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The extent of the devastation of the Black Death pandemic (1346-1353) on European populations is known from documentary sources and its bacterial source illuminated by studies of ancient pathogen DNA. What has remained less understood is the effect of the pandemic on human mobility and genetic diversity at the local scale. Here, we report 275 ancient genomes, including 109 with coverage >0.1×, from later medieval and postmedieval Cambridgeshire of individuals buried before and after the Black Death. Consistent with the function of the institutions, we found a lack of close relatives among the friars and the inmates of the hospital in contrast to their abundance in general urban and rural parish communities. While we detect long-term shifts in local genetic ancestry in Cambridgeshire, we find no evidence of major changes in genetic ancestry nor higher differentiation of immune loci between cohorts living before and after the Black Death.

Yersinia pestis is a historically important vector-borne pathogen causing plague in humans and other mammals. Contemporary zoonotic infections with Y. pestis still occur in sub-Saharan Africa, including Tanzania and Madagascar, but receive relatively little attention. Thus, the role of wildlife reservoirs in maintaining sylvatic plague and spillover risks to humans is largely unknown. The multimammate rodent Mastomys natalensis is the most abundant and widespread rodent in peri-domestic areas in Tanzania, where it plays a major role as a Y. pestis reservoir in endemic foci. Yet, how M. natalensis' immunogenetics contributes to the maintenance of plague has not been investigated to date. Here, we surveyed wild M. natalensis for Y. pestis vectors, i.e., fleas, and tested for the presence of antibodies against Y. pestis using enzyme-linked immunosorbent assays (ELISA) in areas known to be endemic or without previous records of Y. pestis in Tanzania. We characterized the allelic and functional (i.e., supertype) diversity of the major histocompatibility complex (MHC class II) of M. natalensis and investigated links to Y. pestis vectors and infections. We detected antibodies against Y. pestis in rodents inhabiting both endemic areas and areas considered non-endemic. Of the 111 nucleotide MHC alleles, only DRB*016 was associated with an increased infestation with the flea Xenopsylla. Surprisingly, we found no link between MHC alleles or supertypes and antibodies of Y. pestis. Our findings hint, however, at local adaptations towards Y. pestis vectors, an observation that more exhaustive sampling could unwind in the future., (© 2023. The Author(s).)

Nutritional immunity includes sequestration of transition metals from invading pathogens. Yersinia pestis overcomes nutritional immunity by secreting yersiniabactin to acquire iron and zinc during infection. While the mechanisms for yersiniabactin synthesis and import are well-defined, those responsible for yersiniabactin secretion are unknown. Identification of this mechanism has been difficult because conventional mutagenesis approaches are unable to inhibit trans-complementation by secreted factors between mutants. To overcome this obstacle, we utilized a technique called droplet Tn-seq (dTn-seq), which uses microfluidics to isolate individual transposon mutants in oil droplets, eliminating trans-complementation between bacteria. Using this approach, we first demonstrated the applicability of dTn-seq to identify genes with secreted functions. We then applied dTn-seq to identify an AcrAB efflux system as required for growth in metal-limited conditions. Finally, we showed this efflux system is the primary yersiniabactin secretion mechanism and required for virulence during bubonic and pneumonic plague. Together, these studies have revealed the yersiniabactin secretion mechanism that has eluded researchers for over 30 years and identified a potential therapeutic target for bacteria that use yersiniabactin for metal acquisition., (© 2023 The Authors.)

Multiple genetic changes in the enteric pathogen Yersinia pseudotuberculosis have driven the emergence of Yesinia pestis , the arthropod-borne, etiological agent of plague. These include developing the capacity for biofilm-dependent blockage of the flea foregut to enable transmission by flea bite. Previously, we showed that pseudogenization of rcsA , encoding a component of the Rcs signalling pathway, is an important evolutionary step facilitating Y. pestis flea-borne transmission. Additionally, rcsD, another important gene in the Rcs system, harbours a frameshift mutation. Here, we demonstrated that this rcsD mutation resulted in production of a small protein composing the C-terminal RcsD histidine-phosphotransferase domain (designated RcsD-Hpt) and full-length RcsD. Genetic analysis revealed that the rcsD frameshift mutation followed the emergence of rcsA pseudogenization. It further altered the canonical Rcs phosphorylation signal cascade, fine-tuning biofilm production to be conducive with retention of the pgm locus in modern lineages of Y. pestis . Taken together, our findings suggest that a frameshift mutation in rcsD is an important evolutionary step that fine-tuned biofilm production to ensure perpetuation of flea-mammal plague transmission cycles., Competing Interests: XG, HY, WY, ZY, VV, DZ, YS No competing interests declared, (© 2023, Guo, Yan, Yang et al.)

The Black Death, a notorious devastating pandemic caused by Yersinia pestis infection during the 14th century, posed a formidable challenge to human immune defenses. A new article by Klunk et al. reports that a variant in an antigen-processing gene may have favored survival during the plague and may have undergone genomic selection in Europeans at unprecedented speed., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Plague has an enigmatic history as a zoonotic pathogen. This infectious disease will unexpectedly appear in human populations and disappear just as suddenly. As a result, a long-standing line of inquiry has been to estimate when and where plague appeared in the past. However, there have been significant disparities between phylogenetic studies of the causative bacterium, Yersinia pestis, regarding the timing and geographic origins of its reemergence. Here, we curate and contextualize an updated phylogeny of Y. pestis using 601 genome sequences sampled globally. Through a detailed Bayesian evaluation of temporal signal in subsets of these data we demonstrate that a Y. pestis-wide molecular clock is unstable. To resolve this, we developed a new approach in which each Y. pestis population was assessed independently, enabling us to recover substantial temporal signal in five populations, including the ancient pandemic lineages which we now estimate may have emerged decades, or even centuries, before a pandemic was historically documented from European sources. Despite this methodological advancement, we only obtain robust divergence dates from populations sampled over a period of at least 90 years, indicating that genetic evidence alone is insufficient for accurately reconstructing the timing and spread of short-term plague epidemics., (© 2023. The Author(s).)

The magnitude of human affliction brought about by bacterial infections has been on the rise since the mid-5th century. Yersinia pestis is one such notable, gram-negative bacterium that inflicted havoc around the globe three times throughout different millenniums by causing deadly plagues. Despite the unremitting efforts by scientists, different strains of Yersinia pestis are still affecting the populations in various parts of the world by growing resistant to existing antimicrobial agents owing to their overuse. The current scenario, therefore, calls for new therapeutics to further combat the disease. In this study, 3105 core, 387 pathogen-specific unique, 536 choke-point, 796 virulence factors, and 115 antimicrobial resistant proteins were found using a pan-genomic and subtractive genome analysis of nine Yersinia pestis strains that could be instrumental in the development of drugs against Yersinia pestis. Subsequently, 1461 and 1114 essential proteins were identified as non-homologous to human and gut microflora. 535 and 30 proteins were predicted as cytoplasmic and broad-spectrum targets respectively. Finally, four potential targets were selected for their high connectivity in protein-protein interaction network. These selected target proteins are associated with one of the major lipopolysaccharide biosynthesis pathways. Therefore, dismantling their activity might indicate a probable strategy for developing therapeutics to combat bacterial infection caused by Yersinia pestis. However, further experimental validation in the laboratory is needed to consolidate the research findings., Competing Interests: Conflict of Interest The authors report no conflicts of interest in this work., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Human populations have been shaped by catastrophes that may have left long-lasting signatures in their genomes. One notable example is the second plague pandemic that entered Europe in ca. 1,347 CE and repeatedly returned for over 300 years, with typical village and town mortality estimated at 10%-40%. 1 It is assumed that this high mortality affected the gene pools of these populations. First, local population crashes reduced genetic diversity. Second, a change in frequency is expected for sequence variants that may have affected survival or susceptibility to the etiologic agent (Yersinia pestis). 2 Third, mass mortality might alter the local gene pools through its impact on subsequent migration patterns. We explored these factors using the Norwegian city of Trondheim as a model, by sequencing 54 genomes spanning three time periods: (1) prior to the plague striking Trondheim in 1,349 CE, (2) the 17 th -19 th century, and (3) the present. We find that the pandemic period shaped the gene pool by reducing long distance immigration, in particular from the British Isles, and inducing a bottleneck that reduced genetic diversity. Although we also observe an excess of large F ST values at multiple loci in the genome, these are shaped by reference biases introduced by mapping our relatively low genome coverage degraded DNA to the reference genome. This implies that attempts to detect selection using ancient DNA (aDNA) datasets that vary by read length and depth of sequencing coverage may be particularly challenging until methods have been developed to account for the impact of differential reference bias on test statistics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Infectious diseases are among the strongest selective pressures driving human evolution 1,2 . This includes the single greatest mortality event in recorded history, the first outbreak of the second pandemic of plague, commonly called the Black Death, which was caused by the bacterium Yersinia pestis 3 . This pandemic devastated Afro-Eurasia, killing up to 30-50% of the population 4 . To identify loci that may have been under selection during the Black Death, we characterized genetic variation around immune-related genes from 206 ancient DNA extracts, stemming from two different European populations before, during and after the Black Death. Immune loci are strongly enriched for highly differentiated sites relative to a set of non-immune loci, suggesting positive selection. We identify 245 variants that are highly differentiated within the London dataset, four of which were replicated in an independent cohort from Denmark, and represent the strongest candidates for positive selection. The selected allele for one of these variants, rs2549794, is associated with the production of a full-length (versus truncated) ERAP2 transcript, variation in cytokine response to Y. pestis and increased ability to control intracellular Y. pestis in macrophages. Finally, we show that protective variants overlap with alleles that are today associated with increased susceptibility to autoimmune diseases, providing empirical evidence for the role played by past pandemics in shaping present-day susceptibility to disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In a recent article, Immel et al. (Immel A, Key FM, Szolek A, Barquera R, Robinson MK, Harrison GF, Palmer WH, Spyrou MA, Susat J, Krause-Kyora B, et al. 2021. Analysis of genomic DNA from medieval plague victims suggests long-term effect of Yersinia pestis on human immunity genes. Mol Biol Evol. 38:4059-4076) extracted DNA from 36 individuals dead from plague in Ellwangen, Southern Germany, during the 16th century. By comparing their human leukocyte antigen (HLA) genotypes with those of 50 present-day Ellwangen inhabitants, the authors reported a significant decrease of HLA-B*51:01 and HLA-C*06:02 and a significant increase of HLA-DRB1*13:01/13:02 frequencies from ancient to modern populations. After comparing these frequencies with a larger sample of 8,862 modern Germans and performing simulations of natural selection, they concluded that these changes had been driven by natural selection. In an attempt to provide more evidence on such stimulating results, we explored the HLA frequency patterns over all of Europe, we predicted binding affinities of HLA-B/C/DRB1 alleles to 106,515 Yersinia pestis-derived peptides, and we performed forward simulations of HLA genetic profiles under neutrality. Our analyses do not sustain the conclusions of HLA protection or susceptibility to plague based on ancient DNA., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Pathogens and associated outbreaks of infectious disease exert selective pressure on human populations, and any changes in allele frequencies that result may be especially evident for genes involved in immunity. In this regard, the 1346-1353 Yersinia pestis-caused Black Death pandemic, with continued plague outbreaks spanning several hundred years, is one of the most devastating recorded in human history. To investigate the potential impact of Y. pestis on human immunity genes, we extracted DNA from 36 plague victims buried in a mass grave in Ellwangen, Germany in the 16th century. We targeted 488 immune-related genes, including HLA, using a novel in-solution hybridization capture approach. In comparison with 50 modern native inhabitants of Ellwangen, we find differences in allele frequencies for variants of the innate immunity proteins Ficolin-2 and NLRP14 at sites involved in determining specificity. We also observed that HLA-DRB1*13 is more than twice as frequent in the modern population, whereas HLA-B alleles encoding an isoleucine at position 80 (I-80+), HLA C*06:02 and HLA-DPB1 alleles encoding histidine at position 9 are half as frequent in the modern population. Simulations show that natural selection has likely driven these allele frequency changes. Thus, our data suggest that allele frequencies of HLA genes involved in innate and adaptive immunity responsible for extracellular and intracellular responses to pathogenic bacteria, such as Y. pestis, could have been affected by the historical epidemics that occurred in Europe., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The bacterial pathogen, Yersinia pestis , has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised caf1R cloned in three different expression plasmids was examined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised caf1R . Transcriptional- lacZ reporter fusions defined the P M promoter and Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identified Caf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of Y. pestis .

The second plague pandemic started in Europe with the Black Death in 1346 and lasted until the 19th century. Based on ancient DNA studies, there is a scientific disagreement over whether the bacterium, Yersinia pestis , came into Europe once (Hypothesis 1) or repeatedly over the following four centuries (Hypothesis 2). Here, we synthesize the most updated phylogeny together with historical, archeological, evolutionary, and ecological information. On the basis of this holistic view, we conclude that Hypothesis 2 is the most plausible. We also suggest that Y. pestis lineages might have developed attenuated virulence during transmission, which can explain the convergent evolutionary signals, including pla decay, that appeared at the end of the pandemics., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Plague is a highly dangerous vector-borne infectious disease that has left a significant mark on history of humankind. There are 13 natural plague foci in the Caucasus, located on the territory of the Russian Federation, Azerbaijan, Armenia and Georgia. We performed whole-genome sequencing of Y. pestis strains, isolated in the natural foci of the Caucasus and Transcaucasia. Using the data of whole-genome SNP analysis and Bayesian phylogeny methods, we carried out an evolutionary-phylogeographic analysis of modern population of the plague pathogen in order to determine the phylogenetic relationships of Y. pestis strains from the Caucasus with the strains from other countries., Results: We used 345 Y. pestis genomes to construct a global evolutionary phylogenetic reconstruction of species based on whole-genome SNP analysis. The genomes of 16 isolates were sequenced in this study, the remaining 329 genomes were obtained from the GenBank database. Analysis of the core genome revealed 3315 SNPs that allow differentiation of strains. The evolutionary phylogeographic analysis showed that the studied Y. pestis strains belong to the genetic lineages 0.PE2, 2.MED0, and 2.MED1. It was shown that the Y. pestis strains isolated on the territory of the East Caucasian high-mountain, the Transcaucasian high-mountain and the Priaraksinsky low-mountain plague foci belong to the most ancient of all existing genetic lineages - 0.PE2., Conclusions: On the basis of the whole-genome SNP analysis of 345 Y. pestis strains, we describe the modern population structure of the plague pathogen and specify the place of the strains isolated in the natural foci of the Caucasus and Transcaucasia in the structure of the global population of Y. pestis. As a result of the retrospective evolutionary-phylogeographic analysis of the current population of the pathogen, we determined the probable time frame of the divergence of the genetic lineages of Y. pestis, as well as suggested the possible paths of the historical spread of the plague pathogen., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pneumonic plague, caused by Yersinia pestis , is a rapidly progressing bronchopneumonia involving focal bacterial growth, neutrophilic congestion, and alveolar necrosis. Within a short time after inhalation of Y. pestis , inflammatory cytokines are expressed via the Toll/interleukin-1 (IL-1) adaptor myeloid differentiation primary response 88 (MyD88), which facilitates the primary lung infection. We previously showed that Y. pestis lacking the 102-kb chromosomal pigmentation locus ( pgm ) is unable to cause inflammatory damage in the lungs, whereas the wild-type (WT) strain induces the toxic MyD88 pulmonary inflammatory response. In this work, we investigated the involvement of the pgm in skewing the inflammatory response during pneumonic plague. We show that the early MyD88-dependent and -independent cytokine responses to pgm - Y. pestis infection of the lungs are similar yet distinct from those that occur during pgm + infection. Furthermore, we found that MyD88 was necessary to prevent growth of the iron-starved pgm - Y. pestis despite the presence of iron chelators lactoferrin and transferrin. However, while this induced neutrophil recruitment, there was no hyperinflammatory response, and pulmonary disease was mild without MyD88. In contrast, growth in blood and tissues progressed rapidly in the absence of MyD88, due to an almost total loss of serum interferon gamma (IFN-γ). We further show that the expression of MyD88 by myeloid cells is important to control bacteremia but not the primary lung infection. The combined data indicate distinct roles for myeloid and nonmyeloid MyD88 and suggest that expression of the pgm is necessary to skew the inflammatory response in the lungs to cause pneumonic plague., (Copyright © 2021 American Society for Microbiology.)

Formyl peptide receptor 1 (FPR1) is a pattern-recognition receptor that detects bacterial as well as endogenous danger-associated molecular patterns to trigger innate immune responses by myeloid cells. A single nucleotide polymorphism, rs867228 (allelic frequency 19-20%), in the gene coding for FPR1 accelerates the manifestation of multiple carcinomas, likely due to reduced anticancer immunosurveillance secondary to a defect in antigen presentation by dendritic cells. Another polymorphism in FPR1 , rs5030880 (allelic frequency 12-13%), has been involved in the resistance to plague, correlating with the fact that FPR1 is the receptor for Yersinia pestis . Driven by the reported preclinical effects of FPR1 on lung inflammation and fibrosis, we investigated whether rs867228 or rs5030880 would affect the severity of coronavirus disease-19 (COVID-19). Data obtained on patients from two different hospitals in Paris refute the hypothesis that rs867228 or rs5030880 would affect the severity of COVID-19., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla ) had been used as a specific marker of Y. pestis , but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae . Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

Plague continued to afflict Europe for more than five centuries after the Black Death. Yet, by the 17th century, the dynamics of plague had changed, leading to its slow decline in Western Europe over the subsequent 200 y, a period for which only one genome was previously available. Using a multidisciplinary approach, combining genomic and historical data, we assembled Y. pestis genomes from nine individuals covering four Eurasian sites and placed them into an historical context within the established phylogeny. CHE1 (Chechnya, Russia, 18th century) is now the latest Second Plague Pandemic genome and the first non-European sample in the post-Black Death lineage. Its placement in the phylogeny and our synthesis point toward the existence of an extra-European reservoir feeding plague into Western Europe in multiple waves. By considering socioeconomic, ecological, and climatic factors we highlight the importance of a noneurocentric approach for the discussion on Second Plague Pandemic dynamics in Europe., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The Himalayan marmot ( Marmota himalayana ), a natural host and transmitter of plague, is also susceptible to the hepadnavirus infection. To reveal the genetic basis of the hepadnavirus susceptibility and the immune response to plague, we systematically characterized the features of immune genes in Himalayan marmot with those of human and mouse. We found that the entire major histocompatibility complex region and the hepatitis B virus pathway genes of the Himalayan marmot were conserved with those of humans. A Trim (tripartite motif) gene cluster involved in immune response and antiviral activity displays dynamic evolution, which is reflected by the duplication of Trim5 and the absence of Trim22 and Trim34 . Three key regions of Ntcp, which is critical for hepatitis B virus entry, had high identity among seven species of Marmota . Moreover, we observed a severe alveolar hemorrhage, inflammatory infiltrate in the infected lungs and livers from Himalayan marmots after infection of EV76, a live attenuated Yersinia pestis strain. Lots of immune genes were remarkably up-regulated, which several hub genes Il2rγ , Tra29 , and Nlrp7 are placed at the center of the gene network. These findings suggest that Himalayan marmot is a potential animal model for study on the hepadnavirus and plague infection.

Background and Aims: Nucleotide Oligomerisation Domain 2 [NOD2] is a key gene of innate immunity which participates in the host defence against pathogens. Several loss-of-function NOD2 mutations are associated with Crohn's disease [CD]. Their high frequencies in populations of European ancestry suggest a model of balancing selection. Because NOD2 deficiency has been associated with a resistance to Yersinia pseudotuberculosis in mice, we hypothesised that NOD2 mutations have been selected during past plague outbreaks due to the closely related bacterium Yersinia pestis., Methods: Contemporary frequencies of the main CD-associated NOD2 mutations [R702W, G908R, and 1007fs], measured in healthy people from European and Mediterranean countries, were collected from 60 studies via a PubMed search. Plague exposure was calculated from a dataset providing outbreaks from 1346 to 1860 in Europe and the Mediterranean Bassin. A plague index was built to capture the intensity of plague exposure in the studied geographical areas., Results: NOD2 mutation frequencies were associated with the past exposure to plague. Statistical significance was obtained for the most frequent mutation [R702W, p = 0.03] and for the pooled three mutations [p = 0.023]. The association remained significant when putative demographic biases were considered., Conclusions: This result argues for a selection of CD-associated NOD2 mutations by plague outbreaks and further questioned the role of exposure to enteropathogenic Yersinia species in CD., (Copyright © 2019 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Quantitative knowledge about which natural and anthropogenic factors influence the global spread of plague remains sparse. We estimated the worldwide spreading velocity of plague during the Third Pandemic, using more than 200 years of extensive human plague case records and genomic data, and analyzed the association of spatiotemporal environmental factors with spreading velocity. Here, we show that two lineages, 2.MED and 1.ORI3, spread significantly faster than others, possibly reflecting differences among strains in transmission mechanisms and virulence. Plague spread fastest in regions with low population density and high proportion of pasture- or forestland, findings that should be taken into account for effective plague monitoring and control. Temperature exhibited a nonlinear, U-shaped association with spread speed, with a minimum around 20 °C, while precipitation showed a positive association. Our results suggest that global warming may accelerate plague spread in warm, tropical regions and that the projected increased precipitation in the Northern Hemisphere may increase plague spread in relevant regions., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Yersinia pestis causes bubonic, pneumonic, and septicemic plague. Although no longer responsible for pandemic outbreaks, pneumonic plague continues to be a challenge for medical treatment and has been classified as a reemerging disease in some parts of the world. In the early stage of infection, inflammatory responses are believed to be suppressed by Y. pestis virulence factors in order to prevent clearance, while later, the hyperactivation of inflammation contributes to the progression of disease. In this work, we sought to identify the host factors that mediate this process and studied the role of the Toll/interleukin 1 (IL-1) receptor adapter and major inflammatory mediator myeloid differentiation primary response 88 (MyD88) in pneumonic plague. We show that pulmonary challenge of Myd88 -/- mice with wild-type (WT) Y. pestis results in significant loss of pro- and anti-inflammatory cytokines and chemokines, especially gamma interferon (IFN-γ) and KC, in the lungs compared to that in WT mice. Bacterial growth in the lungs occurred more rapidly in the WT mice, however, indicating a role for the MyD88 response in facilitating the primary lung infection. Nevertheless, Myd88 -/- mice were more sensitive to lethality from secondary septicemic plague. Together these findings indicate a central role for MyD88 during the biphasic inflammatory response to pulmonary Y. pestis infection. In the early phase, low-level MyD88-dependent chemokine expression limits initial growth but facilitates Y. pestis access to a protected replicative niche. The later hyperinflammatory phase is partially MyD88 dependent and ineffective in the lungs but controls systemic infection and reduces the progression of secondary septicemic plague., (Copyright © 2019 Olson et al.)

Initiation of treatment during the pre-symptomatic phase of Yersinia pestis (Y. pestis) infection is particularly critical. The rapid proliferation of Y. pestis typically couples with the manifestation of common flu-like early symptoms that often misguides the medical intervention. Our study used African green monkeys (AGM) that did not exhibit clear clinical symptoms for nearly two days after intranasal challenge with Y. pestis and succumbed within a day after showing the first signs of clinical symptoms. The lung, and mediastinal and submandibular lymph nodes (LN) accumulated significant Y. pestis colonization immediately after the intranasal challenge. Hence, organ-specific molecular investigations are deemed to be the key to elucidating mechanisms of the initial host response. Our previous study focused on the whole blood of AGM, and we found early perturbations in the ubiquitin-microtubule-mediated host defense. Altered expression of the genes present in ubiquitin and microtubule networks indicated an early suppression of these networks in the submandibular lymph nodes. In concert, the upstream toll-like receptor signaling and downstream NFκB signaling were inhibited at the multi-omics level. The inflammatory response was suppressed in the lungs, submandibular lymph nodes and mediastinal lymph nodes. We posited a causal chain of molecular mechanisms that indicated Y. pestis was probably able to impair host-mediated proteolysis activities and evade autophagosome capture by dysregulating both ubiquitin and microtubule networks in submandibular lymph nodes. Targeting these networks in a submandibular LN-specific and time-resolved fashion could be essential for development of the next generation therapeutics for pneumonic plague., Competing Interests: The authors have declared that no competing interests exist.

It is generally accepted that human immunodeficiency virus (HIV) is the etiological agent of acquired immune deficiency syndrome. According to this claim, HIV was transferred to humans from contact with monkeys around 35-50 years ago. However, this claim has not been sufficiently confirmed epidemiologically. The spread and incubation period of the plague epidemic has led to the theory that the Black Death was caused by hemorrhagic viruses. Having examined detailed historical data, we have concluded that the bacterium Yersenia pestis was an infectious agent in the epidemic, together with another agent which we suggest was HIV. Our considerations were mainly based on the existence of the CCR5 delta 32 mutation, which protects against HIV infection and has been present in the Caucasian population for over 2000 years. The combination of two infectious agents led to the devastation of the Black Death, the removal of HIV carriers, and an increase in the number of CCR5Δ32 mutations in the Caucasian population. In sub-Saharan Africa, this epidemic and subsequent sanitation process did not occur, which explains the much higher level of HIV genetic information in this part of the world.

Yersinia pestis causes bubonic, pneumonic, and septicemic plague, diseases that are rapidly lethal to most mammals, including humans. Plague develops as a consequence of bacterial neutralization of the host's innate immune response, which permits uncontrolled growth and causes the systemic hyperactivation of the inflammatory response. We previously found that host type I interferon (IFN) signaling is induced during Y. pestis infection and contributes to neutrophil depletion and disease. In this work, we show that type I IFN expression is derived from the recognition of intracellular Y. pestis by host Toll-like receptor 7 (TLR7). Type I IFN expression proceeded independent of myeloid differentiation factor 88 (MyD88), which is the only known signaling adaptor for TLR7, suggesting that a noncanonical mechanism occurs in Y. pestis -infected macrophages. In the murine plague model, TLR7 was a significant contributor to the expression of serum IFN-β, whereas MyD88 was not. Furthermore, like the type I IFN response, TLR7 contributed to the lethality of septicemic plague and was associated with the suppression of neutrophilic inflammation. In contrast, TLR7 was important for defense against disease in the lungs. Together, these data demonstrate that an atypical TLR7 signaling pathway contributes to type I IFN expression during Y. pestis infection and suggest that the TLR7-driven type I IFN response plays an important role in determining the outcome of plague., (Copyright © 2017 Dhariwala et al.)

The primary host of plague in the Qinghai-Tibet Plateau (QTP), China, is Marmota himalayana, which plays an essential role in the maintenance, transmission, and prevalence of plague. To achieve a more clear insight into the differentiation of M. himalayana, complete cytochrome b (cyt b) gene and 11 microsatellite loci were analyzed for a total of 423 individuals from 43 localities in the northeast of the QTP. Phylogenetic analyses with maximum likelihood and Bayesian inference methods showed that all derived haplotypes diverged into two primary well-supported monophyletic lineages, I and II, which corresponded to the referential sequences of two recognized subspecies, M. h. himalayana and M. h. robusta, respectively. The divergence between the two lineages was estimated to be at about 1.03 million years ago, nearly synchronously with the divergence between M. baibacina and M. kastschenkoi and much earlier than that between M. vancouverensis and M. caligata. Genetic structure analyses based on the microsatellite dataset detected significant admixture between the two lineages in the mixed region, which verified the intraspecies level of the differentiation between the two lineages. Our results for the first time demonstrated the coexistence of M. h. himalayana and M. h. robusta, and also, determined the distribution range of the two subspecies in the northeast of QTP. We provided fundamental information for more effective plague control in the QTP.

Objective: To investigate the CRISPR genotypes (clusters) and regional distribution of Yersinia pestis in Qinghai-plateau. Methods: One hundred and two isolates of Y. pestis isolated from human plague patients, host animal and insect vectors from Qinghai-plateau were selected. The DNAs were extracted using the traditional sodium dodecyl sulfate decomposition and phenol-chloroform method. Three CRISPR loci YPa, YPb and YPc of 102 isolates of Y. pesits were amplified and sequenced, and then the CRISPR sequence analysis was carried out by comparing the latest published CRISPR spacer dictionary and the NCBI database to identify the spacer and spacer array. CRISPR genotyping of isolates of Y. pesits were finally conducted according to the polymorphism of the spacer arrays and the regional distribution pattern of isolates of Y. pesits in Qinghai-plateau was described. Results: Forty spacers including 22 of YPa, 13 of YPb and 5 of YPc were observed among 102 isolates of Y. pestis in Qinghai-plateau, of which 5 spacers (a1', a103, a104, b4'' and b4''') were firstly identified. Meanwhile, 16, 10, and 5 different spacer arrays were obtained in YPa, YPb and YPc respectively, including 11 new spacer arrays detected in this study. One hundred and two isolates were divided into 24 CRISPR genotypes and classified into 9 CRISPR clusters (Cb4, Cb4', Cb2, Ca37, Ca7, Ca7', CaΔ5', Ca35' and Cc3'). Each dominant cluster presented significant aggregation geographically: Ca7 were found in Yushu, Nangqian, Chenduo, Zaduo, Zhiduo and Qumalai countries. Ca7' were found in Xunhua, Tongren, Zeku, Tongde, Maqin and Guinan countries. CaΔ5' were restricted to Qilian, Gangcha, Menyuan and Datong countries. CaΔ35' were found in Huangyuan, Haiyan, Gangcha, Tianjun, Delingha, Wulan, Doulan, Gonghe, Xinghai, Guide and Tongde countries. Conclusion: CRISPR-based genotyping analyses showed complicated population of Y. pestis in Qinghai-plateau. Four clusters, Ca7, Ca7', CaΔ5' and Ca35' were the most epidemic dominant four clusters and presented obvious regional distribution patterns, which instructed us to strengthen the surveillance and prevention and control by CRISPR-genotyping technique.

To perform their functions proteins must interact with each other, but how these interactions influence bacterial infection remains elusive. Here we demonstrate that connectivity in the host-pathogen interactome is directly related to pathogen fitness during infection. Using Y. pestis as a model organism, we show that the centrality-lethality rule holds for pathogen fitness during infection but only when the host-pathogen interactome is considered. Our results suggest that the importance of pathogen proteins during infection is directly related to their number of interactions with the host. We also show that pathogen proteins causing an extensive rewiring of the host interactome have a higher impact in pathogen fitness during infection. Hence, we conclude that hubs in the host-pathogen interactome should be explored as promising targets for antimicrobial drug design.

Aim: Determination of the degree of phylogenetic relationship of Yersinia pestis strains iso- lated from the territories of natural foci of plague from the Caucasus using VNTR-typing by 25 loci (MLVA25)., Materials and Methods: 26 strains of Y pestis from Russian natural foci of the Caucasus were used in the study. 25 loci of tandem repeats in Y pestis genome by Le Fleche scheme were used for execution of multi-locus VNTR-analysis. Deciphering of nucleotide sequences was carried out in automatic sequencer ABI 3130 Genetic Analyser. Analysis of confinement of clusters to certain territories, objects and time of isolation of strains was carried out. using Arc GIS 10.1 program., Results: Groups of MLVA25-types of various levels of discrimination were formed: clusters, groups and subgroups. Clusters were formed by strains ofvarious taxonomic membership: main and subspecies of Y pestis. Subgroups reflect membership of strains in certain foci, and MLVA25-types - the degree of genetic relationship., Conclusion: Genetic <> of plague causative agents obtained using MLVA25-types circulating in various natural-focal territories allow to solve problems ofboth theoretical and practical character: from interpretation of microevolution processes to the search of the source of infection and ways of its spread during possible epidemic complications.

Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence., Competing Interests: The authors have declared that no competing interests exist.

This paper studies a full nucleotide sequence of cryptic plasmid pTP33, which was isolated from the typical plague strain of the Tuvinian natural focus, Yersinia pestis I-2638. Sequencing was carried out using the 454 GS Junior platform (Roche). In analysis using the software package GS De Novo Assembler v. 2.7 (Roche) and the algorithm Newbler v. 2.7, 1855 nucleotide reads, which contained 1101246 nucleotides, were assembled to a contig of 33 978 bp. The GC content of the obtained nucleotide sequence was 50.25%. During annotation, we found 56 open reading frames. Homologs of the predicted reading frames were sought in the BLAST databases. We detected 22 reading frames coding hypothetical proteins, 23 frames coding phagerelated proteins, and 11 frames coding proteins with known functions, including toxin–antitoxin system YefM-YoeB, nucleic acids and polysaccharides metabolism proteins (exopolysaccharide production protein ExoZ, exodeoxyribonuclease VIII), and replication proteins (ParA). Some predicted pTP33 proteins were found to be homologs (from 45 to 75%) with sequences of phage-related proteins of certain microorganisms—endosymbionts of insects (Sodalis glossinidius) and endosymbionts of entomopathogenic nematodes (Photorhabdus luminescens, P. asymbiotica, Xenorhabdus bovienii).

Ancient DNA (aDNA) recovered from plague victims of the second plague pandemic (14th to 17th century), excavated from two different burial sites in Germany, and spanning a time period of more than 300 years, was characterized using single nucleotide polymorphism (SNP) analysis. Of 30 tested skeletons 8 were positive for Yersinia pestis-specific nucleic acid, as determined by qPCR targeting the pla gene. In one individual (MP-19-II), the pla copy number in DNA extracted from tooth pulp was as high as 700 gene copies/μl, indicating severe generalized infection. All positive individuals were identical in all 16 SNP positions, separating phylogenetic branches within nodes N07&#95;N10 (14 SNPs), N07&#95;N08 (SNP s19) and N06&#95;N07 (s545), and were highly similar to previously investigated plague victims from other European countries. Thus, beside the assumed continuous reintroduction of Y. pestis from central Asia in multiple waves during the second pandemic, long-term persistence of Y. pestis in Europe in a yet unknown reservoir host has also to be considered.

Background: Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections., Principal Findings: In vitro treatment of Y. pestis culture with sodium tellurite (Na2TeO3) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype., Conclusions: These findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a bacterial adaptive strategy to macrophage associated stresses.

Yersinia pestis has evolved as a clonal variant of Yersinia pseudotuberculosis to cause flea-borne biofilm-mediated transmission of the bubonic plague. The LysR-type transcriptional regulator, RovM, is highly induced only during Y. pestis infection of the flea host. RovM homologs in other pathogens regulate biofilm formation, nutrient sensing, and virulence; including in Y. pseudotuberculosis, where RovM represses the major virulence factor, RovA. Here the role that RovM plays during flea infection was investigated using a Y. pestis KIM6+ strain deleted of rovM, ΔrovM. The ΔrovM mutant strain was not affected in characteristic biofilm gut blockage, growth, or survival during single infection of fleas. Nonetheless, during a co-infection of fleas, the ΔrovM mutant exhibited a significant competitive fitness defect relative to the wild type strain. This competitive fitness defect was restored as a fitness advantage relative to the wild type in a ΔrovM mutant complemented in trans to over-express rovM. Consistent with this, Y. pestis strains, producing elevated transcriptional levels of rovM, displayed higher growth rates, and differential ability to form biofilm in response to specific nutrients in comparison to the wild type. In addition, we demonstrated that rovA was not repressed by RovM in fleas, but that elevated transcriptional levels of rovM in vitro correlated with repression of rovA under specific nutritional conditions. Collectively, these findings suggest that RovM likely senses specific nutrient cues in the flea gut environment, and accordingly directs metabolic adaptation to enhance flea gut colonization by Y. pestis.

An ecological scenario is proposed for the origin of causative agent of the plague (the bacterium Yersenia pestis) from the clone of pseudotuberculous microbe of the first serotype Y. pseudotuberculosis O:1b. Disclosed are the conditions of gradual intrusion of psychrophile saprozoonosis ancestor into the blood of the primary host, Mongolian tarbagan marmot Marmota sibirica. As an inductor of speciation acted the Sartan cooling that occurred in the end of late Pleistocene under conditions of arid ultra-continental climate in Central Asia. Soil freezing down to the level of hibernating chambers in marmot burrows initiated the transition of marmot flea, Oropsylla silantiewi, larvae to optional hemophagy on the mucous coat inside the mouth cavity of sleeping marmots. In its turn, this promoted the conditions of mass traumatic intrusion of Y pseudotuberculosis into marmots bloodstream from faecal particles getting in their mouth cavity in course of building up a plug in a burrow for hibernating. In marmot populations, the selection of bacteria underwent under conditions of heterothermy with repeated changes of hibernating marmots body temperature within the range of 5-37 degrees C (torpor-euthermy). During the warm season, when pseudotuberculous microbes are totally eliminated from the bloodstream of healthy marmots with body temperature about 37 degrees C, bacteria could survive in fleas' digestive tract in the form of biofilm developing in proventriculus as a so called blockage. Final isolation between ancestral and daughter species was helped by the development of intrapopulation antagonism related with the beginning of full-scale synthesis of bacteriocin pesticin. Population-genetic processes in the "marmot-flea" system have led to a macroevolutionary event, that is, to passage of bacteria in a new ecological niche and adaptive zone that are principally different from those of the ancestor. All the present intraspecies forms of Y. pestis that appeared due to microevolution, have originated with the subspecies Y. pestis tarbagani that has formed in Central Asia during the Sartan cooling.

The genetic diversity of Yersinia pestis strains from the Mongolian natural plague foci has been investigated. A total of 32 strains isolated from western, eastern, and central aimaks, as well as from the territory of the Gobi region, have been studied. Twenty-four strains belong to the main Y. pestis subspecies, while eight belong to other subspecies. There is only one strain of biovar medievalis (genovariant 2.MED1) among the strains of the main subspecies, while the rest of the subspecies belong to the biovar antiqua. Biovar antiqua strains are split into three groups. Strains from the eastern part of the country were classified as genovariant 2.ANT3, and those from the western and central regions were classified as genovariant 3.ANT2, which was endemic for Mongolia. One strain from the Bayan-Ulegeiskii aimak had the rare genovariant 4.ANT. None of the strains of the biovar antiqua belonged to its ancient 0.ANT branch, which is inconsistent with the commonly accepted idea that ancient marmot's plague agent race originates from Mongolia. Six out of eight strains of the minor subspecies belonged to the ulegeica subspecies, which are endemic to Mongolia, one strain belonged to the microtus group, and the last belonged to a previously uncharacterized variant of the minor subspecies.

The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

Yersinia pestis is a human pathogen and can cause serious disease. Biosafety level 3 (BSL3) is required when handling this microorganism and all work requires a biological safety cabinet. For pulsed-field gel electrophoresis (PFGE), dedicated BSL3 PFGE equipment or a documented procedure that ensures that all viable bacteria are inactivated is required. All plasticware and glassware that comes into contact with the cultures should be disinfected/sterilized or disposed of in a safe manner, according to the guidelines of institution. This includes decontamination of pipettes, spatulas, etc. that were in contact with the cell suspensions or plugs. Disinfection of reusable plug molds should be done before they are washed; the disposable plug molds, including the tape and the tab that was used to push the plugs out of the wells, are also contaminated and should be disinfected with 10 % bleach for at least 30 min if they will be washed and reused.

Background: We propose the computational reconstruction of a whole bacterial ancestral genome at the nucleotide scale, and its validation by a sequence of ancient DNA. This rare possibility is offered by an ancient sequence of the late middle ages plague agent. It has been hypothesized to be ancestral to extant Yersinia pestis strains based on the pattern of nucleotide substitutions. But the dynamics of indels, duplications, insertion sequences and rearrangements has impacted all genomes much more than the substitution process, which makes the ancestral reconstruction task challenging., Results: We use a set of gene families from 13 Yersinia species, construct reconciled phylogenies for all of them, and determine gene orders in ancestral species. Gene trees integrate information from the sequence, the species tree and gene order. We reconstruct ancestral sequences for ancestral genic and intergenic regions, providing nearly a complete genome sequence for the ancestor, containing a chromosome and three plasmids., Conclusion: The comparison of the ancestral and ancient sequences provides a unique opportunity to assess the quality of ancestral genome reconstruction methods. But the quality of the sequencing and assembly of the ancient sequence can also be questioned by this comparison.

The arthropod-borne transmission route of Yersinia pestis, the bacterial agent of plague, is a recent evolutionary adaptation. Yersinia pseudotuberculosis, the closely related food-and water-borne enteric species from which Y. pestis diverged less than 6,400 y ago, exhibits significant oral toxicity to the flea vectors of plague, whereas Y. pestis does not. In this study, we identify the Yersinia urease enzyme as the responsible oral toxin. All Y. pestis strains, including those phylogenetically closest to the Y. pseudotuberculosis progenitor, contain a mutated ureD allele that eliminated urease activity. Restoration of a functional ureD was sufficient to make Y. pestis orally toxic to fleas. Conversely, deletion of the urease operon in Y. pseudotuberculosis rendered it nontoxic. Enzymatic activity was required for toxicity. Because urease-related mortality eliminates 30-40% of infective flea vectors, ureD mutation early in the evolution of Y. pestis was likely subject to strong positive selection because it significantly increased transmission potential.

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

The study of sampling of strains of Y. pestis of main and altaic subspecies was implemented. The modern technique of identification of microorganisms was applied using MALDI-TOF mass spectrometry analysis. The evaluation of biological safety of method of sampling preparation was implemented. To supplement the identification base "BioTyper" the spectrum of typical strains of Y. pestis were obtained. The enhanced identification base was used to evaluate possibilities of application of MALDI-TOF technology for identification and taxonomic differentiation of Y. pestis from other representatives of genus of Yersinia. In the process of study a complete concordance of results of mass spectrometry identification and classic cultural method was observed. On the basis of mass spectrometry characteristic of analyzed sampling the differentiation between strains of Y. pestis of subspecies pestis and strains of subspecies altaica was implemented. The study results testify the effectiveness of application of mass spectrometry analysis for reliable interspecies and intraspecific differentiation of plague agent. The simplicity and velocity of sampling preparation and implementation of analysis and low cost of active storage allow considering the MALDI-TOF technology of mass spectrometry identification as highly perspective method for laboratory diagnostic of plague agent.

Bacterial pathogens have evolved various mechanisms to modulate host immune responses for successful infection. In this study, RNA-sequencing technology was used to analyze the responses of human monocytes THP1 to Yersinia pestis infection. Over 6000 genes were differentially expressed over the 12 h infection. Kinetic responses of pathogen recognition receptor signaling pathways, apoptosis, antigen processing, and presentation pathway and coagulation system were analyzed in detail. Among them, RIG-I-like receptor (RLR) signaling pathway, which was established for antiviral defense, was significantly affected. Mice lacking MAVS, the adaptor of the RLR signaling pathway, were less sensitive to infection and exhibited lower IFN-β production, higher Th1-type cytokines IFN-γ and IL-12 production, and lower Th2-type cytokines IL-4 and IL-13 production in the serum compared with wild-type mice. Moreover, infection of pathogenic bacteria other than Y. pestis also altered the expression of the RLR pathway, suggesting that the response of RLR pathway to bacterial infection is a universal mechanism., (Copyright © 2014 The Society of Chinese Scholars on Exercise Physiology and Fitness. Published by Elsevier Ltd. All rights reserved.)

It is recognised that the genetic profiles that give rise to chronic inflammatory diseases, under the influence of environmental agents, might have been implicated in the host defence mechanism against lethal infections in the past. Behçet's disease (BD) is an immune-mediated inflammatory disease, expressed as vasculitis, triggered by environmental factors in genetically susceptible individuals. We carried out a review of published data to draw up an evolutionary adaptation model, as Author's perspective, for genetic susceptibility factors and inflammatory immune response involved in BD pathogenesis. Two lethal infectious agents, Plasmodium Falciparum and Yersinia Pestis, are proposed as the putative driving forces that favoured the fixing of the major genetic susceptibility factors to BD, thus determining its geoepidemiology. Further studies are needed to confirm the validity of this evolutionary model which includes and integrates the key insights of previous hypotheses.

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69-77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69-77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69-77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69-77-mediated protection. In contrast, YopE69-77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.

Among the 13 TLRs in the vertebrate systems, only TLR4 utilizes both Myeloid differentiation factor 88 (MyD88) and Toll/Interleukin-1 receptor (TIR)-domain-containing adapter interferon-β-inducing Factor (TRIF) adaptors to transduce signals triggering host-protective immune responses. Earlier studies on the pathway combined various experimental data in the form of one comprehensive map of TLR signaling. But in the absence of adequate kinetic parameters quantitative mathematical models that reveal emerging systems level properties and dynamic inter-regulation among the kinases/phosphatases of the TLR4 network are not yet available. So, here we used reaction stoichiometry-based and parameter independent logical modeling formalism to build the TLR4 signaling network model that captured the feedback regulations, interdependencies between signaling kinases and phosphatases and the outcome of simulated infections. The analyses of the TLR4 signaling network revealed 360 feedback loops, 157 negative and 203 positive; of which, 334 loops had the phosphatase PP1 as an essential component. The network elements' interdependency (positive or negative dependencies) in perturbation conditions such as the phosphatase knockout conditions revealed interdependencies between the dual-specific phosphatases MKP-1 and MKP-3 and the kinases in MAPK modules and the role of PP2A in the auto-regulation of Calmodulin kinase-II. Our simulations under the specific kinase or phosphatase gene-deficiency or inhibition conditions corroborated with several previously reported experimental data. The simulations to mimic Yersinia pestis and E. coli infections identified the key perturbation in the network and potential drug targets. Thus, our analyses of TLR4 signaling highlights the role of phosphatases as key regulatory factors in determining the global interdependencies among the network elements; uncovers novel signaling connections; identifies potential drug targets for infections.

Background: Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14-17th centuries) and third (19-20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6-8th centuries). To address this gap, we sequenced and analysed draft genomes of Y pestis obtained from two individuals who died in the first pandemic., Methods: Teeth were removed from two individuals (known as A120 and A76) from the early medieval Aschheim-Bajuwarenring cemetery (Aschheim, Bavaria, Germany). We isolated DNA from the teeth using a modified phenol-chloroform method. We screened DNA extracts for the presence of the Y pestis-specific pla gene on the pPCP1 plasmid using primers and standards from an established assay, enriched the DNA, and then sequenced it. We reconstructed draft genomes of the infectious Y pestis strains, compared them with a database of genomes from 131 Y pestis strains from the second and third pandemics, and constructed a maximum likelihood phylogenetic tree., Findings: Radiocarbon dating of both individuals (A120 to 533 AD [plus or minus 98 years]; A76 to 504 AD [plus or minus 61 years]) places them in the timeframe of the first pandemic. Our phylogeny contains a novel branch (100% bootstrap at all relevant nodes) leading to the two Justinian samples. This branch has no known contemporary representatives, and thus is either extinct or unsampled in wild rodent reservoirs. The Justinian branch is interleaved between two extant groups, 0.ANT1 and 0.ANT2, and is distant from strains associated with the second and third pandemics., Interpretation: We conclude that the Y pestis lineages that caused the Plague of Justinian and the Black Death 800 years later were independent emergences from rodents into human beings. These results show that rodent species worldwide represent important reservoirs for the repeated emergence of diverse lineages of Y pestis into human populations., Funding: McMaster University, Northern Arizona University, Social Sciences and Humanities Research Council of Canada, Canada Research Chairs Program, US Department of Homeland Security, US National Institutes of Health, Australian National Health and Medical Research Council., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Bubonic plague (a fatal, flea-transmitted disease) remains an international public health concern. Although our understanding of the pathogenesis of bubonic plague has improved significantly over the last few decades, researchers have still not been able to define the complete set of Y. pestis genes needed for disease or to characterize the mechanisms that enable infection. Here, we generated a library of Y. pestis mutants, each lacking one or more of the genes previously identified as being up-regulated in vivo. We then screened the library for attenuated virulence in rodent models of bubonic plague. Importantly, we tested mutants both individually and using a novel, "per-pool" screening method that we have developed. Our data showed that in addition to genes involved in physiological adaptation and resistance to the stress generated by the host, several previously uncharacterized genes are required for virulence. One of these genes (ympt1.66c, which encodes a putative helicase) has been acquired by horizontal gene transfer. Deletion of ympt1.66c reduced Y. pestis' ability to spread to the lymph nodes draining the dermal inoculation site--probably because loss of this gene decreased the bacteria's ability to survive inside macrophages. Our results suggest that (i) intracellular survival during the early stage of infection is important for plague and (ii) horizontal gene transfer was crucial in the acquisition of this ability.