Identification and characterisation of a novel adhesin Ifp in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

Background  

In order to identify new virulence determinants in Y. pseudotuberculosis a comparison between its genome and that of Yersinia pestis was undertaken. This reveals dozens of pseudogenes in Y. pestis, which are still putatively functional in Y. pseudotuberculosis and may be important in the enteric lifestyle. One such gene, YPTB1572 in the Y. pseudotuberculosis IP32953 genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic (EPEC) and enterohaemorraghic (EHEC) Escherichia coli.     

Immunodiffusion test for diagnosing basidiobolomycosis

An immunodiffusion test was developed for the diagnosis of basidiobolomycosis. When culture filtrate antigen (CFA) from Basidiobolus ranarum was reacted against two human patient and two rabbit antisera, 2 precipitin bands, inner (N) and outer (Y), were revealed for both patient and rabbit antisera. A line of identity was also observed between precipitin bands obtained with patient and rabbit sera. When CFA from B. ranarum (B CFA) was reacted against rabbit sera which contained antibody to Conidiobolus coronatus and Pythium insidiosum, 1 precipitin band corresponding to inner band (N) was observed. This finding showed that B. ranarum, C. coronatus and P. insidiosum shared at least one common antigen. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera of Pythium and Conidiobolus disappeared. However, with Basidiobolus rabbit antiserum, the result did not change. The antigens which could be demonstrated by inner (N) and outer (Y) precipitin bands were heat stable at 56 ° C for 30 min. The titer of the antibodies specific to these antigens decreased as the lesions subsided. When B. ranarum CFA was reacted against sera from 20 apparently normal persons, 20 diabetes mellitus patients, 5 aspergillosis patients, 2 candidosis patients and 3 pythiosis patients, no precipitin band was found. B. ranarum CFA was also treated with each rabbit antiserum specific to Candida albicans, Malassezia furfur and Aspergillus fumigatus. No precipitin bands occurred with any of these antisera. Thus, this test was found to be practical, sensitive and specific, and can be used to monitor patients infected with Basidiobolus ranarum.     

Comparison of 16S rDNA analysis and rep-PCR genomic fingerprinting for molecular identification of Yersinia pseudotuberculosis

16S rDNA sequence analysis and repetitive element sequence-based PCR (rep-PCR) genomic fingerprinting were evaluated on 11 type strains of the genus Yersinia and 17 recognized serotype strains of Y. pseudotuberculosis to investigate their genetic relatedness and to establish the value of techniques for the identification of Y. pseudotuberculosis. A phylogenetic tree constructed from 16S rDNA sequences showed that the type strains of Yersinia species formed distinct clusters with the exception of Y. pestis and Y. pseudotuberculosis. Moreover, Y. pestis NCTC 5923^T was found to be closely related to Y. pseudotuberculosis serotypes 1b, 3, and 7. Dendrograms generated from REP-PCR, and ERIC-PCR data revealed that members of the genus Yersinia differed from each other with the degree of similarity 62% and 58%, respectively. However, the BOX-PCR results showed that Y. pestis 5923^T clustered with the Y. pseudotuberculosis group with a degree of similarity 74%. According to these findings, 16S rDNA sequence analysis was unable to reliably discriminate Y. pseudotuberculosis from Y. pestis. However, REP-PCR and especially ERIC-PCR provided an effective means of differentiating between members of the taxa. This revised version was published online in June 2006 with corrections to the Cover Date.     

Th1-type immune response to infection by pYV-cured <Emphasis Type="Italic">phoP-phoQ</Emphasis> null mutant of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> is defective in mouse model

The PhoP-PhoQ two-component system of Yersinia pseudotuberculosis, a Gram-negative enteric pathogen which causes a variety of gastrointestinal and extraintestinal infections in humans, has been shown to be necessary for virulence. A phoP-phoQ null mutant of a strain of Y. pseudotuberculosis cured of its native plasmid pYV was obtained and studied for generation of immune response in mouse model following intravenous inoculation. The phoP-phoQ null mutant elicited much weaker IgG antibody response to whole cell sonicated (WCS) antigen, in particular that of IgG2a isotype. Interferon-γ levels were also significantly reduced in cultured splenocytes of mice immunized with phoP-phoQ null mutant. The null mutant was found to be about 72-fold less virulent than the parent isogenic strain of Y. pseudotuberculosis. Average counts in spleen of mice inoculated with the null mutant were observed to reduce by at least four logs when compared with the counts in the spleen of mice inoculated with parent isogenic strain. We can thus suggest that the Th1-type immune response of the phoP-phoQ null mutant of Y. pseudotuberculosis is diminished in mice.     

Intra- and interspecies relatedness ofYersinia pestis by DNA hybridization and its relationship toYersinia pseudotuberculosis

The biochemical characteristics ofYersinia pestis are presented and compared with those ofY. pseudotuberculosis. Motility at 28°C, urease, fermentation of rhamnose, and growth rate on nutrient agar are the best means of separating these organisms. DNA hybridization studies demonstrated thatY. pestis strains are 90% or more interrelated and thatY. pestis andY. pseudotuberculosis are indistinguishable by DNA relatedness. On the basis of DNA data and biochemical and antigenic similarity, these organisms should be treated as two separate subspecies of the same species.Y. pseudotuberculosis was described beforeY. pestis and therefore has priority.Y. pseudotuberculosis subsp.pseudotuberculosis andY. pseudotuberculosis subsp.pestis are recommended as new designations forY. pseudotuberculosis andY. pestis. For medical purposes,Y. pestis andY. pseudotuberculosis can and should continue to be used.     

Genomic comparison of Yersinia pestis and Yersinia pseudotuberculosis by combination of suppression subtractive hybridization and DNA microarray

In order to further figure out the genetic differences between Yersinia pestis and Yersinia pseudotuberculosis, and to provide novel insights into the evolution of Y. pestis, we compared the genomes of Y. pseudotuberculosis serogroup I strain ATCC29833 and Y. pestis Antiqua strain 49006 using a combination of suppression subtractive hybridization (SSH) and comparative genomic hybridization with DNAs from a diverse panel of Y. pestis and Y. pseudotuberculosis strains. SSH followed by BLAST analysis revealed 112 SSH fragments specific to strain ATCC29833, compared to the genomic sequence data of Y. pestis strains CO92, KIM and 91001. We identified 17 SSH fragments that appeared to be newly determined genetic contents of Y. pseudotuberculosis. The combination of SSH and microarray analysis showed that the parallel loss of genes contributed greatly not only to the significant genomic divergence between Y. pestis and Y. pseudotuberculosis but also to the intra-species microevolution of both of species. The results confirmed our earlier hypothesis that Y. pestis Antiqua isolates from the natural plague focus B in China represented the most ancestral strains in China, hence phylogenetically the closest isolates to Y. pseudotuberculosis.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Xiaoyi Wang and Dongsheng Zhou contributed equally to this work.     

Yersinia pseudotubetculosis in Italy. Attempted Recovery from 37,666 Samples

From 1981 to 1991, 37,666 human, animal, food and environmental samples were cultured for Yersinia pseudotuberculosis using direct plating methods and/or cold enhancement techniques. Despite an intensive surveillance and adequate culture methods, Y. pseudotuberculosis was isolated from stools of 0.05% (5/9,720) of patients with acute enteritis, and alimentary tracts of 0.1% (10/6,849) of apparently healthy animals. No Y. pseudotuberculosis strains were recovered from stools of 4,726 healthy controls nor from the appendices (656), mesenteric lymph nodes (84), and stools (421) of 656 patients operated for suspected appendicitis. Of the 10,842 food and 4,368 environmental samples, none yielded positive cultures for Y. pseudotuberculosis.     

A Highly Specific One-step PCR Assay for the Rapid Discrimination of Enteropathogenic Yersinia enterocolitica from Pathogenic Yersinia pseudotuberculosis and Yersinia pestis

Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y. pseudotuberculosis and Y. pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y. enterocolitica was developed. By this method pathogenic isolates of Y. enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined.     

Characterization of the <Emphasis Type="Italic">ompL1</Emphasis> gene of pathogenic <Emphasis Type="Italic">Leptospira species</Emphasis> in China and cross-immunogenicity of the OmpL1 protein

Background  

The usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp. Identification of genus-specific protein antigens (GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1, a transmembrane porin of pathogenic leptospires, was identified as a possible GP-Ag, but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown.     

Characterization of 13 multi-drug resistant <Emphasis Type="Italic">Salmonella</Emphasis> serovars from different broiler chickens associated with those of human isolates

Background  

Salmonella are frequently isolated from chickens and their products. Prevalent serogroups and serovars of Salmonella as well as their genotypes and antibiograms were determined for cloacal samples from 1595 chickens. To understand the possible serovar and H antigens for transmission between chicken and human, serovars and their H antigens of 164 chicken and 5314 human isolates were compared.     

Immunochemical characteristics of synthetic peptides with T-cellular and B-cellular epitopes of nonspecific porins of pathogenic <Emphasis Type="Italic">Yersinia</Emphasis>

Multiple antigenic peptides (MAPs) that included the common antigenic epitopes of porins from the outer membranes (OM) of bacteria from the Yersinia genus (Y. pseudotuberculosis, Y. enterocolitica, and Y. pestis that are pathogenic for humans) were synthesized. Mice of the BALB/c line were immunized with these peptides, and antisera to the peptides were obtained. It was demonstrated by EIA that these sera interacted with the porins that were isolated from the OM of pathogenic Yersinia. MAPs were shown to be bound to the antibodies in the blood sera of rabbits immunized with the individual porins and to the antibodies in the blood sera of humans suffering from intestinal yersiniosis and pseudotuberculosis.     

The presence of professional phagocytes dictates the number of host cells targeted for Yop translocation during infection

Type III secretion systems deliver effector proteins from Gram‐negative bacterial pathogens into host cells, where they disarm host defences, allowing the pathogens to establish infection. Although Yersinia pseudotuberculosis delivers its effector proteins, called Yops, into numerous cell types grown in culture, we show that during infection Y. pseudotuberculosis selectively targets Yops to professional phagocytes in Peyer's patches, mesenteric lymph nodes and spleen, although it colocalizes with B and T cells as well as professional phagocytes. Strikingly, in the absence of neutrophils, the number of cells with translocated Yops was significantly reduced although the bacterial loads were similar, indicating that Y. pseudotuberculosis did not arbitrarily deliver Yops to the available cells. Using isolated splenocytes, selective binding and selective targeting to professional phagocytes when bacteria were limiting was also observed, indicating that tissue architecture was not required for the tropism for professional phagocytes. In isolated splenocytes, YadA and Invasin increased the number of all cells types with translocated Yops, but professional phagocytes were still preferentially translocated with Yops in the absence of these adhesins. Together these results indicate that Y. pseudotuberculosis discriminates among cells it encounters during infection and selectively delivers Yops to phagocytes while refraining from translocation to other cell types.     

Yersinia ironomics: comparison of iron transporters among Yersiniapestis biotypes and its nearest neighbor, Yersinia pseudotuberculosis

Although Yersinia pestis epidemic biovars and Yersinia pseudotuberculosis are recently diverged, highly related species, they cause different diseases via disparate transmission routes. Since iron transport systems are important for iron acquisition from hosts and for survival in the environment, we have analyzed potential iron transport systems encoded by epidemic and non-epidemic or endemic strains of Y. pestis as well as two virulent Y. pseudotuberculosis strains. Computational biology analysis of these genomes showed a high degree of identity/similarity among 16 proven or possible iron/heme transporters identified. Of these, 7 systems were essentially the same in all seven genomes analyzed. The remaining 9 loci had 2–6 genetic variations among these genomes. Two untested, potential siderophore-dependent systems appear intact in Y. pseudotuberculosis but are disrupted or absent in all the endemic Y. pestis strains as well as the epidemic strains from the antiqua and mediaevalis biovars. Only one of these two loci are obviously disrupted in Y. pestis CO92 (epidemic orientalis biovar). Experimental studies failed to identify a role for hemin uptake systems in the virulence of pneumonic plague and suggest that Y. pestis CO92 does not make a siderophore other than Ybt.     

Autophagosomes can support Yersinia pseudotuberculosis replication in macrophages

Yersinia pseudotuberculosis is able to replicate inside macrophages. However, the intracellular trafficking of the pathogen after its entry into the macrophage remains poorly understood. Using in vitro infected bone marrow‐derived macrophages, we show that Y. pseudotuberculosis activates the autophagy pathway. Host cell autophagosomes subverted by bacteria do not become acidified and sustain bacteria replication. Moreover, we report that autophagy inhibition correlated with bacterial trafficking inside an acidic compartment. This study indicates that Y. pseudotuberculosis hijacks the autophagy pathway for its replication and also opens up new opportunities for deciphering the molecular basis of the host cell signalling response to intracellular Yersinia infection.     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.     

Characteristics and Pathogenicity of Non-Melibiose-Fermenting Strains of Yersinia pseudotuberculosis O3

The biological properties of non-melibiose-fermenting (NMF) strains of Yersinia pseudotuberculosis 03 were investigated. These strains were clearly distinguished from representative melibiose-fermenting (MF) strains of Y. pseudotuberculosis 03 by their pathogenicity in mice, sensitivity to some phages, production of catalase, restriction endonuclease analysis of virulence plasmid DNA with BamHI, detection of specific yersinia outer-membrane proteins with SDS-PAGE, antigenicity of the outer-membrane proteins and neutrophil resistance to phagocytosis. The pathogenicity of NMF strains was clearly less than that of MF strains. In addition, the resistance of NMF strains to phagocytosis and catalase activity was evidently weaker than that of MF strains. These results suggested that the difference of pathogenicity was due to the ability of catalase production. Although the relationship between the above characteristics and melibiose-fermentation was not analysed, the pathogenicity of Y. pseudotuberculosis 03 strains can probably be predicted by testing melibiose-fermentation and catalase production.     

Structural basis for Myf and Psa fimbriae‐mediated tropism of pathogenic strains of Yersinia for host tissues

Three pathogenic species of the genus Yersinia assemble adhesive fimbriae via the FGL‐chaperone/usher pathway. Closely related Y. pestis and Y. pseudotuberculosis elaborate the pH6 antigen (Psa), which mediates bacterial attachment to alveolar cells of the lung. Y. enterocolitica, instead, assembles the homologous fimbriae Myf of unknown function. Here, we discovered that Myf, like Psa, specifically recognizes β1‐3– or β1‐4–linked galactose in glycosphingolipids, but completely lacks affinity for phosphatidylcholine, the main receptor for Psa in alveolar cells. The crystal structure of a subunit of Psa (PsaA) complexed with choline together with mutagenesis experiments revealed that PsaA has four phosphatidylcholine binding pockets that enable super‐high‐avidity binding of Psa‐fibres to cell membranes. The pockets are arranged as six tyrosine residues, which are all missing in the MyfA subunit of Myf. Conversely, the crystal structure of the MyfA‐galactose complex revealed that the galactose‐binding site is more extended in MyfA, enabling tighter binding to lactosyl moieties. Our results suggest that during evolution, Psa has acquired a tyrosine‐rich surface that enables it to bind to phosphatidylcholine and mediate adhesion of Y. pestis/pseudotuberculosis to alveolar cells, whereas Myf has specialized as a carbohydrate‐binding adhesin, facilitating the attachment of Y. enterocolitica to intestinal cells.     

Ail Proteins of Yersinia pestis and Y. pseudotuberculosis Have Different Cell Binding and Invasion Activities

The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ∼50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30–50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.     

Transmission of Yersinia pseudotuberculosis in the Pork Production Chain from Farm to Slaughterhouse

The transmission of Yersinia pseudotuberculosis in the pork production chain was followed from farm to slaughterhouse by studying the same 364 pigs from different production systems at farm and slaughterhouse levels. In all, 1,785 samples were collected, and the isolated Y. pseudotuberculosis strains were analyzed by pulsed-field gel electrophoresis. The results of microbial sampling were combined with data from an on-farm observation and questionnaire study to elucidate the associations between farm factors and the prevalence of Y. pseudotuberculosis. Following the same pigs in the production chain from farm to slaughterhouse, we were able to show similar Y. pseudotuberculosis genotypes in live animals, pluck sets (containing tongue, tonsils, esophagus, trachea, heart, lungs, diaphragm, liver, and kidneys), and carcasses and to conclude that Y. pseudotuberculosis contamination originates from the farms, is transported to slaughterhouses with pigs, and transfers to pluck sets and carcasses in the slaughter process. The study also showed that the high prevalence of Y. pseudotuberculosis in live pigs predisposes carcasses and pluck sets to contamination. When production types and capacities were compared, the prevalence of Y. pseudotuberculosis was higher in organic production than in conventional production and on conventional farms with high rather than low production capacity. We were also able to associate specific farm factors with the prevalence of Y. pseudotuberculosis by using a questionnaire and on-farm observations. On farms, contact with pest animals and the outside environment and a rise in the number of pigs on the farm appear to increase the prevalence of Y. pseudotuberculosis.     

Addition of new serogroups and improvement of the antigenic designs ofYersinia pseudotuberculosis

Eight strains ofYersinia pseudotuberculosis, untypable for any established serogroups, were studied. They were isolated from humans (three), dogs (two), cat (one), and rats (two), and the general characteristics of the strains were identical with those ofY. pseudotuberculosis. On the basis of results of O-agglutinin absorption tests, these strains were classified into three new serogroups. The new serogroups proposed were serogroups IIC, VII, and VIII, and a new O-antigenic scheme ofY. pseudotuberculosis was designed.