Genome Wide Search for Biomarkers to Diagnose Yersinia Infections

Bacterial identification on the basis of the highly conserved 16S rRNA (rrs) gene is limited by its presence in multiple copies and a very high level of similarity among them. The need is to look for other genes with unique characteristics to be used as biomarkers. Fifty-one sequenced genomes belonging to 10 different Yersinia species were used for searching genes common to all the genomes. Out of 304 common genes, 34 genes of sizes varying from 0.11 to 4.42 kb, were selected and subjected to in silico digestion with 10 different Restriction endonucleases (RE) (4–6 base cutters). Yersinia species have 6–7 copies of rrs per genome, which are difficult to distinguish by multiple sequence alignments or their RE digestion patterns. However, certain unique combinations of other common gene sequences—carB, fadJ, gluM, gltX, ileS, malE, nusA, ribD, and rlmL and their RE digestion patterns can be used as markers for identifying 21 strains belonging to 10 Yersinia species: Y. aldovae, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. pestis, Y. pseudotuberculosis, Y. rohdei, Y. ruckeri, and Y. similis. This approach can be applied for rapid diagnostic applications.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0552-6) contains supplementary material, which is available to authorized users.     

Neutrophils Are Resistant to Yersinia YopJ/P-Induced Apoptosis and Are Protected from ROS-Mediated Cell Death by the Type III Secretion System

Background

The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs) for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis) and YopP (Y. enterocolitica) rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined.

Methodology/Principal Findings

In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM) and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death. PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.

Conclusions

Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis.     

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.     

Rapid identification and typing of <Emphasis Type="Italic">Yersinia pestis</Emphasis> and other <Emphasis Type="Italic">Yersinia</Emphasis> species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

Background  

Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species.     

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.     

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.     

Development of phage-based single chain Fv antibody reagents for detection of Yersinia pestis

Background

Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1)^. F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.

Methods

Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA.

Results

Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.

Conclusions

Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.     

Selective isolation of Yersinia pestis from plague-infected fleas

We evaluated Yersinia CIN agar for the isolation of Yersinia pestis from infected fleas. CIN media is effective for the differentiation of Y. pestis from flea commensal flora and is sufficiently inhibitory to other bacteria that typically outcompete Y. pestis after 48 h of growth using less selective media.     

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.     

Identification of Novel Protein-Protein Interactions of Yersinia pestis Type III Secretion System by Yeast Two Hybrid System

Type III secretion system (T3SS) of the plague bacterium Y. pestis encodes a syringe-like structure consisting of more than 20 proteins, which can inject virulence effectors into host cells to modulate the cellular functions. Here in this report, interactions among the possible components in T3SS of Yersinia pestis were identified using yeast mating technique. A total of 57 genes, including all the pCD1-encoded genes except those involved in plasmid replication and partition, pseudogenes, and the putative transposase genes, were subjected to yeast mating analysis. 21 pairs of interaction proteins were identified, among which 9 pairs had been previously reported and 12 novel pairs were identified in this study. Six of them were tested by GST pull down assay, and interaction pairs of YscG-SycD, YscG-TyeA, YscI-YscF, and YopN-YpCD1.09c were successfully validated, suggesting that these interactions might play potential roles in function of Yersinia T3SS. Several potential new interactions among T3SS components could help to understand the assembly and regulation of Yersinia T3SS.     

Identification of Flagellar Motility Genes in Yersinia ruckeri by Transposon Mutagenesis

Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype 2 Y. ruckeri through the mutational loss of flagellar secretion.Yersinia ruckeri is the etiologic agent of enteric redmouth disease, a disease of salmonid fish species that is found worldwide in areas where salmonid fish species are farmed (3, 6, 18, 20). Vaccines for enteric redmouth disease have been used successfully for nearly 3 decades and consist of immersion-applied, killed whole-cell preparations of motile serovar 1 Y. ruckeri strains (22). Recently though, outbreaks have been reported in vaccinated fish at trout farms in the United Kingdom (2), Spain (9), and the United States (1). The Y. ruckeri strains isolated from these outbreaks are uniformly atypical serovar 1 isolates lacking both flagellar motility and secreted lipase activity. These variants have been classified as Y. ruckeri biotype 2 (BT2) and are believed to have a reduced sensitivity to immersion vaccination (2). The objective of this study was to obtain a better understanding of the emergence of BT2 Y. ruckeri by identifying genetic elements necessary for expression of the Y. ruckeri flagellum and determining the role that the flagellum plays in virulence by using a rainbow trout infection model.     

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice

Background

Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy.

Methodology/Principal Findings

The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence.

Conclusions/Significance

We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.     

Caspase-12 and the Inflammatory Response to Yersinia pestis

Background

Caspase-12 functions as an antiinflammatory enzyme inhibiting caspase-1 and the NOD2/RIP2 pathways. Due to increased susceptibility to sepsis in individuals with functional caspase-12, an early-stop mutation leading to the loss of caspase-12 has replaced the ancient genotype in Eurasia and a significant proportion of individuals from African populations. In African-Americans, it has been shown that caspase-12 inhibits the pro-inflammatory cytokine production.

Methodology/Principal Findings

We assessed whether similar mechanisms are present in African individuals, and whether evolutionary pressures due to plague may have led to the present caspase-12 genotype population frequencies. No difference in cytokine induction through the caspase-1 and/or NOD2/RIP2 pathways was observed in two independent African populations, among individuals with either an intact or absent caspase-12. In addition, stimulations with Yersinia pestis and two other species of Yersinia were preformed to investigate whether caspase-12 modulates the inflammatory reaction induced by Yersinia. We found that caspase-12 did not modulate cytokine production induced by Yersinia spp.

Conclusions

Our experiments demonstrate for the first time the involvement of the NOD2/RIP2 pathway for recognition of Yersinia. However, caspase-12 does not modulate innate host defense against Y. pestis and alternative explanations for the geographical distribution of caspase-12 should be sought.     

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.