Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry.

Plague, caused by the bacterium Yersinia pestis, is still present in several countries worldwide. Besides, Y. pestis has been designated as Tier 1 agent, the highest rank of bioterrorism agents. In this context, reliable diagnostic methods are of great importance. Here, we have developed an original workflow based upon dried blood spot for simplified sampling of clinical specimens, and specific immuno-mass spectrometry monitoring of Y. pestis biomarkers. Targeted proteins were selectively enriched from dried blood spot extracts by multiplex immunocapture using antibody-coated magnetic beads. After accelerated on-beads digestion, proteotypic peptides were monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode. The DBS-IC-MS assay was designed to quantify both F1 and LcrV antigens, although 10-fold lower sensitivity was observed with LcrV. The assay was successfully validated for F1 with a lower limit of quantification at 5 ng·mL-1 in spiked blood, corresponding to only 0.1 ng on spots. In vivo quantification of F1 in blood and organ samples was demonstrated in the mouse model of pneumonic plague. The new assay could help to simplify the laboratory confirmation of positive point of care F1 dipstick.

Detection of Yersinia pestis in Complex Matrices by Intact Cell Immunocapture and Targeted Mass Spectrometry.

We describe an immunoaffinity-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) protocol for the direct (i.e., without prior culture), sensitive and specific detection of Yersinia pestis in complex matrices. Immunoaffinity enables isolation and concentration of intact bacterial cells from food and environmental samples. After protein extraction and digestion, suitable proteotypic peptides corresponding to three Y. pestis-specific protein markers (murine toxine, plasminogen activator and pesticin) are monitored by targeted LC-MS/MS using the selected reaction monitoring (SRM) mode. This immuno-LC-MS/MS assay has a limit of detection of 2 × 104 CFU/mL in milk or tap water, and 4.5 × 105 CFU in 10 mg of soil.

Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague.

Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host.

Detection of Yersinia pestis in environmental and food samples by intact cell immunocapture and liquid chromatography-tandem mass spectrometry.

Yersinia pestis is the causative agent of bubonic and pneumonic plague, an acute and often fatal disease in humans. In addition to the risk of natural exposure to plague, there is also the threat of a bioterrorist act, leading to the deliberate spread of the bacteria in the environment or food. We report here an immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) method for the direct (i.e., without prior culture), sensitive, and specific detection of Y. pestis in such complex samples. In the first step, a bottom-up proteomics approach highlighted three relevant protein markers encoded by the Y. pestis-specific plasmids pFra (murine toxin) and pPla (plasminogen activator and pesticin). Suitable proteotypic peptides were thoroughly selected to monitor the three protein markers by targeted MS using the selected reaction monitoring (SRM) mode. Immunocapture conditions were optimized for the isolation and concentration of intact bacterial cells from complex samples. The immuno-LC-SRM assay has a limit of detection of 2 × 10(4) CFU/mL in milk or tap water, which compares well with those of state-of-the-art immunoassays. Moreover, we report the first direct detection of Y. pestis in soil, which could be extremely useful in confirming Y. pestis persistence in the ground.

The Yersinia pseudotuberculosis complex: characterization and delineation of a new species, Yersinia wautersii.

The genus Yersinia contains three species pathogenic for humans, one of which is the enteropathogen Yersinia pseudotuberculosis. A recent analysis by Multi Locus Sequence Typing (MLST) of the 'Y. pseudotuberculosis complex' revealed that this complex comprises three distinct populations: the Y. pestis/Y. pseudotuberculosis group, the recently described species Yersinia similis, and a third not yet characterized population designated 'Korean Group', because most strains were isolated in Korea. The aim of this study was to perform an in depth phenotypic and genetic characterization of the three populations composing the Y. pseudotuberculosis complex (excluding Y. pestis, which belonged to the Y. pseudotuberculosis cluster in the MLST analysis). Using a set of strains representative of each group, we found that the three populations had close metabolic properties, but were nonetheless distinguishable based on D-raffinose and D-melibiose fermentation, and on pyrazinamidase activity. Moreover, high-resolution electrospray mass spectrometry highlighted protein peaks characteristic of each population. Their 16S rRNA gene sequences shared high identity (≥99.5%), but specific nucleotide signatures for each group were identified. Multi-Locus Sequence Analysis also identified three genetically closely related but distinct populations. Finally, an Average Nucleotide Identity (ANI) analysis performed after sequencing the genomes of a subset of strains of each group also showed that intragroup identity (average for each group ≥99%) was higher than intergroup diversity (94.6-97.4%). Therefore, all phenotypic and genotypic traits studied concurred with the initial MLST data indicating that the Y. pseudotuberculosis complex comprises a third and clearly distinct population of strains forming a novel Yersinia species that we propose to designate Yersinia wautersii sp. nov. The isolation of some strains from humans, the detection of virulence genes (on the pYV and pVM82 plasmids, or encoding the superantigen ypmA) in some isolates, and the absence of pyrazinamidase activity (a hallmark of pathogenicity in the genus Yersinia) argue for the pathogenic potential of Y. wautersii.

Fast and simple detection of Yersinia pestis applicable to field investigation of plague foci.

Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37°C. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20°C and 37°C, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples.

Imaging of bubonic plague dynamics by in vivo tracking of bioluminescent Yersinia pestis.

Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.

Insights into the infective properties of YpfΦ, the Yersinia pestis filamentous phage.

YpfΦ is a filamentous phage that infected Yersinia pestis, the plague bacillus, during its emergence. Using an experimental transduction approach, we show here that this phage has the capacity to infect with variable efficiencies, all three pathogenic Yersinia species as well as Escherichia coli. Like other Inovirus phages, its genetic organization comprises three functional modules necessary for the production of infectious virions. Upon infection, YpfΦ integrates into the chromosomal dif site, but extrachromosomal forms are also frequently observed. Several pieces of evidence suggest that the absence of chromosomal YpfΦ in natural non-Orientalis Y. pestis isolates results from a higher chromosomal excision rate rather than from a defective integration machinery. A resident YpfΦ confers some protection against a superinfection. In contrast to other filamentous phages, the incoming YpfΦ genome inserts itself between two copies of the resident prophage. This analysis thus unravels infective properties specific to YpfΦ.