to puncture mammalian cell membranes to facilitate delivery of Yersinia outer protein effectors into host cells. To evaluate the potential immunogenicity and protective efficacy of YscF against Y. pestis, we used a purified recombinant YscF protein as a potential vaccine candidate in a mouse subcutaneous infection model. YscF was expressed and purified from Escherichia coli by immobilized metal-ion affinity chromatography and protein identity was confirmed by ion trap mass spectrometry. The recombinant protein was highly α-helical and formed relatively stable aggregates under physiological conditions. The properties were consistent with behavior expected for the native YscF, suggesting that the antigen was properly folded. Ten mice were inoculated subcutaneously, administered booster injections after one month, and challenged with 130 LD 50 of wild type Y. pestis CO92. Six animals in the vaccinated group but none in the control group survived the challenge. The vaccinated animals produced high levels of specific antibodies against YscF as determined by Western blot. The data were statistically significant ( P = 0.053 by two-tailed Fisher’s test), suggesting that the YscF protein can provide a protective immune response against lethal plague challenge during subcutaneous plague infection.

Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica

The length of the needle ending the Yersinia Ysc injectisome is determined by YscP, a protein acting as a molecular ruler. In addition, YscP is required for Yop secretion. In the present paper, by a systematic deletion analysis, we localized accurately the region required for Yop secretion between residues 405 and 500. As this C-terminal region of YscP has also been shown to control needle length it probably represents the substrate specificity switch of the machinery. By a bioinformatics analysis, we show that this region has a globular structure, an original alpha/beta fold, a P-x-L-G signature and presumably no catalytic activity. In spite of very limited sequence similarities, this structure is conserved among the proteins that are presumed to control the needle length in many different injectisomes and also among members of the FliK family, which control the flagellar hook length. This region thus represents a new protein domain that we called T3S4 for Type III secretion substrate specificity switch. The T3S4 domain of YscP can be replaced by the T3S4 domain of AscP (Aeromonas salmonicida) or PscP (Pseudomonas aeruginosa) but not by the one from FliK, indicating that in spite of a common global structure, these domains need to fit their partner proteins in the secretion apparatus.

Novel Protein-Protein Interactions of the Yersinia pestis Type III Secretion System Elucidated with a Matrix Analysis by Surface Plasmon Resonance and Mass Spectrometry

Binary complexes formed by components of the Yersinia pestis type III secretion system were investigated by surface plasmon resonance (SPR) and matrix-assisted laser desorption time-of-flight mass spectrometry. Pairwise interactions between 15 recombinant Yersinia outer proteins (Yops), regulators, and chaperones were first identified by SPR. Mass spectrometry confirmed over 80% of the protein-protein interactions suggested by SPR, and new binding partners were further characterized. The Yop secretion protein (Ysc) M2 of Yersinia enterocolitica and LcrQ of Y. pestis, formerly described as ligands only for the specific Yop chaperone (Syc) H, formed stable complexes with SycE. Additional previously unreported complexes of YscE with the translocation regulator protein TyeA and the thermal regulator protein YmoA and multiple potential protein contacts by YscE, YopK, YopH, and LcrH were also identified. Because only stably folded proteins were examined, the interactions we identified are likely to occur either before or after transfer through the injectosome to mammalian host cells and may have relevance to understanding disease processes initiated by the plague bacterium.

Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB.

The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

Defensive function of phagocytes in pseudotuberculosis.

The results of our investigations showed that plasmid-harbouring Y. pseudotuberculosis actively suppress the phagocytic activity of neutrophils and macrophages. It is likely that deactivation of phagocytes is caused by secretion of Yops. Moreover, bacteriological studies showed that only a few of the ingested bacteria were destroyed and the remaining killed the macrophages.

Characterisation of the type III secretion protein YscU in Yersinia pseudotuberculosis. YscU cleavage--dispensable for TTSS but essential for survival.

The YscU/FlhB protein family is very highly conserved, amino acid sequence homologies between the cytoplasmic domains being particularly high. Here we investigated a feature common to YscU and FlhB, i. e. the cleavage of the proteins within the C-terminal cytoplasmic tail. We could conclude that the cleavage is not connected to TTSS regulation. Nor is it required for YscU function in Yersinia type III secretion, since the uncleavable YscU variants are able to restore Yop secretion to a yscU null mutant strain. However, if YscU is not cleaved within the cytoplasmic tail, it has a toxic effect on the bacteria. The fact that Yersinia strains expressing the uncleavable YscU point mutant proteins are seriously inhibited for growth shows that the cleavage is required for survival of the bacterium.

Yersinia enterocolitica type III secretion: Evidence for the ability to transport proteins that are folded prior to secretion

BackgroundPathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2–3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs).ResultsIn this study we have fused YopE secretion signals of various length to the mouse dihydrofolate reductase (DHFR) in order to analyse the DHFR folding state prior to secretion. We could demonstrate that secretion-deficient as well as secretion-competent YopE-DHFR fusions complexed to SycE can be efficiently purified from Yersinia cytosol by affinity chromatography using methotrexate-agarose. This implies the folding of the DHFR fusion moiety despite SycE binding and contradicts the previously presented model of folding inhibition by chaperone binding. Secretion-deficient YopE-DHFR fusions caused severe jamming of the TTSS. This observation contradicts the co-translational secretion model.ConclusionsWe present evidence that the Yersinia TTSS is familiar with the processing of transport substrates which are folded prior to secretion. We therefore predict that an unfoldase is involved in type III secretion.

YscP and YscU regulate substrate specificity of the Yersinia type III secretion system.

Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.

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.

Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies.

Yersinia pestis, the causative agent of plague, exports a set of virulence proteins called Yops upon contact with eukaryotic cells. A subset of these Yops is translocated directly into the cytosol of host cells. In this study, a novel protein tag-based reporter system is used to measure the translocation of Yops into cultured eukaryotic cells. The reporter system uses a small bipartite phosphorylatable peptide tag, termed the Elk tag. Translocation of an Elk-tagged protein into eukaryotic cells results in host cell protein kinase-dependent phosphorylation of the tag at a specific serine residue, which can subsequently be detected with phosphospecific antibodies. The YopN, TyeA, SycN, YscB and LcrG proteins function to prevent Yop secretion before host cell contact. The role of these proteins was investigated in the translocation of Elk-tagged YopE (YopE129-Elk) and YopN (YopN293-Elk) into HeLa cells. Y. pestis yopN, tyeA, sycN and yscB deletion mutants showed reduced levels of YopE129-Elk phosphorylation compared with the parent strain, indicating that these mutants translocate reduced amounts of YopE. We also demonstrate that YopN293-Elk is translocated into HeLa cells and that this process is more efficient in a Yersinia yop polymutant strain lacking the six translocated effector Yops. Y. pestis sycN and yscB mutants translocated reduced amounts of YopN293-Elk; however, tyeA and lcrG mutants translocated higher amounts of YopN293-Elk compared with the parent strain. These data suggest that TyeA and LcrG function to suppress the secretion of YopN before host cell contact, whereas SycN and YscB facilitate YopN secretion and subsequent translocation.

Analysis of chaperone-dependent Yop secretion/translocation and effector function using a mini-virulence plasmid of Yersinia enterocolitica

AbstractWe have constructed a mini-pYV plasmid (pTTSS) harboring the Yersinia type three secretion system (TTSS) and the adhesin yadA on a low-copy vector. Using this system we could demonstrate for the first time that YopO, YopP, YopM, and YopQ do not require any of the known or orphan chaperones for efficient secretion/translocation. Y. enterocolitica harboring pTTSS, (WA-C(pTTSS)) was able to secrete and translocate single Yop effector proteins in trans. WA-C(pTTSS) proved to be stable and secretion of Yops was Ca2+ and temperature dependent as is the case for the parental strain. This shows that all genes necessary for translocation and expression of the Ca2+-dependent phenotype are contained within the cloned region. In contrast to previously published multiple yop mutants which were constructed by sequential deletion of yops, our system which harbors only the TTSS region without yops, chaperones, and unknown ORFs can be sequentially complemented with yops and sycs of choice. WA-C(pTTSS) was able to translocate YopE, YopM and YopT into HeLa cells as demonstrated by Western blotting. Translocation of YopE and YopT was strictly dependent on the presence of their respective chaperones, whereas YopM did not require a chaperone for translocation. WA-C(pTTSS) harboring yopT and sycT was shown to translocate active YopT by demonstrating modification of the small GTP-binding protein RhoA. This shows for the first time that RhoA modification is strictly dependent on YopT and does not require additional effector Yops. WA-C(pTTSS) harboring YopP was shown to induce apoptosis. This system is ideal to study chaperone-dependent Yop secretion/translocation without the background of other effector Yops (YopE, YopM, YopO, YopP, YopT, YopH), chaperones (SycE, SycH, SycT) and unknown ORFs. In addition this system can secrete heterologous proteins fused to the N-terminal secretion/translocation domain of YopE.

Evidence for targeting of Yop effectors by the chromosomally encoded Ysa type III secretion system of Yersinia enterocolitica.

Yersinia enterocolitica O:8 has two contact-dependent type III secretion systems (TTSSs). The Ysa TTSS is encoded by a set of genes located on the chromosome and exports Ysp proteins. The Ysc TTSS and the Yop effector proteins it exports are encoded by genes located on plasmid pYVe8081. In this study, secretion of YspG, YspH, and YspJ by the Ysa TTSS was shown to require pYVe8081. Furthermore, mutations that blocked the function of the Ysc TTSS did not affect YspG, YspH, and YspJ production. This indicated that YspG, YspH, and YspJ are encoded by genes located on pYVe8081 and that they may correspond to Yops. A comparison of Ysps with Yop effectors secreted by Y. enterocolitica indicated that YspG, YspH, and YspJ have apparent molecular masses similar to those of YopN, YopP, and YopE, respectively. Immunoblot analysis demonstrated that antibodies directed against YopN, YopP, and YopE recognized YspG, YspH, and YspJ. Furthermore, mutations in yopN, yopP, and yopE specifically blocked YopN, YopP, and YopE secretion by the Ysc TTSS and YspG, YspH, and YspJ secretion by the Ysa TTSS. These results indicate YspG, YspH, and YspJ are actually YopN, YopP, and YopE. Additional analysis demonstrated that YopP and YspH secretion was restored to yopP mutants by complementation in trans with a wild-type copy of the yopP gene. Examination of Y. enterocolitica-infected J774A.1 macrophages revealed that both the Ysc and Ysa TTSSs contribute to YopP-dependent suppression of tumor necrosis factor alpha production. This indicates that both the Ysa and Ysc TTSSs are capable of targeting YopP and that they influence Y. enterocolitica interactions with macrophages. Taken together, these results suggest that the Ysa and Ysc TTSSs contribute to Y. enterocolitica virulence by exporting both unique and common subsets of 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.

Up-regulation of the Yersinia enterocolitica yop regulon by deletion of the flagellum master operon flhDC.

The Yop virulon enables extracellularly located Yersinia, in close contact with a eukaryotic target cell, to inject bacterial toxic proteins directly into the cytosol of this cell. Several Ysc proteins, forming the Yop secretion apparatus, display homology with proteins of the flagellar basal body. To determine whether this relationship could extend to the regulatory pathways, we analyzed the influence of flhDC, the master regulatory operon of the flagellum, on the yop regulon. In an flhDC mutant, the yop regulon was up-regulated. The transcription of virF and the steady-state level of the transcriptional activator VirF were enhanced. yop transcription was increased at 37 degrees C and could also be detected at a low temperature. Yop secretion was increased at 37 degrees C and occurred even at a low temperature. The Ysc secretion machinery was thus functional at room temperature in the absence of flagella, implying that in wild-type bacteria, FlhD and/or FlhC, or the product of a gene downstream of flhDC, represses the yop regulon. In agreement with this notion, increased expression of flhDC in wild-type bacteria resulted in the oversecretion of flagellins at room temperature and in decreased Yop secretion at 37 degrees C.

DNA adenine methylase overproduction in Yersinia pseudotuberculosis alters YopE expression and secretion and host immune responses to infection.

Yersinia pseudotuberculosis mutants that overproduce the DNA adenine methylase (Dam) are highly attenuated, confer fully protective immune responses, and secrete several Yersinia virulence proteins (Yersinia outer proteins [Yops]) under conditions that are nonpermissive for secretion in wild-type strains. We examined here the effects of Dam overproduction on Yersinia virulence determinant expression and secretion, as well as the host immune response to Yersinia antigens. Western blot analysis with convalescent antisera identified several low-calcium-responsive antigens whose synthesis was affected by Dam overproduction. One of these antigens was shown to be the type III secretion effector protein, YopE, a cytotoxin involved in antiphagocytosis. Dam overproduction disrupted both the thermal and calcium regulation of YopE synthesis and relaxed the thermal but not the calcium dependence of YopE secretion. Altered expression and/or secretion of Yersinia proteins in Dam-overproducing strains may contribute to the decreased virulence and heightened immunity observed in vaccinated hosts and may provide a means by which to deliver heterologous antigens and/or immune modulators of the inflammatory response.