Direct detection of Yersinia pestis from the infected animal specimens by a fiber optic biosensor

The FOB-3, anew type fiber optic biosensor, is designed to rapidly detect a variety of biological agents or analytes with better stability, sensitivity and specificity. In order to detect Y. Pestis, a sandwich immunoassay was developed by using the purified antibody against antigen FI immobilized on polystyrene probes as the capture antibody and the monoclonal antibody-Cy5 conjugate as the detector. After a series of optimization for the stability, sensitivity and specificity of the FOB-3, 50-1000 ng/ml of antigen FI and 6 x 10(1)-6 x 10(7) CFU/ml Y. pestis could be detected constantly in about 20 min, and Y pestis could be detected specifically from Y. pseudotuberculosis, Y. enterocolitica, B. anthracis and E. coli. Then, 39 blind samples, including 27 tissues of mice infected with Y pestis and 12 tissues of healthy mice as negative control, were detected with the FOB-3. 92.6% infected tissues were identified from the tissues of healthy mice and the tissues containing more than 100 CFU/ml bacteria could be detected by the biosensor. The results demonstrated the feasibility of the FOB-3 as an effective method to detect Y. pestis rapidly and directly from the infected animal specimens with the advantage of portability, simple-operation as well as high sensitivity and specificity. (c) 2006 Elsevier B.V. All rights reserved.

Yersinia high pathogenicity island genes modify the Escherichia coli primary metabolome independently of siderophore production

Bacterial siderophores may enhance pathogenicity by scavenging iron, but their expression has been proposed to exert a substantial metabolic cost. Here we describe a combined metabolomic-genetic approach to determine how mutations affecting the virulence-associated siderophore yersiniabactin affect the Escherichia coli primary metabolome. Contrary to expectations, we did not find yersiniabactin biosynthesis to correspond to consistent metabolomic shifts. Instead, we found that targeted deletion of ybtU or ybtA, dissimilar genes with similar roles in regulating yersiniabactin expression, were associated with a specific shift in arginine pathway metabolites during growth in minimal media. This interaction was associated with high arginine levels in the model uropathogen Escherichia coli UTI89 compared to its ybtU and ybtA mutants and the K12 strain MG1655, which lacks yersiniabactin-associated genes. Because arginine is not a direct yersiniabactin biosynthetic substrate, these findings show that virulence-associated secondary metabolite systems may shape bacterial primary metabolism independently of substrate consumption

Molecular Details of Serum Resistance of Yersinia enterocolitica

In complement activation, Factor H (FH) and C4b-binding protein (C4bp) are the key regulators that prevent the complement cascade from attacking host tissues. Some bacteria may bind and deposit these regulators on their own surfaces and thus provide themselves with an efficient means to avoid complement activation. In consequence, bacteria resist complement-mediated lysis and opsonin-dependent phagocytosis. This study has demonstrated that Y. enterocolitica, similar to many other pathogens, recruits both FH and C4bp to its surface to ensure protection against the complement-mediated killing. YadA and Ail, the most crucial serum resistance factors of Y.enterocolitica, mediate the binding of FH and C4bp. FH - YadA interaction involves multiple higher structural motifs on the YadA stalk and the short consensus repeats (SCRs) of the entire polypeptide chain of FH. The Ail binding site on FH has been located to SCRs 6 and 7. The binding site for FH on Ail, however, remains undetermined. Both YadA- and Ail-bound regulators display full cofactor activity for FI-mediated cleavage of C3b/C4b. FH/C4bp-binding characteristics do, however, differ between YadA and Ail. In addition, Ail captures the regulators only in the absence of blocking lipopolysaccharide O-antigen and outer core, whereas YadA binds FH/C4bp independent of the presence of other surface factors Independent of mode of binding, however, YadA and Ail provide Y. enterocolitica a means to avoid complement-mediated lysis, enhancing chances for the bacteria to survive in the host during various phases of infection.

Functional studies of purified transmembrane proteases, omptins, of Yersinia pestis and Salmonella enterica.

Surface proteolysis is important in migration of cells through tissue barriers. In the case of prokaryotes, surface proteolysis has been associated with invasiveness of pathogenic bacteria from the primary infection site into circulation and secondary infection sites in the host. This study addressed surface proteases of two important bacterial pathogens, Yersinia pestis which is the causative agent of the lethal systemic zoonosis, plague, and Salmonella enterica serovar Typhimurium which is an oral-faecal pathogen that annually causes millions of cases of gastoenteritis that may develop to septicaemia. Both bacterial species express an ortholog of the omptin family of transmembrane β-barrel, outer membrane proteases/adhesins. This thesis work addressed the functions of isolated plasminogen activator Pla of Y. pestis and the PgtE omptin of S. enterica. Pla and PgtE were isolated as His6-fusion proteins in denaturing conditions from recombinant Escherichia coli and activated by adding lipopolysaccharide (LPS). The structural features in LPS that enhance plasminogen activation by His6-Pla were determined, and it was found that the lack of O-specifi c chain, the presence of outer core oligosaccharide, the presence of phosphates in lipid A, as well as a low level of acylation in lipid A influence the enhancement of Pla activity by LPS. A conserved lipid A phosphate binding motif in Pla and PgtE was found important for the enhancement of enzymatic activity by LPS. The results help to explain the biological signifi cance of the genetic loss of the O-specifi c chain biosynthesis in Y. pestis as well as the variations in LPS structure upon entry of Y. pestis into the human host. Expression of Pla in Y. pestis is associated with adhesiveness to lamin of basement membranes. Here, isolated and LPS-activated His6-Pla was coated onto fluorescent microparticles. The coating conferred specifi c adhesiveness of the particles to laminin and reconstituted basement membrane, thus confi rming the intrinsic adhesive characteristics of the Pla protein. The adhesiveness is thought to direct plasmin proteolysis at tissue barriers, thus increasing tissue damage and bacterial spread. Gelatinase activity has not been previously reported in enteric bacteria. Expression of PgtE in S. enterica was associated with cleavage of porcine skin gelatin, denaturated human type I collagen, as well as DQ-gelatin. Purifi ed His6-PgtE also degraded porcine skin gelatin and human type I gelatin but did not react with DQ-gelatin, indicating that minor differences are seen in proteolysis by isolated and cell-bound PgtE. Pla was less effective in gelatin degradation. The novel gelatinase activity in S. enterica is likely to enhance bacterial dissemination during infection.

Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor

Up-converting phosphor technology (UPT)-based lateral-flow immunoassay has been developed for quantitative detection of Yersinia pestis rapidly and specifically. In this assay, 400 nm up-converting phosphor particles were used as the reporter. A sandwich immumoassay was employed by using a polyclonal antibody against F1 antigen of Y. pestis immobilized on the nitrocellulose membrane and the same antibody conjugated to the UPT particles. The signal detection of the strips was performed by the UPT-based biosensor that could provide a 980 nm IR laser to excite the phosphor particles, then collect the visible luminescence emitted by the UPT particles and finally convert it to the voltage as a signal. V-T and V-c stand for the multiplied voltage units for the test and the control line, respectively, and the ratio V-T/V-C is directly proportional to the number of Y pestis in a sample. We observed a good linearity between the ratio and log CFU/ml of Y pestis above the detection limit, which was approximately 10(4) CFU/mI. The precision of the intra- and inter-assay was below 15% (coefficient of variation, CV). Cross-reactivity with related Gram-negative enteric bacteria was not found. The UPT-LF immunoassay system presented here takes less than 30 min to perform from the sample treatment to the data analysis. The current paper includes only preliminary data concerning the biomedical aspects of the assay, but is more concentrated on the technical details of establishing a rapid manual assay using a state-of-the-art label chemistry. (c) 2006 Elsevier B.V. All rights reserved.

Estudo das características fenotípicas e genotípicas de amostras de Corynebacterium pseudotuberculosis

Uma vez que C. pseudotuberculosis é o agente etiológico de processos infecciosos em animais caprinos e ovinos e que também pode ser isolado de processos infecciosos em seres humanos as investigações direcionadas para a espécie em questão são necessárias, visto que a escassez de dados epidemiológicos e de conhecimento relativo ao comportamento do microrganismo em hospedeiros animais e humanos em nosso país dificulta o diagnóstico laboratorial da espécie, à semelhança do observado com outra espécie de transmissão zoonótica, o C. ulcerans. Uma preocupação adicional é o fato da espécie em questão também ser capaz de albergar bacteriófagos codificadores da toxina diftérica, representando uma ameaça à circulação dos bacteriófagos. Assim, o presente estudo tem como objetivo geral analisar as características fenotípicas e genotípicas de amostras de C. pseudotuberculosis. Neste sentido, foram propostos os seguintes objetivos: Avaliar as características bioquímicas das amostras através de testes bioquímicos convencionais; avaliar as características bioquímicas das amostras utilizando o sistema semi-automatizado API Coryne; diferenciar amostras de C. pseudotuberculosis de C. ulcerans utilizando a técnica de PCR multiplex; pesquisar a presença de gene tox. Os resultados demonstraram que amostras de C. diphtheriae, C. ulcerans e C. pseudotuberculosis podem ser caracterizadas por métodos bioquímicos convencionais e por taxonomia numérica (API Coryne System). C. ulcerans e C. pseudotuberculosis, com potencial de circulação zoonótica, da mesma forma que C. diphtheriae são capazes de albergar o gene da toxina diftérica. A reação m-PCR foi capaz de discernir as amostras de C. diphtheriae, C. ulcerans e C. pseudotuberculosis e ainda definir o potencial das amostras em produzir a toxina diftérica. Os dados enfatizam a necessidade da técnica multiplex PCR para o diagnostico e para o controle de espécies associadas a quadros de difteria em populações humana.

鲢、鳙鱼一种新的传染病——Yersinia ruckeri,一种新的鲢、鳙鱼病原菌

<正> 历来以少病易养、产量高著称的鲢、鳙鱼近年来却爆发了一种新的流行病,而且流行面很广,从南方各省至京津地区均有发生。罹病鱼一周之内便会出现大批死亡,甚至可以死光。 据我们观察该鱼病以体表发炎充血为特征,特别在体表两侧,以及腹鳍下和尾柄处、眼眶、鳃盖、头、嘴等部位出现,但每条病鱼症状并不完全相同。有的还可见突眼和鳃丝贫血。内部

Análise da variabilidade intra-espécie dos genes pld e rpoB de Corynebacterium pseudotuberculosis por meio de marcadores PCR-RFLP.

Corynebacterium pseudotuberculosis é o agente etiológico da linfadenite caseosa em ovinos e caprinos, uma doença infectocontagiosa crônica caracterizada por abscessos em linfonodos superficiais ou internos. Os animais acometidos podem apresentar lesões internas ou externas, o que representa perdas econômicas graves na caprinovinocultura, como o comprometimento da produção de lã em ovinos, deficiência reprodutiva, condenação da carne ou morte do animal. Uma vez estabelecida a infecção no rebanho, torna-se difícil sua erradicação. Com o objetivo de avaliar a variabilidade genética intra-espécie em C. pseudotuberculosis, 31 isolados provenientes de seis municípios do Estado de Pernambuco, sendo 23 de caprinos e oito de ovinos, tiveram os seus DNAs genômicos extraídos e analisados por reação em cadeia da polimerase e análise de polimorfismos por tamanho de fragmentos de restrição (PCR-RFLP). Os locis escolhidos foram os genes pld e rpoB, cujas seqüências dos oligonucleotídeos amplificaram fragmentos de 924 e 447 pares de bases (pb), respectivamente. Os produtos da amplificação foram digeridos com as enzimas Pst I e Msp I (gene pld) e HpyCh4 IV e Msp I (gene rpoB). Os fragmentos de restrição foram corridos em gel de poliacrilamida a 15%, os quais foram corados com brometo de etídeo. Para o gene pld, digerido com a enzima Pst I, foram obtidos fragmentos de 566, 195, 91 e 72 pb; e com a enzima Msp I, fragmentos de 395, 369, 108 e 52 pb. O gene rpoB digerido com a enzima HpyCh4 IV apresentou fragmentos de 235, 126 e 86 pb; e com a enzima Msp I apresentou fragmentos de 222, 93, 78 e 54 pb. O padrão de bandas obtido para os 31 isolados de C. pseudotuberculosis, analisados neste estudo, foi monomórfico, não havendo, portanto, polimorfismo entre os diferentes isolados, independentes da espécie hospedeira ou da área geográfica estudada, o que corrobora com o padrão homogêneo da infecção para a região.

Epidemiology and comparative analysis of Yersinia in Ireland

Yersiniosis is an acute or chronic enteric zoonosis caused by enteropathogenic Yersinia species. Although yersiniosis is predominantly associated with gastroenteric forms of infection, extraintestinal forms are often reported from the elderly or patients with predisposing factors. Yersiniosis is often reported in countries with cold and mild climates (Northern and Central Europe, New Zealand and North of Russian Federation). The Irish Health Protection Surveillance Centre (HPSC) currently records only 3-7 notified cases of yersiniosis per year. At the same time pathogenic Yersinia enterocolitica is recovered from pigs (main source of pathogenic Y. enterocolitica) at the levels similar to that observed in Yersinia endemic countries. Introduction of Yersinia selective culture procedures may increase Yersinia isolation rates. To establish whether the small number of notifications of human disease was an underestimate due to lack of specific selective culture for Yersinia we carried out a prospective culture study of faecal samples from outpatients with diarrhoea, with additional culture of appendix and throat swabs. Higher levels of anti-Yersinia seroprevalence than yersiniosis notification rates in endemic countries suggests that most yersiniosis cases are unrecognised by culture. Subsequently, in addition to a prospective culture study of clinical specimens, we carried out serological screening of Irish blood donors and environmental screening of human sewage. Pathogenic Yersinia strains were not isolated from 1,189 faeces samples, nor from 297 throat swabs, or 23 appendix swabs. This suggested that current low notification rates in Ireland are not due to the lack of specific Yersinia culture procedures. Molecular screening detected a wider variety of Y. enterocolitica-specific targets in pig slurry than in human sewage. A serological survey for antibodies against Yersinia YOP (Yersinia Outer Proteins) proteins in Irish blood donors found antibodies in 25%, with an age-related trend to increased seropositivity, compatible with the hypothesis that yersiniosis may have been more prevalent in Ireland in the recent past. Y. enterocolitica is a heterogeneous group of microorganisms that comprises strains with different degree of pathogenicity. Although non-pathogenic Y. enterocolitica lack conventional virulence factors, these strains can be isolated from patients with diarrhoea. Insecticidal Toxin Complex (ITC) and Cytolethal Distending Toxins can potentially contribute to the virulence of non-pathogenic Y. enterocolitica in the absence of other virulence factors. We compared distribution of ITC and CDT loci among pathogenic and non-pathogenic Y. enterocolitica. Additionally, to demonstrate potential pathogenicity of non-pathogenic Y. enterocolitica we compared their virulence towards Galleria mellonella larvae (a non-mammalian model of human bacterial infections) with the virulence of highly and mildly pathogenic Y. enterocolitica strains. Surprisingly, virulence of pathogenic and non-pathogenic Y. enterocolitica in Galleria mellonella larvae observed at 37°C did not correlate with their pathogenic potential towards humans. Comparative phylogenomic analysis detects predicted coding sequences (CDSs) that define host-pathogen interactions and hence providing insights into molecular evolution of bacterial virulence. Comparative phylogenomic analysis of microarray data generated in Y. enterocolitica strains isolated in the Great Britain from humans with diarrhoea and domestic animals revealed high genetic heterogeneity of these species. Because of the extensive human, animal and food exchanges between the UK and Ireland the objective of this study was to gain further insight into genetic heterogeneity and relationships among clinical and non-clinical Y. enterocolitica strains of various pathogenic potential isolated in Ireland and Great Britain. No evidence of direct transfer of strains between the two countries was found.

Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2)-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.

Molecular basis of Yersinia enterocolitica temperature-dependent resistance to antimicrobial peptides

Antimicrobial peptides (APs) belong to the arsenal of weapons of the innate immune system against infections. In the case of gram-negative bacteria, APs interact with the anionic lipid A moiety of the lipopolysaccharide (LPS). In yersiniae most virulence factors are temperature regulated. Studies from our laboratory demonstrated that Yersinia enterocolitica is more susceptible to polymyxin B, a model AP, when grown at 37°C than at 22°C (J. A. Bengoechea, R. Díaz, and I. Moriyón, Infect. Immun. 64:4891-4899, 1996), and here we have extended this observation to other APs, not structurally related to polymyxin B. Mechanistically, we demonstrate that the lipid A modifications with aminoarabinose and palmitate are downregulated at 37°C and that they contribute to AP resistance together with the LPS O-polysaccharide. Bacterial loads of lipid A mutants in Peyer's patches, liver, and spleen of orogastrically infected mice were lower than those of the wild-type strain at 3 and 7 days postinfection. PhoPQ and PmrAB two-component systems govern the expression of the loci required to modify lipid A with aminoarabinose and palmitate, and their expressions are also temperature regulated. Our findings support the notion that the temperature-dependent regulation of loci controlling lipid A modifications could be explained by H-NS-dependent negative regulation alleviated by RovA. In turn, our data also demonstrate that PhoPQ and PmrAB regulate positively the expression of rovA, the effect of PhoPQ being more important. However, rovA expression reached wild-type levels in the phoPQ pmrAB mutant background, hence indicating the existence of an unknown regulatory network controlling rovA expression in this background.

Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core

Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.