The exoenzyme S regulon of Pseudomonas aeruginosa

Pseudomonas aeruginosa can cause severe life-threatening infections in which the bacterium disseminates rapidly from epithelial colonization sites to the bloodstream. In experimental models, the ability of P . aeruginosa to disseminate is linked to epithelial injury, in vitro cytotoxicity and expression of the exoenzyme S regulon. Using the expression of ExoS as a model, a series of genes that are important for regulation, secretion and, perhaps, intoxication of eukaryotic cells have been identified. Proteins encoded by the exoenzyme S regulon and the Yersinia Yop virulon show a high level of amino acid homology, suggesting that P . aeruginosa may use a contact-mediated translocation mechanism to transfer anti-host factors directly into eukaryotic cells. Potential anti-host factors that may disrupt eukaryotic signal transduction through ADP-ribosylation include ExoS and ExoT. Expression of ExoU, another candidate anti-host factor, has been correlated with acute cytotoxicity and lung epithelial injury. Members of the exoenzyme S regulon represent only a portion of the virulence factor arsenal possessed by P . aeruginosa . It will be important to understand how the exoenzyme S regulon contributes to pathogenesis and whether these factors could serve as potential therapeutic targets.     

微量法鼠疫菌质粒DNA抽提的优化

旨在分析微量法抽提鼠疫菌质粒DNA的效果,探讨其在鼠疫菌分子生物学实验研究中的应用价值.采用微量法分别提取鼠疫菌EV76株,假结核耶尔森菌PstII株及大肠杆菌V517株质粒DNA,琼脂糖凝胶电泳对质粒DNA抽提结果进行分析.结果显示,微量法能在较短时间内获取开环较少的闭合环状鼠疫菌质粒DNA,经琼脂糖凝胶电泳图示其电泳条带清晰、亮度均一.微量法鼠疫菌质粒DNA抽提效率和纯度较好,抽提结果稳定,重复性良好.经微量法抽提的质粒DNA符合多数鼠疫菌分子生物学试验的要求,可广泛应用于鼠疫菌分子生物学试验研究中.     

Minimal YopB and YopD translocator secretion by Yersinia is sufficient for Yop-effector delivery into target cells

Pathogenic Yersinia sp. utilise a common type III secretion system to translocate several anti-host Yop effectors into the cytosol of target eukaryotic cells. The secreted YopB and YopD translocator proteins are essential for this process, forming pores in biological membranes through which the effectors are thought to gain access to the cell interior. The non-secreted cognate chaperone, LcrH, also plays an important role by ensuring pre-secretory stabilisation and efficient secretion of YopB and YopD. This suggests that LcrH-regulated secretion of the translocators could be used by Yersinia to control effector translocation levels. We collected several LcrH mutants impaired in chaperone activity. These poorly bound, stabilised and/or secreted YopB and YopD in vitro. However, these mutants generally maintained stable substrates during a HeLa cell infection and these infected cells were intoxicated by translocated effectors. Surprisingly, this occurred in the absence of detectable YopB- and YopD-dependent pores in eukaryotic membranes. A functional type III translocon must therefore only require minuscule amounts of secreted translocator proteins. Based on these observations, LcrH dependent control of translocation via regulated YopB and YopD secretion would need to be exquisitely tight.     

Depolymerization of beta-1,6-N-acetyl-D-glucosamine disrupts the integrity of diverse bacterial biofilms

Polymeric beta-1,6-N-acetyl-D-glucosamine (poly-beta-1,6-GlcNAc) has been implicated as an Escherichia coli and Staphylococcus epidermidis biofilm adhesin, the formation of which requires the pgaABCD and icaABCD loci, respectively. Enzymatic hydrolysis of poly-beta-1,6-GlcNAc, demonstrated for the first time by chromatography and mass spectrometry, disrupts biofilm formation by these species and by Yersinia pestis and Pseudomonas fluorescens, which possess pgaABCD homologues.     

Yersinia protein kinase YopO is activated by a novel G-actin binding process

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject effector proteins (Yops) into host cells. The Yops down-regulate host cell functions through unique biochemical activities. YopO, a serine/threonine kinase required for Yersinia virulence, is activated by host cell actin via an unknown process. Here we show that YopO kinase is activated by formation of a 1:1 complex with monomeric (G) actin but is unresponsive to filamentous (F) actin. Two separate G-actin binding sites, one in the N-terminal kinase region (amino acids 89-440) and one in the C-terminal guanine nucleotide dissociation inhibitor-like region (amino acids 441-729) of YopO, were identified. Actin binding to both of these sites was necessary for effective autophosphorylation of YopO on amino acids Ser-90 and Ser-95. A S90A/S95A YopO mutant was strongly reduced in substrate phosphorylation, suggesting that autophosphorylation activates YopO kinase activity. In cells the kinase activity of YopO regulated rounding/arborization and was specifically required for inhibition of Yersinia YadA-dependent phagocytosis. Thus, YopO kinase is activated by a novel G-actin binding process, and this appears to be crucial for its anti-host cell functions.     

The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.     

DNA probe for detecting Yersinia pestis and serovariant I of Yersinia pseudotuberculosis by detecting specific DNA repeating sequences]

In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.     

The role of virulence antigens (VW) in the protection of mice againstYersinia pestis infection

Subcutaneous infection withYersinia enterocolitica harboring plasmid responsible for Ca²⁺ dependence at 37°C induced cell-mediated protective immunity against a lethal challenge withYersinia pestis; the isogenic derivative strain cured from this plasmid subverted the immunity in mice. This is the first identification of the antigen(s) responsible for the induction of cell-mediated protective immunity against the facultatively intracellular bacteria.     

Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR

Pathogenic yersiniae secrete the Yop anti-host proteins using a type-III secretion pathway. The components of the secretion machinery are encoded by three loci on the pYV plasmid: virA, virB, and virC . In this paper we describe the characterization of eight non-polar mutants of the virC locus, constructed by allelic exchange. The yscE, FG, I, J and K mutants were defective in Yop secretion and independent of Ca²⁺ (Cl) for their growth at 37°C. Substitution of the 12 N-terminal amino-acid residues of YscF impaired secretion of YopB and YopD only and led also to a Cl phenotype. The culture supernatant of the yscH mutant contained all the Yops except the 18 kDa YopR. Complementation experiments and an immunoblot analysis confirmed that YopR is encoded by the yscH gene. The LD₅₀ for the mouse of the yscH mutant was 10-fold higher than that of the parental strain indicating that YopR is involved in pathogenesis. The phenotype of the yscM mutant was similar to that of the wild-type strain. However, overproduction of YscM from a multicopy plasmid in wild-type Yersinia enterocolitica prevented Yop secretion and synthesis. A hybrid YopH—LacZ' protein, encoded by a gene transcribed from the lac promoter, was secreted by a strain overexpressing YscM, showing that the secretion machinery was still functional. These results indicate that YscM plays a role in the feedback inhibition of Yop synthesis when secretion is compromised by acting as a negative regulator of Yop synthesis.     

Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.     

Homologous DNA sequences on the virulence plasmids of pathogenic Yersinia and Salmonella dublin Lane

Yersinia and Salmonella harbour plasmids that encode traits important for virulence, enabling both pathogenic genera to survive and grow in cells of the reticulo-endothelial organs during systemic infections. We have detected DNA homology between the Salmonella dublin virulence plasmid pSDL2 and the plasmids of the pathogenic Yersinia species pestis, pseudotuberculosis, and enterocolitica. Three regions of pSDL2 were found to share homology with the virulence plasmid pIB1 of Yersinia pseudotuberculosis. Two separate hybridizing segments mapped within the previously characterized 6.4 kb vir region of pSDL2 in the SalI B fragment. The third homologous region involved the replicon of pIB1, which hybridized to the SalI C2 fragment of pSDL2. The virulence plasmid pCD1 from Y. pestis showed similar homology with the three regions of pSDL2. Homologies to the vir and SalI C2 regions of pSDL2 were also found on plasmids from Yersinia enterocolitica serotypes 0:9, 0:3 and 0:5, 27. The discovery of separate homologous regions on the virulence plasmids of Salmonella and Yersinia suggests a distant evolutionary relationship.     

Incompatibility of the Cad plasmid from Yersinia pestis with plasmids of the IncFI group

The relation of Yersinia pestis calcium dependence plasmid (pCad) to known Inc FI (F'lac, R386, pOX38) and IncFV (F0lac) plasmids has been studied. Evidence that plasmid pCad of Yersinia pestis belongs to FI incompatibility group is presented.     

The effect of the bacterial genome on the expression and behavior of the plasmid determining the Ca2+-dependence of the plague pathogen]

The conjugative cointegrate containing the 47 Md plasmid of Yersinia pestis has been transferred into the strains of the different Yersinia (Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica) and Escherichia coli CA. There appeared in the populations of recombinant Yersinia under the conditions of Ca2+ deficit at 37 degrees C the cells coming into the stasis stage or dying. It was shown on the model of Yersinia enterocolitica that bacterial lethality might be prevented by exclusion of the sheep blood from Ca2+ deficient medium. Ca(2+)-dependence was not expressed in Escherichia coli cells in which the cointegrates were prone to deletions although the cad-genes were preserved intact. The latter conclusion is based on the positive reciprocal transfer of the Cad(+)-marker into Yersinia pestis cells.     

Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia.

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278-300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic alpha helix ranging from Phe280 to Tyr291, followed by a type I beta turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278-292 and YopD271-292, lacked helical structure, implicating the beta turn in helix stability. An interaction between YopD278-300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide-LcrH complex. These effects were not observed for the unstructured peptide, YopD278-292, which confirms that the alpha helical structure of the YopD amphipathic domain is a critical binding region of LcrH.     

Haemagglutinins and Fimbriae in Different Serotypes and Biotypes of Yersinia enterocolitica

When cultured in static broth at 20°C, 46 of 115 strains of Yersinia enterocolitica , diverse in biotype and serotype, produced a broad-spectrum mannose-resistant (MR) adhesin that agglutinated the erythrocytes of all of 10 animal species examined. The production of haemagglutinin (HA) was associated with the presence of fimbriae of S nm diameter. Culture of HA+ strains at 37° resulted in the disappearance of haemagglutinating ability and loss of fimbrial production. Strains of Y. enterocolitica with K1 antigen produced an MR adhesin that agglutinated only fowl erythrocytes and was associated with fimbriae of 4–4.5 nm diameter. None of 14 strains of Y. pseudotuberculosis was haemagglutinating.     

Design of a species-specific DNA probe based on the pFra plasmid of the plague pathogen]

The recombinant plasmid pBS1 carrying a 2 kb SalGI fragment of Yersinia pestis pFra plasmid was constructed by insertion of the fragment into a vector plasmid pBR327. SalGI-BspRI 400 bp subfragment was recloned into a pBR322 vector plasmid. Open reading frame was found in the fragment by DNA sequencing technique. The subfragment designated F1-probe permits one to identify specifically the Yersinia pestis strains harbouring pFra plasmid, thus, differing them from closely related Yersiniea and other representatives of Enterobacteriaceae family.     

Hijacking of the pleiotropic cytokine interferon-γ by the type III secretion system of Yersinia pestis

Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague.     

Quorum sensing and the lifestyle of Yersinia

Bacterial cell-to-cell communication ('quorum sensing') is mediated by structurally diverse, small diffusible signal molecules which regulate gene expression as a function of cell population density. Many different Gram-negative animal, plant and fish pathogens employ N-acylhomoserine lactones (AHLs) as quorum sensing signal molecules which control diverse physiological processes including bioluminescence, swarming, antibiotic biosynthesis, plasmid conjugal transfer, biofilm development and virulence. AHL-dependent quorum sensing is highly conserved in both pathogenic and non-pathogenic members of the genus Yersinia. Yersinia pseudotuberculosis for example, produces at least eight different AHLs and possesses two homologues of the LuxI family of AHL synthases and two members of the LuxR family of AHL-dependent response regulators. In all Yersinia species so far examined, the genes coding for LuxR and LuxI homologues are characteristically arranged convergently and overlapping. In Y. pseudotuberculosis AHL-dependent quorum sensing is involved in the control of cell aggregation and swimming motility, the latter via the flagellar regulatory cascade. This is also the case for swimming and also swarming motility in Yersinia enterocolitica. Howeverthe role of AHL-dependent quorum sensing in Yersinia pestis remains to be determined.     

BamHI restriction endonuclease analysis of Yersinia ruckeri plasmids and their relatedness to the genus Yersinia 42- to 47-megadalton plasmid

The plasmid profile and BamHI restriction pattern of 17 sorbitol-negative and 1 sorbitol-positive French Yersinia ruckeri strain of the American type strain were studied. The 17 sorbitol-negative strains and the American strain harbored a 62-megadalton (MDa) plasmid with an identical BamHI restriction pattern. Southern hybridization indicated that this 62-MDa plasmid is common among these various strains. The sorbitol-positive strain had four plasmid bands (70, 62, 32, and 25 MDa), and there was no comigration of the DNA fragments of these cleaved plasmids with the fragments of the 62-MDa plasmid. Hybridization of these restricted plasmids with the common 62-MDa plasmid showed a weak DNA homology. The Y. ruckeri plasmid (62 MDa) had a different molecular weight than the virulence plasmid (42 to 47 MDa) of the genus Yersinia, and they had different BamHI restriction patterns. Furthermore, no sequence of the Y. ruckeri plasmid DNA was recognized after Southern hybridization when the 47-MDa plasmid of Y. enterocolitica was used as a probe.