Rapid genotyping assays for the identification and differentiation of Yersinia ruckeri biotype 2 strains

Aims: To establish PCR‐based assays for the rapid identification and differentiation of each of four known biotype 2 (BT2) phenotype‐causing alleles in Yersinia ruckeri strains currently circulating in Europe and the United States. Methods and Results: Novel assays were developed relying on detection of mutant allele‐specific changes in restriction enzyme cleavage sites within targeted PCR products. The developed assays were validated against isolates previously genotyped by DNA sequencing. Conclusions: The described methods were specific, rapid and simple to perform and interpret. Significance and Impact of the Study: The developed genotyping assays provide a valuable tool for identification and differentiation of specific BT2 strains of Y. ruckeri. These assays will be critical for the design and validation of new vaccines or other measures meant to control BT2 strains.     

Crystal structure of ferric-yersiniabactin, a virulence factor of Yersinia pestis

Yersiniabactin (Ybt), the siderophore produced by Yersinia pestis, has been crystallized successfully in the ferric complex form and the crystal structure has been determined. The crystals are orthorhombic with a space group of P2(1)2(1)2(1) and four distinct molecules per unit cell with cell dimensions of a=11.3271(+/-0.0003)A, b=22.3556(+/-0.0006)A, and c=39.8991(+/-0.0011)A. The crystal structure of ferric Ybt shows that the ferric ion is coordinated as a 1:1 complex by three nitrogen electron pairs and three negatively charged oxygen atoms with a distorted octahedral coordination. The molecule displays a Delta absolute configuration with chiral centers at N2, C9, C10, C12, C13, and C19 in R, R, R, R, S, S configurations, respectively. Few of the crystal structures of siderophores have been solved, and those which have been are of simple hydroxamate and catechol types such as ferrioxamine B and agrobactin. To our knowledge this is the first report of the ferric crystal structure of 5-member heterocycle siderophore.     

The yctCBA operon of Yersinia ruckeri, involved in in vivo citrate uptake, is not required for virulence

A three-gene operon, named yctCBA (Yersinia citrate transporter), induced by citrate and repressed by glucose was identified from a previously selected in vivo-induced (ivi) clone in the fish pathogen Yersinia ruckeri. Interestingly, despite being an ivi clone, the drastic growth reduction of the yctC mutant in the presence of citrate, and the relatively high content of this compound in rainbow trout serum, the operon was not required for virulence.     

Analysis of the gyrA gene of clinical Yersinia ruckeri isolates with reduced susceptibility to quinolones

Antimicrobial susceptibility of seven clinical strains of Yersinia ruckeri representative of those isolated between 1994 and 2002 from a fish farm with endemic enteric redmouth disease was studied. All isolates displayed indistinguishable pulsed-field gel electrophoresis restriction patterns, indicating that they represented a single strain. However, considering both inhibition zone diameters (IZD) and MICs, the isolates recovered in 2001-2002 formed a separate cluster with lower levels of susceptibility to all the quinolones tested, especially nalidixic acid (NA) and oxolinic acid (OA), compared with the isolates recovered between 1994 and 1998. Analysis of the PCR product of the quinolone resistance-determining region of the gyrA gene from clinical isolates of Y. ruckeri with reduced susceptibility to OA and NA revealed a single amino acid substitution, Ser-83 to Arg-83 (Escherichia coli numbering). Identical substitution was observed in induced OA-resistant mutant strains, which displayed IZD and MICs of quinolones similar to those of the clinical isolates of Y. ruckeri with reduced susceptibility to these antimicrobial agents. These data indicate in that for Y. ruckeri, the substitution of Ser by Arg at position 83 of the gyrA gene is associated with reduced susceptibility to quinolones.     

Investigation of virulence genes in clinical isolates of Yersinia enterocolitica

In this study, we aimed to investigate the distribution of virulence genes in clinical isolates of pathogenic Yersinia enterocolitica. Two thousand six hundred stool samples were collected from 2600 patients with diarrhea, and were tested using the culture method and real-time PCR. Then, all isolates of pathogenic Y. enterocolitica cultured from the culture method were examined for virulence genes (inv, ail, ystA, ystB, ystC, yadA, virF) by PCR and for the presence of plasmid by four phenotypic tests. As a result, 160 pathogenic strains were successfully detected by the culture method, including bio/serotype 1A/unknown (4), 1B/unknown (8), 2/O:9 (39), 2/unknown (7), 3/O:3 (22), 3/unknown (6), 4/O:3 (55), 4/unknown (10) and 5/unknown (9). The positive rate of virulence genes tested in 160 isolates was inv (100%), ail (94%), ystA (93%), ystB (7.5%), ystC (5%), yadA (89%) and virF (82%) while the phenotypic test included autoagglutination (87%), binding of crystal violet (89%), calcium-dependent growth (74%) and Congo red absorption (78%), respectively. Finally, we found that not all pathogenic Y. enterocolitica necessarily carry all traditional virulence genes in both chromosomes and plasmids to cause illness. Perhaps, some of them, lacking some traditional virulence genes, contain other unknown virulence markers that interact with each other and play an important role in the diverse pathogenesis of pathogenic Y. enterocolitica.     

PCR detection of virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and investigation of virulence gene distribution

PCR-based assays were developed for the detection of plasmid- and chromosome-borne virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis, to investigate the distribution of these genes in isolates from various sources. The results of PCR genotyping, based on 5 virulence-associated genes of 140 strains of Y. enterocolitica, were compared to phenotypic tests, such as biotyping and serotyping, and to virulence plasmid-associated properties such as calcium-dependent growth at 37 degrees C and Congo red uptake. The specificity of the PCR results was validated by hybridization. Genotyping data correlated well with biotype data, and most biotypes resulted in (nearly) homogeneous genotypes for the chromosomal virulence genes (ystA, ystB, and ail); however, plasmid-borne genes (yadA and virF) were detected with variable efficiency, due to heterogeneity within the bacterial population for the presence of the virulence plasmid. Of the virulence genes, only ystB was present in biotype 1A; however, within this biotype, pathogenic and apathogenic isolates could not be distinguished based on the detection of virulence genes. Forty Y. pseudotuberculosis isolates were tested by PCR for the presence of inv, yadA, and lcrF. All isolates were inv positive, and 88% of the isolates contained the virulence plasmid genes yadA and lcrF. In conclusion, this study shows that genotyping of Yersinia spp., based on both chromosome- and plasmid-borne virulence genes, is feasible and informative and can provide a rapid and reliable genotypic characterization of field isolates.     

Construction of a virulent, green fluorescent protein-tagged Yersinia ruckeri and detection in trout tissues after intraperitoneal and immersion challenge

A green fluorescent protein (GFP) expressing strain of Yersinia ruckeri was created by the transposition of a Tn10-GFP-kan cassette into the genome of Y. ruckeri Strain YRNC10. The derivative, YRNC10-gfp, was highly GFP fluorescent, retained the gfp-km marker in the absence of kanamycin selection, and exhibited in vitro growth kinetics similar to those of the wild type strain. YRNC10-gfp colonized and caused mortality in immersion and intraperitoneally challenged rainbow trout Oncorhynchus mykiss, although it was modestly attenuated compared to the wild type strain. The distribution and location of YRNC10-gfp in infected fish was visualized by epifluorescence microscopy. Abundant extracellular bacteria and a small number of intracellular bacteria were observed in the kidney, spleen and peripheral blood. To determine the percentage of trout cells containing intracellular bacteria, GFP fluorescence was measured by flow cytometry. A small population of GFP positive leukocytes was detected in peripheral blood (1.6%), spleen (1.1%) and anterior kidney (0.4%) tissues. In summary, this is the first report of the construction of a virulent, GFP-tagged Y. ruckeri, which may be a useful model for detecting and imaging the interactions between an aquatic pathogen and the natural salmonid host.     

Highly sensitive detection and quantification of the pathogen Yersinia ruckeri in fish tissues by using real-time PCR

Yersinia ruckeri is the causative agent of enteric redmouth diseases (ERM) and one of the major bacterial pathogens causing losses in salmonid aquaculture. Since recent ERM vaccine breakdowns have been described mostly attributed to emergence of Y. ruckeri biotype 2 strains, rapid, reproducible, and sensitive methods for detection are needed. In this study, a real-time polymerase chain reaction (PCR) primer/probe set based on recombination protein A (recA) gene was designed and optimized to improve the detection of Y. ruckeri. The primer/probe set proved to have a 100 % analytical specificity and a sensitivity of 1.8 ag μl^?1, equivalent to 1.7 colony-forming units (CFU)?ml^?1, for purified DNA, 3.4 CFU g^?1 for seeded liver, kidney, and spleen tissues, and 0.34 CFU/100 μl^?1 for seeded blood, respectively. The assay was highly reproducible with low variation coefficient values for intra- and inter-run experiments (2.9 % and 9.5 %, respectively). Following optimization, the assay was used to detect changes in the bacterial load during experimental infection. Rainbow trout (Onchorhynchus mykiss) were exposed to two strains of Y. ruckeri (biotype 1 and biotype 2) by intraperitoneal inoculation. Internal organs (liver, kidney, spleen) and blood were biopsied from dead fish daily for 15 days to quantify copies of pathogen DNA per gram of tissue. The findings showed the efficacy of this real-time PCR assay to quantify Y. ruckeri cells in the fish tissues and also confirmed this assay as a non-lethal method for the detection of this pathogen in blood samples.     

Simultaneous detection of Aeromonas salmonicida,Flavobacterium psychrophilum,and Yersinia ruckeri,three major fish pathogens,by multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.     

The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor

The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.     

Pathological activities of Yersinia ruckeri, the Enteric Redmouth (ERM) bacterium

Abstract The adherence and invasive capacities as well as the pathobiological activities exhibited by Yersinia ruckeri were examined. Although adhesive ability was dependent on the cell-line employed, all the strains showed moderate adhesion and invasiveness in the salmon cell-line CHSE-214. With regard to the extracellular products (ECP) all of them were strongly toxic for fish with LD₅₀ ranging from 2 to 9.12 μg protein per g fish. In addition, all the ECP samples showed caseinase, gelatinase, amylase, lipase and phospholipase activities, hydrolysed esculin and displayed hemolytic activities for trout, salmon, sheep and human erythrocytes. Heat treatment (100°C for 10 min) caused the loss of all these biological activities except the hydrolysis of gelatin. On the other hand, SDS-PAGE analysis of the LPS and protein components of the ECP revealed variations among strains depending on the serotype. The lack of lethal effects of the LPS present in the ECP was also demonstrated.     

Purification and Characterization of an Extracellular Protease from the Fish Pathogen Yersinia ruckeri and Effect of Culture Conditions on Production

A novel protease, hydrolyzing azocasein, was identified, purified, and characterized from the culture supernatant of the fish pathogen Yersinia ruckeri. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity was detected in peptone but not in Casamino Acid medium. Its synthesis appeared to be under catabolite repression and ammonium control. The protease was purified in a simple two-step procedure involving ammonium sulfate precipitation and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein indicated an estimated molecular mass of 47 kDa. The protease had characteristics of a cold-adapted protein, i.e., it was more active in the range of 25 to 42°C and had an optimum activity at 37°C. The activation energy for the hydrolysis of azocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42°C. The enzyme had an optimum pH of around 8. Characterization of the protease showed that it required certain cations such as Mg²⁺ or Ca²⁺ for maximal activity and was inhibited by EDTA, 1,10-phenanthroline, and EGTA but not by phenylmethylsulfonyl fluoride. Two N-methyl-N-nitro-N-nitrosoguanidine mutants were isolated and analyzed; one did not show caseinolytic activity and lacked the 47-kDa protein, while the other was hyperproteolytic and produced increased amounts of the 47-kDa protein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal anti-47-kDa-protease serum, and zymogram analyses showed that protease activity was present in 8 of 14 strains tested and that two Y. ruckeri groups could be established based on the presence or absence of the 47-kDa protease.     

Real-time characterization of virulence factor expression in Yersinia pestis using a GFP reporter system

A real-time reporter system was developed to monitor the thermal induction of virulence factors in Yersinia pestis, the etiological agent of plague. The reporter system consists of a plasmid in Y. pestis in which the expression of green fluorescent protein (GFP) is under the control of the promoters for six virulence factors, yopE, sycE, yopK, yopT, yscN, and lcrE yopN, which are all components of the Type III secretion virulence mechanism of Y. pestis. Induction of the expression of these genes in vivo was determined by the increase in fluorescence intensity of GFP in real time, in 96-well format. Different basal levels of expression at 26 degrees C were observed for the Y. pestis promoters. Expressed as percentages of the level measured for the lac promoter (positive control), the basal expression levels before temperature shift were: yopE (15%), sycE (15%), yopK (13%), yopT (4%), lcrE (3.3%), and yscN (0.8%). Following the shift in temperature from 26 to 37 degrees C, the rates of expression of these genes increased with the yopE reporter showing the strongest degree of induction. The rates of induction of the other virulence factors after the temperature, expressed as percentages of yopE induction, were: yopK (57%), sycE (9%), yscN (3%), lcrE (3%), and yopT (2%). The thermal induction of each of these promoter fusions was repressed by calcium, and the ratios of the initial rates of thermal induction without calcium supplementation compared to the rate with calcium supplementation were: yopE (11-fold), yscN (7-fold), yopK (6-fold), lcrE (3-fold), yopT (2-fold), and sycE (1-fold). This work demonstrates a novel approach to quantify gene induction and provides a method to rapidly determine the effects of external stimuli on expression of Y. pestis virulence factors in real time, in living cells, as a means to characterize virulence determinants.     

The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function

The iron-repressible outer membrane protein FyuA of Yersinia enterocolitica operates as a receptor with dual function: (i) as a receptor for the Y. pestis bacterlocin pesticin, and (ii) as a receptor for yersiniabactin, a siderophore that is produced by mouse-viruient Y. enterocolitica strains of biogroup IB. Cloning of the FyuA-encoding gene was achieved by mobilization of a genomic cosmid library of the pesticin-sensitive and mouse-virulent Y. enterocolitica O:8 strain WA into the pesticin-reslstant WA fyuA mutant and subsequent in vivo selection of transconjugants for the ability to survive and multiply in mice (phenotype mouse viruience). The reisolated transconjugants which survived in mice for 3d harboured a unique cosmid and phenotypicaity were pesticin sensitive. From this cosmid a 2650 bp SalI-PstI fragment conferring pesticin sensitivity was subcioned. Sequencing of this DNA fragment revealed a single open reading frame of 2022 bp, which encodes a deduced polypeptide of 673 amino acids with a predicted molecular mass of 73 677 Da. Cleavage of a putative signal sequence composed of 22 amino acids should lead to a mature protein of 651 amino acids with a molecular mass of 71 368 Da. The open reading frame is preceded by a sequence which shares homoiogy with the postulated consensus Fur iron-repressor protein-binding site. FyuA shows homology to other iron-regulated TonB-dependent outer membrane proteins with receptor functions (e.g. BtuB, CirA, FepA, lutA, FhuA, FoxA, FcuA). On the basis of multiple alignment of amino acid sequences of FyuA and other TonB-dependent receptors, a phylogenetic tree was constructed, demonstrating that FyuA probably belongs to the citrate subfamily or represents a new subfamily of TonB-dependent receptors. Moreover, by complementation of the WA fyuA mutant by the cioned fyuA gene.     

Biochemical, structural and molecular dynamics analyses of the potential virulence factor RipA from Yersinia pestis

Human diseases are attributed in part to the ability of pathogens to evade the eukaryotic immune systems. A subset of these pathogens has developed mechanisms to survive in human macrophages. Yersinia pestis, the causative agent of the bubonic plague, is a predominately extracellular pathogen with the ability to survive and replicate intracellularly. A previous study has shown that a novel rip (required for intracellular proliferation) operon (ripA, ripB and ripC) is essential for replication and survival of Y. pestis in postactivated macrophages, by playing a role in lowering macrophage-produced nitric oxide (NO) levels. A bioinformatics analysis indicates that the rip operon is conserved among a distally related subset of macrophage-residing pathogens, including Burkholderia and Salmonella species, and suggests that this previously uncharacterized pathway is also required for intracellular survival of these pathogens. The focus of this study is ripA, which encodes for a protein highly homologous to 4-hydroxybutyrate-CoA transferase; however, biochemical analysis suggests that RipA functions as a butyryl-CoA transferase. The 1.9 Å X-ray crystal structure reveals that RipA belongs to the class of Family I CoA transferases and exhibits a unique tetrameric state. Molecular dynamics simulations are consistent with RipA tetramer formation and suggest a possible gating mechanism for CoA binding mediated by Val227. Together, our structural characterization and molecular dynamic simulations offer insights into acyl-CoA specificity within the active site binding pocket, and support biochemical results that RipA is a butyryl-CoA transferase. We hypothesize that the end product of the rip operon is butyrate, a known anti-inflammatory, which has been shown to lower NO levels in macrophages. Thus, the results of this molecular study of Y. pestis RipA provide a structural platform for rational inhibitor design, which may lead to a greater understanding of the role of RipA in this unique virulence pathway.     

Multilocus Electrophoretic Assessment of the Genetic Structure and Diversity of Yersinia ruckeri

Multilocus isoenzyme electrophoresis was used to screen 47 field isolates of Yersinia ruckeri for electrophoretic variation at 15 enzyme loci. Only four electrophoretic types were observed, thus indicating that the genetic structure of Y. ruckeri is clonal. Forty-two isolates were of one electrophoretic type, a reflection of the low amount of genetic diversity extant in this species. Although sorbitol fermentation has been considered to be indicative of a second biotype, no significant gene frequency differences were found between the group of 20 isolates that readily used sorbitol as the sole carbon source and the group of 27 that did not.     

Virulence of Yersinia ruckeri serotype I strains is associated with a heat sensitive factor (HSF) in cell extracts

Cell extracts of Yersinia ruckeri (serotype I) were examined by SDS-PAGE and Western blotting. An unusual band, termed heat-sensitive factor (HSF) was observed in extracts of virulent strains only. It is thought to be lipid in nature; no differences could be detected in the region of the band in protein profiles of virulent and avirulent strains. When trout were infected either by intraperitoneal injection or bath immersion, mortalities occurred only with HSF+ strains. The HSF appears to be an important virulence determinant of Y. ruckeri.     

鼠疫菌毒力调控研究

鼠疫菌通过一系列转录调控子(如CRP、PhoP、RovA和Fur)控制着一些关键毒力因子(如Pla、强毒力岛、III型分泌系统等)的基因表达。鼠疫菌可感应宿主体内信号刺激,紧密调控毒力因子的表达。在这个紧密调控过程中,调控子、毒力相关基因构成了一个动态网络。鼠疫菌在从假结核菌祖先演化的进程中,基因表达调控网络的重塑在鼠疫菌毒力进化过程中发挥着不可取代的作用。     

Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aims: The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. Methods and Results: The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole‐cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron‐regulated outer membrane proteins (IROMP) of c. 90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. Conclusions: The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. Significance and Impact of the Study: There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.