Individual chaperones required for Yop secretion by Yersinia.

Pathogenic yersiniae secrete anti-host proteins called Yops, by a recently discovered Sec-independent pathway. The Yops do not have a classical signal peptide at their N terminus and they are not processed during membrane translocation. The secretion domain is nevertheless contained in their N-terminal part but these domains do not resemble each other in the different Yops. We have previously shown that YopE secretion requires SycE, a 15-kDa acidic protein acting as a specific cytosolic chaperone. Here we show that the gene downstream from yopH encodes a 16-kDa acidic protein that binds to hybrid proteins made of the N-terminal part of YopH and either the bacterial alkaline phosphatase or the cholera toxin B subunit. Loss of this protein by mutagenesis led to accumulation of YopH in the cytoplasm and to a severe and selective reduction of YopH secretion. This protein thus behaves like the counterpart of SycE and we called it SycH. We also engineered a mutation in lcrH, the gene upstream from yopB and yopD, known to encode a 19-kDa acidic protein. Although this mutation was nonpolar, the mutant no longer secreted YopB and YopD. The product of lcrH could be immunoprecipitated together with cytoplasmic YopD. lcrH therefore seems to encode a YopD-specific chaperone, which we called SycD. Determination of the dependence of YopB on SycD requires further investigation. SycE, SycH, and SycD appear to be members of a new family of cytosolic chaperones required for Yop secretion.     

Walking exercise in patients with intermittent claudication. Experience in routine clinical practice.

BACKGROUND: In patients with intermittent claudication, exercise in the form of walking is effective in reducing pain and maximising achievable walking distance. However, data are lacking on the implementation of walking exercise in these patients. AIMS: To explore the current behaviour and views of patients with intermittent claudication towards taking walking exercise. DESIGN OF STUDY: Postal questionnaire and focus group meetings. SETTING: Two academic general practice networks (Utrecht and Maastricht Universities) in The Netherlands. METHOD: Three hundred and seventy-five patients with intermittent claudication, selected from the files of general practitioners participating in two academic general practice networks, were sent a postal questionnaire; 216 (58%) were returned. Nine of these responders also attended a focus group meeting. RESULTS: Seventy per cent (151/216) of the patients reported having received advice about walking exercise. If specified, the advice given most often recommended walking in the local neighbourhood (56%, 84/151). Fifty-two per cent (113/216) of all patients actually performed walking exercise and only 32%of them received any kind of supervision. Among the barriers for taking walking exercise, 'comorbidity', 'lack of (specific) advice' and 'lack of supervision' were often mentioned. Among the stimuli to start and continue walking, 'following the doctor's advice', 'relief of complaints' and 'a better general condition' were often mentioned by patients. CONCLUSIONS: Walking exercise was not carried out by almost half of patients with intermittent claudication in this study. Lack of specific advice and supervision were found to be important barriers to taking walking exercise.     

Rat Cytomegalovirus-Accelerated Transplant Vascular Sclerosis Is Reduced with Mutation of the Chemokine-Receptor R33

Cytomegalovirus (CMV) infection accelerates transplant vascular sclerosis (TVS) and chronic rejection (CR) in both human and animal solid organ transplantation models. The host/viral mechanisms involved in this process are unclear. We examine the role of the rat CMV (RCMV)-encoded chemokine-receptor R33 in the development of TVS using a rat heart transplantation/CR model. F344 heart grafts were transplanted heterotopically into Lewis recipients. The ability of RCMV lacking the R33 gene (RCMV-Deltar33) to accelerate CR/TVS (neointimal index, NI) was compared to wild-type (WT) RCMV. Allograft recipients were infected with 1 x 10(5) pfu RCMV or RCMV-Deltar33 on postoperative day (POD) 1. Grafts from RCMV-Deltar33-infected recipients demonstrated an accelerated time to allograft CR compared to grafts from uninfected recipients (POD = 56 vs. 90), this was slower than that seen in grafts from WT-RCMV-infected recipients (POD = 45). Similarly, the degree of graft TVS formation at terminal rejection in RMCV-Deltar33 infected recipients was more severe than uninfected recipients (NI = 63 vs. 45), yet not as severe as in WT-RCMV infected recipients (NI = 83). In parallel, RCMV-Deltar33 failed to induce vascular smooth muscle cell (SMC) migration in vitro, whereas WT-RCMV induced substantial migration. The RCMV-encoded chemokine-receptor r33 is critical for RCMV-accelerated TVS/CR and vascular SMC migration.     

Specific antibody responses to three schistosome-related carbohydrate structures in recently exposed immigrants and established residents in an area of Schistosoma mansoni endemicity

By the use of surface plasmon resonance spectroscopy, immunoglobulin G (IgG) subclass and IgM antibodies against three schistosome-derived carbohydrate structures, FLDN (Fucalpha1-3GalNAcbeta1-4GlcNAcbeta1-3Galalpha1), LDN-DF [GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1], and LDNF [GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galalpha1], were measured in 184 previously unexposed Kenyan immigrants who moved into the Masongaleni area, where Schistosoma mansoni is endemic. They were sampled within their first year of exposure and again 2 years later. A cohort selected out of the original residents of the area, who had been exposed for many years, served as controls. Associations with responses to S. mansoni worm, egg (SEA), and cercarial (CERC) antigens were examined. In addition, we measured responses to keyhole limpet hemocyanin, a glycoprotein which carries glycan epitopes that are also expressed by schistosomes. Specific IgG1 responses were most pronounced against FLDN and LDN-DF and strongly associated with those previously measured to SEA and CERC. Similarly to previously published age profiles of IgG1 and IgG2 responses to SEA, levels of IgG1 against LDN-DF decreased with age. In contrast, specific IgM responses against the three schistosome-derived carbohydrate structures were most marked against LDNF. Our results indicate that, of the three glycan structures tested, the acute response against schistosome glycoconjugate antigens in young children is mainly directed against the LDN-DF epitope. The response to LDN-DF in older individuals and the responses to the two other epitopes were similar in the two cohorts, suggesting that these antigens are recognized in the early stages of infection and that the immune response persists. The biological significance of these observations needs further elucidation.     

Molecular epidemiology of Pseudomonas aeruginosa colonization in a burn unit: persistence of a multidrug-resistant clone and a silver sulfadiazine-resistant clone

To study the epidemiology of Pseudomonas aeruginosa colonization in a 32-bed burn wound center (BWC), 321 clinical and 45 environmental P. aeruginosa isolates were collected by prospective surveillance culture over a 1-year period and analyzed by serotyping, drug susceptibility testing, and amplified fragment length polymorphism (AFLP) analysis. Among 441 patients treated at the center, 70 (16%) were colonized with P. aeruginosa, including 12 (17%) patients who were colonized on admission and 58 (83%) patients who acquired the organism during their stay. Of the 48 distinct AFLP genotypes found, 21 were found exclusively in the environment, 15 were isolated from individual patients only, and 12 were responsible for the colonization of 57 patients, of which 2 were also isolated from the environment, but secondary to patient carriage. Polyclonal P. aeruginosa colonization with strains of two to four genotypes, often with different antibiotic susceptibility patterns, was observed in 19 patients (27%). Two predominant genotypes were responsible for recurrent outbreaks and the colonization of 42 patients (60% of all colonized patients). The strain with one of those genotypes appeared to be endemic to the BWC and developed multidrug resistance (MDR) at the end of the study period, whereas the strain with the other genotype was antibiotic susceptible but resistant to silver sulfadiazine (SSD(r)). The MDR strain was found at a higher frequency in sputum samples than the SSD(r) strain, which showed a higher prevalence in burn wound samples, suggesting that anatomic habitat selection was associated with adaptive resistance to antimicrobial drugs. Repeated and thorough surveys of the hospital environment failed to detect a primary reservoir for any of those genotypes. Cross-acquisition, resulting from insufficient compliance with infection control measures, was the major route of colonization in our BWC. In addition to the AFLP pattern and serotype, analysis of the nucleotide sequences of three (lipo)protein genes (oprI, oprL, and oprD) and the pyoverdine type revealed that all predominant strains except the SSD(r) strain belonged to recently identified clonal complexes. These successful clones are widespread in nature and therefore predominate in the patient population, in whom variants accumulate drug resistance mechanisms that allow their transmission and persistence in the BWC.     

Nucleotide sequence of yst, the Yersinia enterocolitica gene encoding the heat-stable enterotoxin, and prevalence of the gene among pathogenic and nonpathogenic yersiniae.

The gene encoding the heat-stable enterotoxin (yst) was cloned from the chromosome of Yersinia enterocolitica W1024 (serotype O:9), and the nucleotide sequence was determined. The yst gene encodes a 71-amino-acid polypeptide. The C-terminal 30 amino acids of the predicted protein exactly correspond to the amino acid sequence of the toxin extracted from culture supernatants (T. Takao, N. Tominaga, and Y. Shimonishi, Biochem. Biophys. Res. Commun. 125:845-851, 1984). The N-terminal 18 amino acids have the properties of a signal sequence. The central 22 residues are removed during or after the secretion process. This organization in three domains (Pre, Pro, and mature Yst) resembles that of the enterotoxin STa of Escherichia coli. The degree of conservation between the E. coli and Y. enterocolitica toxins is much lower in the Pre and the Pro domains than in the mature proteins. The mature toxin of Y. enterocolitica is much larger than that of E. coli, but the active domain appears to be highly conserved. The yst gene of Y. enterocolitica introduced in E. coli K-12 directed the secretion of an active toxin. The cloned yst gene was used as an epidemiological probe among a collection of 174 strains representative of all Yersinia species except Yersinia pestis and numerous Y. enterocolitica subgroups. In Y. enterocolitica, there was a clear-cut difference between pathogenic and nonpathogenic strains: 89 of 89 pathogenic and none of 51 nonpathogenic strains contained yst-homologous DNA, suggesting that Yst is involved in pathogenesis. Among the other Yersinia species, only four strains of Yersinia kristensenii had DNA homologous to yst.     

Detection of pYV+ Yersinia enterocolitica isolates by P1 slide agglutination.

Rabbit polyclonal antisera were raised against the pYV-encoded outer membrane protein P1 of five Yersinia enterocolitica strains belonging to serogroups O:3, O:5,27, O:8, and O:9. Analysis of these strains with the sera showed that P1 presented at least six different antigenic factors. Two of the serum specimens were chosen to test the P1 agglutinability of 797 strains isolated from various sources. This technique appeared to be more reliable than autoagglutination and Ca2+ dependency to monitor the presence of the pYV plasmid. Hence, we propose this P1-mediated agglutination as a new and easy virulence test.