Involvement of the virulence gene products of Yersinia enterocolitica in the immune response of infected mice

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P-) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD50 of P+ strain and those with 1/10 LD50 of P- strain produced a high level of gamma interferon (IFN-gamma) upon stimulation with heat-killed bacteria, and CD4+ T cells were exclusively responsible for IFN-gamma production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-gamma response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-gamma-producing CD8+ T cells as well as the increase in IFN-gamma-producing CD4+ T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.     

Maximizing Plasmid Stability and Production of Released Proteins in Yersinia enterocolitica

Virulent serotypes of Yersinia enterocolitica carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32°C is the lowest temperature at which plasmid-encoded proteins are expressed in quantity. The highest calcium concentration allowing full expression of these proteins was 445 to 545 μM at 32°C. Calcium concentrations of 745 μM and above at 37°C completely prevented the loss of pYV during multiple subcultures, while at 32°C, calcium concentrations of 245 μM and greater were sufficient to stabilize the plasmid. Growth of Y. enterocolitica at pH 5.5 was slower than at neutral pH values, but it also resulted in greatly increased stability of pYV. These studies showed that bacterial growth, retention of pYV, and expression of plasmid-encoded proteins may be maximized at 32°C with 445 μM calcium and that pYV stability is enhanced by growth at low pH. These observations suggest new approaches for isolation of plasmid-bearing virulent strains of Y. enterocolitica from samples contaminated with this organism and also may improve our understanding of pYV retention in vivo.     

YscN, the putative energizer of the Yersinia Yop secretion machinery.

Pathogenic yersiniae secrete a set of 11 antihost proteins called Yops. Yop secretion appears as the archetype of the type III secretion pathway. Several components of this machinery are encoded by the virA (lcrA) and virC (lcrC) loci of the 70-kb pYV plasmid. In this paper, we describe yscN, another gene involved in this pathway. It is the first gene of the virB locus. It encodes a 47.8-kDa protein similar to the catalytic subunits of F0F1 and related ATPases, as well as to products of other genes presumed to be involved in a type III secretion pathway. YscN contains the two consensus nucleotide-binding motifs (boxes A and B) described by Walker et al. (J. E. Walker, M. Saraste, M. J. Runswick, and N. J. Gay, EMBO J. 1:945-951, 1982). We engineered a pYV mutant encoding a modified YscN protein lacking box A. This mutant, impaired in Yop secretion, can be complemented in trans by a cloned yscN gene. We conclude that YscN is a component of the Yop secretion machinery using ATP. We hypothesize that it is either the energizer of this machinery or a part of it.     

Y. enterocolitica inhibits antigen degradation in dendritic cells

Yersinia enterocolitica (Ye) disrupts the ability of dendritic cells (DC) to prime CD4+ T cells suggesting that Ye may subvert uptake and/or processing of soluble antigens (Ag). To investigate this Ye-infected DC were loaded with fluorescently labelled ovalbumins as markers for Ag uptake and processing, and analysed by flow cytometry, fluorometry and microscopy. Wild type pYV+ as well as plasmidless pYV(-) bacteria inhibited Ag degradation in DC by 40% compared to non-infected cells. Microscopic analyses of pYV(-)-infected DC revealed that 40% of DC contained intracellular bacteria, and that DC without intracellular bacteria had degraded more Ag. When internalization of pYV(-) was blocked by cytochalasin D, Ag degradation was no longer inhibited indicating the competition between degradation of bacteria and ovalbumin. In contrast, cytochalasin D pre-treated DC infected with pYV+ inhibited Ag degradation by a mechanism dependent on the presence of virulence plasmid pYV encoding YopE, YopH, YopM, YopP, YopT and YopO. As no single Yop inhibited Ag degradation, interaction of multiple Yops might account for this effect, possibly by inhibiting Rho GTPases, because of a significant decrease of Ag degradation observed in DC incubated with toxin B of C. difficile. However, the contribution of other pYV-encoded factors cannot be excluded.     

Cloning of Yersinia pseudotuberculosis calcium-dependence plasmid pYV6953 BamHI fragments and their analysis in Escherichia coli mini-cells

Calcium dependence plasmid pYV6953 (70.4 kb) in Yersinia pseudotuberculosis cells codes for the major quantities synthesis of 150; 48.5; 19.4 Kd outer membrane proteins and the 51, 38, 27 Kd proteins secreted into the culturing medium. These outer membrane and secreted proteins are synthesized in considerable amounts in Yersinia pseudotuberculosis strains 6953 and 9547 at 37 degrees C and in the absence of calcium ions in the culturing medium. BamHI fragments of the plasmid pYV6953 as components of the recombinant plasmids code for the synthesis of 150; 66.6; 51; 48.5; 47; 38 and 21.5 Kd proteins in Escherichia coli mini cells. The synthesis of 150 and 48.5 Kd proteins is determined by the BamHI fragment 9 of the plasmid pYV6953 (3.3 kb). Addition of up to 8% of ethanol inhibiting the protein synthesis eliminates the 150 Kd protein but not the 48.5 Kd synthesis. The 48.5 Kd protein is concluded to be a subunit of the 150 Kd protein. The plasmid pYV6953 is different from the known plasmids pIB1 and pCD1 plasmids as far as the outer membrane and secreted proteins coded by the plasmids are concerned.     

YopD of Yersinia pestis Plays a Role in Negative Regulation of the Low-Calcium Response in Addition to Its Role in Translocation of Yops

Yersinia pestis produces a set of virulence proteins (Yops and LcrV) that are expressed at high levels and secreted by a type III secretion system (Ysc) upon bacterium-host cell contact, and four of the Yops are vectorially translocated into eukaryotic cells. YopD, YopB, and YopK are required for the translocation process. In vitro, induction and secretion occur at 37°C in the absence of calcium. LcrH (also called SycD), a protein required for the stability and secretion of YopD, had initially been identified as a negative regulator of Yop expression. In this study, we constructed a yopD mutation in both wild-type and secretion-defective (ysc) Y. pestis to determine if the lcrH phenotype could be attributed to the decreased stability of YopD. These mutants were constitutively induced for expression of Yops and LcrV, despite the presence of the secreted negative regulator LcrQ, demonstrating that YopD is involved in negative regulation, regardless of a functioning Ysc system. Normally, secretion of Yops and LcrV is blocked in the presence of calcium. The single yopD mutant was not completely effective in blocking secretion: LcrV was secreted equally well in the presence and absence of calcium, while there was partial secretion of Yops in the presence of calcium. YopD is probably not rate limiting for negative regulation, as increasing levels of YopD did not result in decreased Yop expression. Overexpression of LcrQ in the yopD mutant had no significant effect on Yop expression, whereas increased levels of LcrQ in the parent resulted in decreased levels of Yops. These results indicate that LcrQ requires YopD to function as a negative regulator.     

Yersinia effectors target mammalian signalling pathways

Animals have an immune system to fight off challenges from both viruses and bacteria. The first line of defence is innate immunity, which is composed of cells that engulf pathogens as well as cells that release potent signalling molecules to activate an inflammatory response and the adaptive immune system. Pathogenic bacteria have evolved a set of weapons, or effectors, to ensure survival in the host. Yersinia spp. use a type III secretion system to translocate these effector proteins, called Yops, into the host. This report outlines how Yops thwart the signalling machinery of the host immune system.     

Evaluation of the usefulness of selected virulence markers for the identification of virulent Yersinia enterocolitica strains. I. Phenotypic markders associated with Plasmid pYV

The species Yersinia enterocolitica includes either pathogenic or non-pathogenic strains. Therefore it is necessary to differentiate virulent bacilli from other. It is well known that pathogenic strains of Y. enterocolitica bearing virulence associated plasmid called pYV, which could be demonstrated by its isolation or detected by the presence of specific, phenotypic properties directly related with this plasmid. The aim of the presented paper was to check the ability of some phenotypic virulence markers associated with pYV, to detection of pathogenic Y. enterocolitica strains. In the presented work 152 (130 carrying pYV) clinical strains of Y. enterocolitica O3 isolated mainly from stool were examined for the presence of phenotypic virulence markers such as: calcium dependency, Congo-red binding, autoagglutination and agglutination with Mangifera indica extract. Both first features were detected parallel, on the same plate, using CRMOX (Congo-red, Magnesium Oxalate) agar. The detection of the tested markers in the examined strains was compared with the presence of virulence plasmid. The obtained results confirmed the observations done by other authors that Y. enterocolitica strains, in which bacilli bearing the virulence plasmid predominate, exhibit all tested phenotypic properties whereas the plasmid-cured isogenic strains show no one of these features. Therefore all the tested markers could be useful for detection of virulent Y. enterocolitica strains directly isolated from patients. The most useful virulence markers in bacteriological study seems to be calcium dependency and Congo-red binding, examined together by the use of CRMOX agar, because they confirm the presence of the virulence plasmid by parallel detection of two physiologically different features associated with this plasmid. In addition CRMOX agar allows for the examination rough strains while agglutination tests do not.     

A proton-led model of fast calcium waves

Fast (10-30 microm/s) calcium waves can be propagated through all nucleated eukaryotic cells that have been tested as well as certain cell-free extracts. In a widely used model, they are propagated by a reaction-diffusion cycle in which calcium ions diffuse along the outside of endoplasmic reticula and induce their own release from calsequestrin or calreticulin molecules stored within the reticulum's lumen. Here we propose a new tandem wave model in which they are also propagated by a reaction-diffusion cycle within a reticulum's lumen. In this cycle, increases in luminal [H(+)] induce proton release from luminal calsequestrin or calreticulin. The released protons diffuse ahead to where they release more protons from these luminal storage proteins. What might be called proton induced proton release. They also raise luminal electropositivity. The resultant luminal waves are coordinated with extrareticular ones by movements of calcium and hydrogen ions through the reticular membrane. This model makes five testable predictions which include the autorelease of protons in solutions of calsequestrins or calreticulins as well as waves of increased [H(+)], of increased [Ca(2+)] and of more positive voltage within the reticula of whole cells. Moreover, under some conditions, such luminal waves should cross regions without cytosolic ones.     

LcrG-LcrV interaction is required for control of Yops secretion in Yersinia pestis

Yersinia pestis expresses a set of plasmid-encoded virulence proteins called Yops and LcrV that are secreted and translocated into eukaryotic cells by a type III secretion system. LcrV is a multifunctional protein with antihost and positive regulatory effects on Yops secretion that forms a stable complex with a negative regulatory protein, LcrG. LcrG has been proposed to block the secretion apparatus (Ysc) from the cytoplasmic face of the inner membrane under nonpermissive conditions for Yops secretion, when levels of LcrV in the cell are low. A model has been proposed to describe secretion control based on the relative levels of LcrG and LcrV in the bacterial cytoplasm. This model proposes that under secretion-permissive conditions, levels of LcrV are increased relative to levels of LcrG, so that the excess LcrV titrates LcrG away from the Ysc, allowing secretion of Yops to occur. To further test this model, a mutant LcrG protein that could no longer interact with LcrV was created. Expression of this LcrG variant blocked secretion of Yops and LcrV under secretion permissive conditions in vitro and in a tissue culture model. These results agree with the previously described secretion-blocking activity of LcrG and demonstrate that the interaction of LcrV with LcrG is necessary for controlling Yops secretion.     

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.     

Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops

'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.     

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells

Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia , other pathogens such as Salmonella , Shigella , Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella , Shigella and Listeria , but not Salmonella . Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.     

Secretion of hybrid proteins by the Yersinia Yop export system.

After incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of Yersinia spp. release large amounts of a set of plasmid-encoded proteins called Yops. The secretion of these proteins, involved in pathogenicity, occurs via a mechanism that involves neither the removal of a signal sequence nor the recognition of a C-terminal domain. Analysis of deletion mutants allowed the secretion recognition domain to be localized within the 48 N-terminal amino acids of protein YopH, within the 98 N-terminal residues of protein YopE, and within the 76 N-terminal residues of YopQ. Comparison of these regions failed to reveal any sequence similarity, suggesting that the secretion signal of Yop proteins is conformational rather than sequential. Hybrid proteins containing the amino-terminal part of YopH fused to either the alpha-peptide of beta-galactosidase or to alkaline phosphatase deprived of its signal sequence were efficiently secreted to the Yersinia culture medium. This observation opens new prospects in using Yersinia spp. as chimeric-protein producers and as potential live carriers for foreign antigens.