Bioluminescent tracing of a Yersinia pestis pCD1+-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice

Yersinia pestis has evolved from Yersinia pseudotuberculosis serotype O:1b. A typical Y. pestis contains three plasmids: pCD1, pMT1 and pPCP1. However, some isolates only harbor pCD1 (pCD1⁺-mutant). Y. pestis and Y. pseudotuberculosis share a common plasmid (pCD1 or pYV), but little is known about whether Y. pseudotuberculosis exhibited plague-inducing potential before it was evolved into Y. pestis. Here, the luxCDABE::Tn5::kan was integrated into the chromosome of the pCD1⁺-mutant, Y. pseudotuberculosis or Escherichia coli K12 to construct stable bioluminescent strains for investigation of their dissemination in mice by bioluminescence imaging technology. After subcutaneous infection, the pCD1⁺-mutant entered the lymph nodes, followed by the liver and spleen, and, subsequently, the lungs, causing pathological changes in these organs. Y. pseudotuberculosis entered the lymph nodes, but not the liver, spleen and lungs. It also resided in the lymph nodes for several days, but did not cause lymphadenitis or pathological lesions. By contrast, E. coli K12-lux was not isolatable from mouse lymph nodes, liver, spleen and lungs. These results indicate that the pCD1⁺-mutant can cause typical bubonic and pneumonic plague-like diseases, and Y. pestis has inherited lymphoid tissue tropism from its ancestor rather than acquiring these properties independently.     

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°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.     

The Yersiniabactin Biosynthetic Gene Cluster of Yersinia enterocolitica: Organization and Siderophore-Dependent Regulation

The ability to synthesize and uptake the Yersinia siderophore yersiniabactin is a hallmark of the highly pathogenic, mouse-lethal species Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica 1B. We have identified four genes, irp1, irp3, irp4, and irp5, on a 13-kb chromosomal DNA fragment of Y. enterocolitica O8, WA-314. These genes constitute the yersiniabactin biosynthetic gene cluster together with the previously defined irp2. The irp1 gene consists of 9,486 bp capable of encoding a 3,161-amino-acid high-molecular-weight protein 1 (HMWP1) polypeptide with a predicted mass of 384.6 kDa. The first 3,000 bp of irp1 show similarity to the corresponding regions of the polyketide synthase genes of Bacillus subtilis and Streptomyces antibioticus. The remaining part of irp1 is most similar to irp2, encoding HMWP2, which might be the reason for immunological cross-reactivity of the two polypeptides. Irp4 was found to have 41.7% similarity to thioesterase-like protein of the anguibactin biosynthetic genes of Vibrio anguillarum. Irp5 shows 41% similarity to EntE, the 2,3-dihydroxybenzoic acid-activating enzyme utilized in enterobactin synthesis of Escherichia coli. Irp4 and Irp5 are nearly identical to YbtT and YbtE, recently identified in Y. pestis. irp3 has no similarity to any known gene. Inactivation of either irp1 or irp2 abrogates yersiniabactin synthesis. Mutations in irp1 or fyuA (encoding yersiniabactin/pesticin receptor) result in downregulation of irp2 that can be upregulated by the addition of yersiniabactin. A FyuA-green fluorescent protein translational fusion was downregulated in an irp1 mutant. Upregulation was achieved by addition of yersiniabactin but not desferal, pesticin, or pyochelin, which indicates high specificity of the FyuA receptor and autoregulation of genes involved in synthesis and uptake of yersiniabactin.     

Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10² CFU/ml in pure cultures and ≥10³ CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Study of the incompatibility and replication of the 70-kb virulence plasmid of Yersinia

The 70-kb virulence plasmid, vir, from four Yersinia enterocolitica and one Y. pseudotuberculosis strains are incompatible with IncFI plasmids FLac and R386 while they are compatible with plasmids representing nine other incompatibility groups. Hybridization experiments carried out on one of these virulence plasmids showed that it contains the F incompatibility determinant D, incD. This determinant was cloned onto pACYC184 and the recombinant clone expressed incompatibility with FLac. We conclude that the incompatibility observed between F or R386 and the 70-kb virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis is mediated by incD. Replication genes (rep) from the same plasmid were cloned independently in Escherichia coli. Rep and incD map on two different BamHI fragments. Surprisingly, the replicon isolated is not sensitive to inc D incompatibility. Apart from incD, vir and F share extremely little homology. In particular, there is no evidence for the presence of an F-like transfer operon on vir.     

Genome Sequence of the Deep-Rooted Yersinia pestis Strain Angola Reveals New Insights into the Evolution and Pangenome of the Plague Bacterium

To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis, we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis. Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis, we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies.Yersinia pestis, the causative agent of plague, is a nonmotile Gram-negative bacterial pathogen. The genus Yersinia comprises two other pathogens that cause worldwide infections in humans and animals: Y. pseudotuberculosis and Y. enterocolitica (11, 12, 22, 61, 71). Despite their genetic relationship, these species differ radically in their pathogenicity and transmission. Plague is primarily a disease of wild rodents that is transmitted to other mammals through flea bites. In humans it produces the bubonic form of plague. Y. pestis also can be transmitted from human to human by aerosol, especially during pandemics, causing primarily pneumonic plague. Evolutionarily, it is estimated that Y. pestis diverged from the enteric pathogen Y. pseudotuberculosis within the last 20,000 years, while Y. pseudotuberculosis and Y. enterocolitica lineages separated 0.4 to 1.9 million years ago (2). Y. pestis inhabits a distinct ecological niche, and its transmission is anchored in its unique plasmid inventory: the murine toxin (pMT) and plasminogen activator (pPCP) plasmids. In addition, Y. pestis harbors the low-calcium-response plasmid pCD, which it inherited from its closest relative, Y. pseudotuberculosis (pYV) (12), and it also is found in the more distantly related Y. enterocolitica (71). So-called cryptic plasmids have been described in the literature as part of the Y. pestis mobilome (71), but no sequence data are available to decipher the nature and impact of such plasmids in the epidemiology and pathogenicity of Y. pestis (14). Y. pestis isolates have been historically grouped into the biovars Antiqua (ANT), Medievalis (MED), and Orientalis (ORI), based on metabolic properties such as nitrate reduction and fermentation patterns (72). However, we will use the population-based nomenclature for Y. pestis introduced by Achtman et al. (1), as we believe it better reflects the true evolutionary relationship. Due to its young evolutionary age, only a few genetic polymorphisms have been identified within the Y. pestis genomes sequenced to date (1). Here, we report the comparative analysis of the virulent Y. pestis strain Angola, a representative of one of the most ancient Y. pestis lineages thus far sequenced. We studied adaptive microevolutionary traits Y. pestis has acquired and predicted the global Yersinia pangenome. By comparing the genomes of the three human pathogenic Yersinia species (12, 22), we investigated the global- and species-specific gene reservoir, the genome dynamics, and the degree of genetic diversity that is found within these species. Our genotypic and phenotypic analyses, as well as the refined single-nucleotide polymorphism (SNP)-based phylogeny of Y. pestis, indicate that Angola is a deep-rooted isolate with unique genome characteristics intermediate between modern Y. pestis isolates and Y. pseudotuberculosis.     

Bioluminescent tracking of colonization and clearance dynamics of plasmid-deficient Yersinia pestis strains in a mouse model of septicemic plague

Yersinia pestis 201 contains 4 plasmids pPCP1, pMT1, pCD1 and pCRY, but little is known about the effects of these plasmids on the dissemination of Y. pestis. We developed a plasmid-based luxCDABE bioreporter in Y. pestis 201, Y. pestis 201-pCD1⁺, Y. pestis 201-pMT1⁺, Y. pestis 201-pPCP1⁺, Y. pestis 201-pCRY⁺, Y. pestis 201-p⁻ and Yersinia pseudotuberculosis Pa36060 strains, and investigated their dissemination by bioluminescence imaging during primary septicemic plague in a mouse model. These strains mainly colonized the livers and spleens shortly after intravenous inoculation. Y. pestis 201-pMT1⁺ appeared to have a stronger ability to survive in the livers, spleens and blood, and to be more virulent than other plasmid-deficient strains. Y. pestis 201-pPCP1⁺ appeared to have a stronger ability to colonize lungs than other plasmid-deficient strains. Pa36060 has the strongest ability to colonize intestines and lungs. Y. pestis 201 has the strongest ability to survive in blood, and the strongest virulence. These results indicated that the plasmid pMT1 was an important determinant in the colonization of livers, spleens and blood, whereas the plasmid pPCP1 appeared to correlate with the colonization in lungs. The resistance to killing in mouse blood seemed to be the critical factor causing animal death.     

virF-Positive Yersinia pseudotuberculosis and Yersinia enterocolitica Found in Migratory Birds in Sweden

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total, Yersinia spp. were isolated from 12.8% of collected samples. The most commonly found species was Yersinia enterocolitica, which was isolated from 5.6% of all collected samples, followed by Y. intermedia (3.8%), Y. frederiksenii (3.0%), Y. kristensenii (0.9%), Y. pseudotuberculosis (0.6%), and Y. rohdei (0.4%). The pathogenic, virF-positive Y. pseudotuberculosis strains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis with NotI and SpeI enzymes. In addition, 10 Y. enterocolitica strains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying the virF gene on their plasmids. All pathogenic Y. pseudotuberculosis and Y. enterocolitica strains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.     

Evaluation of a Modified Cefsulodin-Irgasan-Novobiocin Agar for Isolation of Yersinia spp

Y. enterocolitica and Y. pseudotuberculosis are important food borne pathogens. However, the presence of competitive microbiota makes the isolation of Y. enterocolitica and Y. pseudotuberculosis from naturally contaminated foods difficult. We attempted to evaluate the performance of a modified Cefsulodin-Irgasan-Novobiocin (CIN) agar in the differentiation of Y. enterocolitica from non-Yersinia species, particularly the natural intestinal microbiota. The modified CIN enabled the growth of Y. enterocolitica colonies with the same efficiency as CIN and Luria-Bertani agar. The detection limits of the modified CIN for Y. enterocolitica in culture medium (10 cfu/ml) and in artificially contaminated pork (10⁴ cfu/ml) were also comparable to those of CIN. However, the modified CIN provided a better discrimination of Yersinia colonies from other bacteria exhibiting Yersinia-like colonies on CIN (H₂S-producing Citrobacter freundii, C. braakii, Enterobacter cloacae, Aeromonas hydrophila, Providencia rettgeri, and Morganella morganii). The modified CIN exhibited a higher recovery rate of Y. enterocolitica from artificially prepared bacterial cultures and naturally contaminated samples compared with CIN. Our results thus demonstrated that the use of modified CIN may be a valuable means to increase the recovery rate of food borne Yersinia from natural samples, which are usually contaminated by multiple types of bacteria.     

Oligomeric coiled-coil adhesin YadA is a double-edged sword

Yersinia adhesin A (YadA) is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Suprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt) Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs) upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadA_V98D,N99A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions.     

Sequencing and comparative analysis of IncP-1α antibiotic resistance plasmids reveal a highly conserved backbone and differences within accessory regions

Although IncP-1 plasmids are important for horizontal gene transfer among bacteria, in particular antibiotic resistance spread, so far only three plasmids from the subgroup IncP-1α have been completely sequenced. In this study we doubled this number. The three IncP-1α plasmids pB5, pB11 and pSP21 were isolated from bacteria of two different sewage treatment plants and sequenced by a combination of next-generation and capillary sequencing technologies. A comparative analysis including the previously analysed IncP-1α plasmids RK2, pTB11 and pBS228 revealed a highly conserved plasmid backbone (at least 99.9% DNA sequence identity) comprising 54 core genes. The accessory elements of the plasmid pB5 constitute a class 1 integron interrupting the parC gene and an IS6100 copy inserted into the integron. In addition, the tetracycline resistance genes tetAR and the ISTB11-like element are located between the klc operon and the trfA-ssb operon. Plasmid pB11 is loaded with a Tn5053-like mercury resistance transposon between the parCBA and parDE operons and contains tetAR that are identical to those identified in plasmid pB5 and the insertion sequence ISSP21. Plasmid pSP21 harbours an ISPa7 element in a Tn402 transposon including a class 1 integron between the partitioning genes parCBA and parDE. The IS-element ISSP21 (99.89% DNA sequence identity to ISSP21 from pB11), inserted downstream of the tetR gene and a copy of ISTB11 (identical to ISTB11 on pTB11) inserted between the genes pncA and pinR. On all three plasmids the accessory genes are almost always located between the backbone modules confirming the importance of the backbone functions for plasmid maintenance. The striking backbone conservation among the six completely sequenced IncP-1α plasmids is in contrast to the much higher diversity within the IncP-1β subgroup.     

An encapsulated Yersinia pseudotuberculosis is a highly efficient vaccine against pneumonic plague

Background

Plague is still a public health problem in the world and is re-emerging, but no efficient vaccine is available. We previously reported that oral inoculation of a live attenuated Yersinia pseudotuberculosis, the recent ancestor of Yersinia pestis, provided protection against bubonic plague. However, the strain poorly protected against pneumonic plague, the most deadly and contagious form of the disease, and was not genetically defined.

Methodology and Principal Findings

The sequenced Y. pseudotuberculosis IP32953 has been irreversibly attenuated by deletion of genes encoding three essential virulence factors. An encapsulated Y. pseudotuberculosis was generated by cloning the Y. pestis F1-encoding caf operon and expressing it in the attenuated strain. The new V674pF1 strain produced the F1 capsule in vitro and in vivo. Oral inoculation of V674pF1 allowed the colonization of the gut without lesions to Peyer''s patches and the spleen. Vaccination induced both humoral and cellular components of immunity, at the systemic (IgG and Th1 cells) and the mucosal levels (IgA and Th17 cells). A single oral dose conferred 100% protection against a lethal pneumonic plague challenge (33×LD₅₀ of the fully virulent Y. pestis CO92 strain) and 94% against a high challenge dose (3,300×LD₅₀). Both F1 and other Yersinia antigens were recognized and V674pF1 efficiently protected against a F1-negative Y. pestis.

Conclusions and Significance

The encapsulated Y. pseudotuberculosis V674pF1 is an efficient live oral vaccine against pneumonic plague, and could be developed for mass vaccination in tropical endemic areas to control pneumonic plague transmission and mortality.     

Yersinia enterocolitica Isolates from Wild Boars Hunted in Lower Saxony,Germany

Yersiniosis is strongly associated with the consumption of pork contaminated with enteropathogenic Yersinia enterocolitica, which is harbored by domestic pigs without showing clinical signs of disease. In contrast to data on Y. enterocolitica isolated from conventionally reared swine, investigations into the occurrence of Y. enterocolitica in wild boars in Germany are rare. The objectives of the study were to get knowledge about these bacteria and their occurrence in wild boars hunted in northern Germany by isolation of the bacteria from the tonsils, identification of the bioserotypes, determination of selected virulence factors, macrorestriction analysis, multilocus sequence typing (MLST), and testing of antimicrobial susceptibility. Altogether, tonsils from 17.1% of 111 tested wild boars were positive for Y. enterocolitica by culture methods. All but two isolates belonged to biotype (BT) 1A, with the majority of isolates bearing a ystB nucleotide sequence which was revealed to have 85% identity to internal regions of Y. enterocolitica heat-stable enterotoxin type B genes. The remaining Y. enterocolitica isolates were identified to be BT 1B and did not carry the virulence plasmid. However, two BT 1A isolates carried the ail gene. Macrorestriction analysis and results from MLST showed a high degree of genetic diversity of the isolates, although the region where the samples were taken was restricted to Lower Saxony, Germany, and wild boars were shot during one hunting season. In conclusion, most Y. enterocolitica isolates from wild boars investigated in this study belonged to biotype 1A. Enteropathogenic Y. enterocolitica bioserotypes 4/O:3 and 2/O:9, usually harbored by commercially raised pigs in Europe, could not be identified.     

Essential virulence determinants of different Yersinia species are carried on a common plasmid

Plasmids of 44.4–46 Mdal were identified in conditional virulent Yersinia species. All virulent strains studied are unable to grow on oxalate-containing plates at 37 °C (OX^? phenotype) which is a characteristic property of strains producing the essential virulence VW antigens. The phenotopic transition from OX^? to OX⁺ in these strains is concomitant with loss of virulence and loss of this plasmid. The similarity in size and in the DNA fragmentation patterns, generated by HindIII, of the plasmids isolated from either Y. pseudotuberculosis or two conditional virulent Y. pestis strains, suggests that a common plasmid—pSB2—is carried by these strains. A plasmid of a similar size, ~42 Mdal, and function was recently identified (P. Gemski, J. R. Lazere, and T. Casey, 1980, Infect. Immunity27, 682–685; D. L. Zink, J. C. Feeley, J. G. Wells, C. Vanderzant, J. C. Vickery, W. D. Roof, and G. A. O'Donovan, 1980, Nature (London)283, 224–225) in virulent Y. enterocolitica. We conclude that pSB2 in Y. pseudotuberculosis and Y. pestis and its counterpart in Y. enterocolitica carry genetic information essential for virulence common to the Yersinia species, probably related to VW antigen production. Several additional plasmids were identified in several strains of Y. pestis. One of these plasmids, designated pSB3 (12.5 Mdal), appears to be associated with pesticin production.     

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.     

Yersinia enterocolitica Infection and tcaA-Dependent Killing of Caenorhabditis elegans

Caenorhabditis elegans is a validated model to study bacterial pathogenicity. We report that Yersinia enterocolitica strains {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 (biovar 2, serovar O:9) and WA314 (biovar 1B, serovar O:8) kill C. elegans when feeding on the pathogens for at least 15 min before transfer to the feeding strain Escherichia coli OP50. The killing by Yersinia enterocolitica requires viable bacteria and, in contrast to that by Yersinia pestis and Yersinia pseudotuberculosis strains, is biofilm independent. The deletion of tcaA encoding an insecticidal toxin resulted in an OP50-like life span of C. elegans, indicating an essential role of TcaA in the nematocidal activity of Y. enterocolitica. TcaA alone is not sufficient for nematocidal activity because E. coli DH5α overexpressing TcaA did not result in a reduced C. elegans life span. Spatial-temporal analysis of C. elegans infected with green fluorescent protein-labeled Y. enterocolitica strains showed that Y. enterocolitica colonizes the nematode intestine, leading to an extreme expansion of the intestinal lumen. By low-dose infection with {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 or DH5α followed by transfer to E. coli OP50, proliferation of Y. enterocolitica, but not E. coli, in the intestinal lumen of the nematode was observed. The titer of {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 cells within the worm increased to over 10⁶ per worm 4 days after infection while a significantly lower number of a tcaA knockout mutant was recovered. A strong expression of tcaA was observed during the first 5 days of infection. Y. enterocolitica WA314 (biovar 1B, serovar O:8) mutant strains lacking the yadA, inv, yopE, and irp1 genes known to be important for virulence in mammals were not attenuated or only slightly attenuated in their toxicity toward the nematode, suggesting that these factors do not play a significant role in the colonization and persistence of this pathogen in nematodes. In summary, this study supports the hypothesis that C. elegans is a natural host and nutrient source of Y. enterocolitica.Yersinia enterocolitica belongs to the family of Enterobacteriaceae and is a psychrotolerant human pathogen that causes gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis (5). It infects a number of mammals, and swine was identified as a major source for human infection (6). A multiphasic life cycle, which comprises a free-living phase and several host-associated phases, including cold-blooded and warm-blooded hosts, appears to be characteristic for biovars 1B and 2 to 5 of Y. enterocolitica (7, 24).Nonmammalian host organisms including Dictyostelium discoideum, Drosophila melanogaster, or Caenorhabditis elegans are increasingly used to study host-pathogen interactions (16, 26). Due to the obvious parallels between the mammalian and invertebrate defense mechanisms, it has been suggested that the bacteria-invertebrate interaction has shaped the evolution of microbial pathogenicity (53). Several human pathogens including Gram-positive and Gram-negative bacteria infect and kill the soil nematode C. elegans when they are supplied as a nutrient source (42). For example, Streptococcus pneumoniae (4), Listeria monocytogenes (50), extraintestinal Escherichia coli (15), and Staphylococcus aureus (43) but not Bacillus subtilis have been shown to kill the nematode. Upon infection of C. elegans with Enterococcus faecalis, Gram-positive virulence-related factors as well as putative antimicrobials have been identified (20, 35). The extensive conservation in virulence mechanisms directed against invertebrates as well as mammals was demonstrated using a screen with Pseudomonas aeruginosa (30). In this study, 10 of 13 genes whose knockout attenuated the nematode killing were also required for full virulence in a mouse model, confirming the suitability of the C. elegans model to study bacterial pathogenicity. C. elegans is also colonized by Salmonella enterica serovar Typhimurium (S. Typhimurium). This process requires Salmonella virulence factors and was used to study the innate immune response of the nematode (1, 2, 49).The effect of pathogenic Yersinia spp. on C. elegans has also been investigated. It could be demonstrated that both Yersinia pestis and Yersinia pseudotuberculosis block food intake by creating a biofilm around the worm''s mouth (13, 27). This biofilm formation requires the hemin storage locus (hms) and has been suggested to be responsible for the blockage of the digestive tract following uptake by fleas, thus acting as a bacterial defense against predation by invertebrates. In a study with 40 Y. pseudotuberculosis strains, one-quarter of them caused an infection of C. elegans by biofilm formation on the worm head (27). In contrast, a similar effect was not observed following nematode infection with 15 Y. enterocolitica strains. Using a Y. pestis strain lacking the hms genes, it could be demonstrated that this mutant can infect and kill the nematode by a biofilm-independent mechanism that includes the accumulation of Y. pestis in the intestine of the worm (47). This pathogenesis model was applied to show that putative virulence factors such as YapH, OmpT, or a metalloprotease, Y3857, but not the virulence plasmids pCD1 and pPCP1, are required for Y. pestis virulence in C. elegans. Six yet unknown genes required for full virulence in C. elegans were also identified, and one of them appeared to be a virulence factor in the mouse infection model.C. elegans has not been used to study the pathogenicity properties of Y. enterocolitica, mainly due to the fact that many of its virulence factors are upregulated at 37°C in comparison to growth at lower temperatures while C. elegans cannot be cultivated at temperatures above 25°C. In this study, we examined for the first time the infection of C. elegans by Y. enterocolitica strains, demonstrating that this pathogen colonizes and kills C. elegans and that the insecticidal toxin TcaA, which is expressed only at ambient temperature, is required for full nematocidal activity.     

Complete genome sequence of Yersinia pestis strain 91001, an isolate avirulent to humans.

Genomics provides an unprecedented opportunity to probe in minute detail into the genomes of the world's most deadly pathogenic bacteria- Yersinia pestis. Here we report the complete genome sequence of Y. pestis strain 91001, a human-avirulent strain isolated from the rodent Brandt's vole-Microtus brandti. The genome of strain 91001 consists of one chromosome and four plasmids (pPCP1, pCD1, pMT1 and pCRY). The 9609-bp pPCP1 plasmid of strain 91001 is almost identical to the counterparts from reference strains (CO92 and KIM). There are 98 genes in the 70,159-bp range of plasmid pCD1. The 106,642-bp plasmid pMT1 has slightly different architecture compared with the reference ones. pCRY is a novel plasmid discovered in this work. It is 21,742 bp long and harbors a cryptic type IV secretory system. The chromosome of 91001 is 4,595,065 bp in length. Among the 4037 predicted genes, 141 are possible pseudo-genes. Due to the rearrangements mediated by insertion elements, the structure of the 91001 chromosome shows dramatic differences compared with CO92 and KIM. Based on the analysis of plasmids and chromosome architectures, pseudogene distribution, nitrate reduction negative mechanism and gene comparison, we conclude that strain 91001 and other strains isolated from M. brandti might have evolved from ancestral Y. pestis in a different lineage. The large genome fragment deletions in the 91001 chromosome and some pseudogenes may contribute to its unique nonpathogenicity to humans and host-specificity.     

Tapping the potential of intact cell mass spectrometry with a combined data analytical approach applied to Yersinia spp.: detection, differentiation and identification of Y. pestis

In the everyday routine of an analytic lab, one is often confronted with the challenge to identify an unknown microbial sample lacking prior information to set the search limits.In the present work, we propose a workflow, which uses the spectral diversity of a commercial database (SARAMIS) to narrow down the search field at a certain taxonomic level, followed by a refined classification by supervised modelling. As supervised learning algorithm, we have chosen a shrinkage discriminant analysis approach, which takes collinearity of the data into account and provides a scoring system for biomarker ranking. This ranking can be used to tailor specific biomarker subsets, which optimize discrimination between subgroups, allowing a weighting of misclassification.The suitability of the approach was verified based on a dataset containing the mass spectra of three Yersinia species Yersinia enterocolitica, Y. pseudotuberculosis and Yersinia pestis. Thereby, we laid the emphasis on the discrimination between the highly related species Yersinia pseudotuberculosis and Y. pestis.All three species were correctly identified at the genus level by the commercial database. Whereas Y. enterocolitica was correctly identified at the species level, discrimination between the highly related Y. pseudotuberculosis and Y. pestis strains was ambiguous. With the use of the supervised modelling approach, we were able to accurately discriminate all the species even when grown under different culture conditions.     

IS1631 Occurrence in Bradyrhizobium japonicum Highly Reiterated Sequence-Possessing Strains with High Copy Numbers of Repeated Sequences RSα and RSβ

From Bradyrhizobium japonicum highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSα and RSβ (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845–1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of B. japonicum differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of B. japonicum contained many copies of IS1631, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of B. japonicum were examined. Consequently, IS1631 occurrence was well correlated with B. japonicum HRS strains possessing high copy numbers of the repeated sequence RSα or RSβ. DNA sequence analysis indicated that IS1631 is 2,712 bp long. In addition, IS1631 belongs to the IS21 family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS21, and its terminal inverted repeat sequences with multiple short repeats.