Paleolithic to Bronze Age Siberians Reveal Connections with First Americans and across Eurasia.

Modern humans have inhabited the Lake Baikal region since the Upper Paleolithic, though the precise history of its peoples over this long time span is still largely unknown. Here, we report genome-wide data from 19 Upper Paleolithic to Early Bronze Age individuals from this Siberian region. An Upper Paleolithic genome shows a direct link with the First Americans by sharing the admixed ancestry that gave rise to all non-Arctic Native Americans. We also demonstrate the formation of Early Neolithic and Bronze Age Baikal populations as the result of prolonged admixture throughout the eighth to sixth millennium BP. Moreover, we detect genetic interactions with western Eurasian steppe populations and reconstruct Yersinia pestis genomes from two Early Bronze Age individuals without western Eurasian ancestry. Overall, our study demonstrates the most deeply divergent connection between Upper Paleolithic Siberians and the First Americans and reveals human and pathogen mobility across Eurasia during the Bronze Age.

Emergence and Spread of Basal Lineages of Yersinia pestis during the Neolithic Decline.

Between 5,000 and 6,000 years ago, many Neolithic societies declined throughout western Eurasia due to a combination of factors that are still largely debated. Here, we report the discovery and genome reconstruction of Yersinia pestis, the etiological agent of plague, in Neolithic farmers in Sweden, pre-dating and basal to all modern and ancient known strains of this pathogen. We investigated the history of this strain by combining phylogenetic and molecular clock analyses of the bacterial genome, detailed archaeological information, and genomic analyses from infected individuals and hundreds of ancient human samples across Eurasia. These analyses revealed that multiple and independent lineages of Y. pestis branched and expanded across Eurasia during the Neolithic decline, spreading most likely through early trade networks rather than massive human migrations. Our results are consistent with the existence of a prehistoric plague pandemic that likely contributed to the decay of Neolithic populations in Europe.

Droplet Tn-Seq identifies the primary secretion mechanism for yersiniabactin in Yersinia pestis.

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.

Stone Age Yersinia pestis genomes shed light on the early evolution, diversity, and ecology of plague.

The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague's formative years in terms of its early evolution and ecology.

No evidence for persistent natural plague reservoirs in historical and modern Europe.

Caused by Yersinia pestis, plague ravaged the world through three known pandemics: the First or the Justinianic (6th-8th century); the Second (beginning with the Black Death during c.1338-1353 and lasting until the 19th century); and the Third (which became global in 1894). It is debatable whether Y. pestis persisted in European wildlife reservoirs or was repeatedly introduced from outside Europe (as covered by European Union and the British Isles). Here, we analyze environmental data (soil characteristics and climate) from active Chinese plague reservoirs to assess whether such environmental conditions in Europe had ever supported "natural plague reservoirs". We have used new statistical methods which are validated through predicting the presence of modern plague reservoirs in the western United States. We find no support for persistent natural plague reservoirs in either historical or modern Europe. Two factors make Europe unfavorable for long-term plague reservoirs: 1) Soil texture and biochemistry and 2) low rodent diversity. By comparing rodent communities in Europe with those in China and the United States, we conclude that a lack of suitable host species might be the main reason for the absence of plague reservoirs in Europe today. These findings support the hypothesis that long-term plague reservoirs did not exist in Europe and therefore question the importance of wildlife rodent species as the primary plague hosts in Europe.

High genetic diversity of the himalayan marmot relative to plague outbreaks in the Qinghai-Tibet Plateau, China.

Plague, as an ancient zoonotic disease caused by Yersinia pestis, has brought great disasters. The natural plague focus of Marmota himalayana in the Qinghai-Tibet Plateau is the largest, which has been constantly active and the leading source of human plague in China for decades. Understanding the population genetics of M. himalayana and relating that information to the biogeographic distribution of Yersinia pestis and plague outbreaks are greatly beneficial for the knowledge of plague spillover and arecrucial for pandemic prevention. In the present research, we assessed the population genetics of M. himalayana. We carried out a comparative study of plague outbreaks and the population genetics of M. himalayana on the Qinghai-Tibet Plateau. We found that M. himalayana populations are divided into two main clusters located in the south and north of the Qinghai-Tibet Plateau. Fourteen DFR genomovars of Y. pestis were found and exhibited a significant region-specific distribution. Additionally, the increased genetic diversity of plague hosts is positively associated with human plague outbreaks. This insight gained can improve our understanding of biodiversity for pathogen spillover and provide municipally directed targets for One Health surveillance development, which will be an informative next step toward increased monitoring of M. himalayana dynamics.

Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017.

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

Symbiosis of a lytic bacteriophage and Yersinia pestis and characteristics of plague in Marmota himalayana.

Surveillance for animal plague was conducted in the Marmota himalayana plague focus of the Qinghai-Tibet Plateau from 2020 to 2023. A 22.89% positive rate of serum F1 antibody was detected in live-caught marmots, alongside a 43.40% incidence of Yersinia pestis isolation from marmot carcasses. Marmot carcasses infected with plague exhibited a significantly higher spleen-somatic index (P < 0.05). Twenty-one Y. pestis-specific phages were isolated, among which one Y. pestis lytic phage (AKS2022HT87GU_phi) was isolated from the bone marrow of a marmot carcass (no. AKS2022HT87) and was found to be symbiotic with Y. pestis. Microscopy revealed the coexistence of lysed and non-lysed colonies of Y. pestis AKS2022HT87. Genome-wide analysis showed that certain strains of the Y. pestis AKS2022HT87 carried phage DNA fragments consistent with phage AKS2022HT87GU_phi. The rare symbiotic relationship between a lytic phage and Y. pestis observed in vitro was highlighted in this study, laying the basis for further exploring the relationship between Y. pestis and its bacteriophages.IMPORTANCEBacteriophages and host bacteria commonly coexist in vivo or in soil environments through complex and interdependent microbial interactions. However, recapitulating this symbiotic state remains challenging in vitro due to limited medium nutrients. In this work, the natural symbiosis between Yersinia pestis and specific phages has been discovered in a Marmota himalayana specimen. Epidemiological analysis presented the characteristics of the Y. pestis and specific phages in the area with a strong plague epidemic. Crucially, comparative genomics has been conducted to analyze the genetic changes in both the Y. pestis and phages over different periods, revealing the dynamic and evolving nature of their symbiosis. These are the critical steps to study the mechanism of the symbiosis.

Screening and identification of DNA nucleic acid aptamers against F1 protein of Yersinia pestis using SELEX method.

BACKGROUND: Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein. METHODS AND RESULTS: In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E - 7, 2.004E - 8, and 1.68E - 8 M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033 µg/ml for Yer 21, Yer 24, and Yer 25, respectively. CONCLUSION: This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

Lyophilization for bacteria preservation: a promising approach for Yersinia pestis strains from an unique collection in Brazil (Fiocruz-CYP).

Yersinia pestis, the causative agent of plague, is a highly virulent bacterium that poses a significant threat to human health. Preserving this bacterium in a viable state is crucial for research and diagnostic purposes. This paper presents and evaluates a simple lyophilization protocol for the long-term storage of Y. pestis strains from Fiocruz-CYP, aiming to explore its impact on viability and long-term stability, while replacing the currently used methodologies. The lyophilization tests were conducted using the non-virulent Y. pestis strain EV76, subjected to the lyophilization process under vacuum conditions. Viability assessment was performed to evaluate the effects of lyophilization and storage conditions on Y. pestis under multiple temperature conditions (- 80 °C, - 20 °C, 4-8 °C and room temperature). The lyophilization protocol employed in this study consistently demonstrated its efficacy in maintaining high viability rates for Y. pestis samples in a up to one year follow-up. The storage temperature that consistently exhibited the highest recovery rates was - 80 °C, followed by - 20 °C and 4-8 °C. Microscopic analysis of the post-lyophilized cultures revealed preserved morphological features, consistent with viable bacteria. The high viability rates observed in the preserved samples indicate the successful preservation of Y. pestis using this protocol. Overall, the presented lyophilization protocol provides a valuable tool for the long-term storage of Y. pestis, offering stability, viability, and functionality. By refining the currently used methods of lyophilization, this protocol can improve long-term preservation for Y. pestis strains collections, facilitating research efforts, diagnostic procedures, and the development of preventive and therapeutic strategies against plague.

Yersiniabactin contributes to overcoming zinc restriction during Yersinia pestis infection of mammalian and insect hosts.

Yersinia pestis causes human plague and colonizes both a mammalian host and a flea vector during its transmission cycle. A key barrier to bacterial infection is the host's ability to actively sequester key biometals (e.g., iron, zinc, and manganese) required for bacterial growth. This is referred to as nutritional immunity. Mechanisms to overcome nutritional immunity are essential virulence factors for bacterial pathogens. Y. pestis produces an iron-scavenging siderophore called yersiniabactin (Ybt) that is required to overcome iron-mediated nutritional immunity and cause lethal infection. Recently, Ybt has been shown to bind to zinc, and in the absence of the zinc transporter ZnuABC, Ybt improves Y. pestis growth in zinc-limited medium. These data suggest that, in addition to iron acquisition, Ybt may also contribute to overcoming zinc-mediated nutritional immunity. To test this hypothesis, we used a mouse model defective in iron-mediated nutritional immunity to demonstrate that Ybt contributes to virulence in an iron-independent manner. Furthermore, using a combination of bacterial mutants and mice defective in zinc-mediated nutritional immunity, we identified calprotectin as the primary barrier for Y. pestis to acquire zinc during infection and that Y. pestis uses Ybt to compete with calprotectin for zinc. Finally, we discovered that Y. pestis encounters zinc limitation within the flea midgut, and Ybt contributes to overcoming this limitation. Together, these results demonstrate that Ybt is a bona fide zinc acquisition mechanism used by Y. pestis to surmount zinc limitation during the infection of both the mammalian and insect hosts.

Assessing the origins of the European Plagues following the Black Death: A synthesis of genomic, historical, and ecological information.

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.

Dynamics of Changes in the cAMP/cGMP Concentration Ratio in the Thymus and Spleen of Laboratory Mice during Vaccination against Plague and Tularemia against the Background of Immunomodulation.

Vaccine strains Yersinia pestis EV NIIEG at a dose of 103 CFU and Francisella tularensis 15 NIIEG at a dose of 102 CFU induced changes in the concentration of cyclic nucleotides in the thymus and spleen of white mice. Antigen-induced changes in the cAMP/cGMP ratio in immunocompetent organs had a phase or oscillatory character, which seems to be related to the regulation of postvaccination immunoreactivity in the body. Synthetic organoselenium compound 974zh stimulated an increase in the amplitude of cAMP/cGMP oscillations, indicating its stimulating effect on the immunogenic properties of vaccine strains at doses an order of magnitude below the standard doses.

CsrA-Mediated Translational Activation of the hmsE mRNA Enhances HmsD-Dependent C-di-GMP-Enabled Biofilm Production in Yersinia pestis.

The plague bacterium, Yersinia pestis, forms a biofilm-mediated blockage in the flea foregut that enhances its transmission by fleabite. Biofilm formation is positively controlled by cyclic di-GMP (c-di-GMP), which is synthesized by the diguanylate cyclases (DGC), HmsD and HmsT. While HmsD primarily promotes biofilm-mediated blockage of fleas, HmsT plays a more minor role in this process. HmsD is a component of the HmsCDE tripartite signaling system. HmsC and HmsE posttranslationally inhibit or activate HmsD, respectively. HmsT-dependent c-di-GMP levels and biofilm formation are positively regulated by the RNA-binding protein CsrA. In this study we determined whether CsrA positively regulates HmsD-dependent biofilm formation through interactions with the hmsE mRNA. Gel mobility shift assays determined that CsrA binds specifically to the hmsE transcript. RNase T1 footprint assays identified a single CsrA binding site and CsrA-induced structural changes in the hmsE leader region. Translational activation of the hmsE mRNA was confirmed in vivo using plasmid-encoded inducible translational fusion reporters and by HmsE protein expression studies. Furthermore, mutation of the CsrA binding site in the hmsE transcript significantly reduced HmsD-dependent biofilm formation. These results suggest that CsrA binding leads to structural changes in the hmsE mRNA that enhance its translation to enable increased HmsD-dependent biofilm formation. Given the requisite function of HmsD in biofilm-mediated flea blockage, this CsrA-dependent increase in HmsD activity underscores that complex and conditionally defined modulation of c-di-GMP synthesis within the flea gut is required for Y. pestis transmission. IMPORTANCE Mutations enhancing c-di-GMP biosynthesis drove the evolution of Y. pestis to flea-borne transmissibility. c-di-GMP-dependent biofilm-mediated blockage of the flea foregut enables regurgitative transmission of Y. pestis by fleabite. The Y. pestis diguanylate cyclases (DGC), HmsT and HmsD, which synthesize c-di-GMP, play significant roles in transmission. Several regulatory proteins involved in environmental sensing, as well as signal transduction and response regulation, tightly control DGC function. An example is CsrA, a global posttranscriptional regulator that modulates carbon metabolism and biofilm formation. CsrA integrates alternative carbon usage metabolism cues to activate c-di-GMP biosynthesis through HmsT. Here, we demonstrated that CsrA additionally activates hmsE translation to promote c-di-GMP biosynthesis through HmsD. This emphasizes that a highly evolved regulatory network controls c-di-GMP synthesis and Y. pestis transmission.

Sialoglycan-binding patterns of bacterial AB5 toxin B subunits correlate with host range and toxicity, indicating evolution independent of A subunits.

Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.

Pulmonary Expression of Interleukin-17 Contributes to Neutrophil Infiltration into the Lungs during Pneumonic Plague.

Inhalation of respiratory droplets infected with Yersinia pestis results in a rapidly progressing and lethal necrotic pneumonia called primary pneumonic plague. Disease manifests as biphasic, with an initial preinflammatory phase with rapid bacterial replication in the lungs absent readily detectable host immune responses. This is followed by the onset of a proinflammatory phase that sees the dramatic upregulation of proinflammatory cytokines and extensive neutrophil accumulation in the lungs. The plasminogen activator protease (Pla) is an essential virulence factor that is responsible for survival of Y. pestis in the lungs. Our lab recently showed that Pla functions as an adhesin that promotes binding to alveolar macrophages to facilitate translocation of effector proteins called Yops into the cytosol of target host cells via a type 3 secretion system (T3SS). Loss of Pla-mediated adherence disrupted the preinflammatory phase of disease and resulted in early neutrophil migration to the lungs. While it is established that Yersinia broadly suppresses host innate immune responses, it is not clear precisely which signals need to be inhibited to establish a preinflammatory stage of infection. Here, we show that early Pla-mediated suppression of Interleukin-17 (IL-17) expression in alveolar macrophages and pulmonary neutrophils limits neutrophil migration to the lungs and aids in establishing a preinflammatory phase of disease. In addition, IL-17 ultimately contributes to neutrophil migration to the airways that defines the later proinflammatory phase of infection. These results suggest that the pattern of IL-17 expression contributes to the progression of primary pneumonic plague.

Challenging Ancient DNA Results About Putative HLA Protection or Susceptibility to Yersinia pestis.

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.

Immunogenetics, sylvatic plague and its vectors: insights from the pathogen reservoir Mastomys natalensis in Tanzania.

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.

Ancient Yersinia pestis genomes lack the virulence-associated YpfΦ prophage present in modern pandemic strains.

Yersinia pestis is the causative agent of at least three major plague pandemics (Justinianic, Medieval and Modern). Previous studies on ancient Y. pestis genomes revealed that several genomic alterations had occurred approximately 5000-3000 years ago and contributed to the remarkable virulence of this pathogen. How a subset of strains evolved to cause the Modern pandemic is less well-understood. Here, we examined the virulence-associated prophage (YpfΦ), which had been postulated to be exclusively present in the genomes of strains associated with the Modern pandemic. The analysis of two new Y. pestis genomes from medieval/early modern Denmark confirmed that the phage is absent from the genome of strains dating to this time period. An extended comparative genome analysis of over 300 strains spanning more than 5000 years showed that the prophage is found in the genomes of modern strains only and suggests an integration into the genome during recent Y. pestis evolution. The phage-encoded Zot protein showed structural homology to a virulence factor of Vibrio cholerae. Similar to modern Y. pestis, we observed phages with a common origin to YpfΦ in individual strains of other bacterial species. Our findings present an updated view on the prevalence of YpfΦ, which might contribute to our understanding of the host spectrum, geographical spread and virulence of Y. pestis responsible for the Modern pandemic.

Yersinia pestis Δail Mutants Are Not Susceptible to Human Complement Bactericidal Activity in the Flea.

Ail confers serum resistance in humans and is a critical virulence factor of Y. pestis, the causative agent of plague. Here, the contribution of Ail for Y. pestis survival in the flea vector was examined. Rat or human but not mouse sera were bactericidal against a Y. pestis Δail mutant at 28°C in vitro. Complement components deposited rapidly on the Y. pestis surface as measured by immunofluorescent microscopy. Ail reduced the amount of active C3b on the Y. pestis surface. Human sera retained bactericidal activity against a Y. pestis Δail mutant in the presence of mouse sera. However, in the flea vector, the serum protective properties of Ail were not required. Flea colonization studies using murine sera and Y. pestis KIM6+ wild type, a Δail mutant, and the Δail/ail+ control showed no differences in bacterial prevalence or numbers during the early stage of flea colonization. Similarly, flea studies with human blood showed Ail was not required for serum resistance. Finally, a variant of Ail (AilF100V E108_S109insS) from a human serum-sensitive Y. pestis subsp. microtus bv. Caucasica 1146 conferred resistance to human complement when expressed in the Y. pestis KIM6+ Δail mutant. This indicated that Ail activity was somehow blocked, most likely by lipooligosaccharide, in this serum sensitive strain. IMPORTANCE This work contributes to our understanding of how highly virulent Y. pestis evolved from its innocuous enteric predecessor. Among identified virulence factors is the attachment invasion locus protein, Ail, that is required to protect Y. pestis from serum complement in all mammals tested except mice. Murine sera is not bactericidal. In this study, we asked, is bactericidal sera from humans active in Y. pestis colonized fleas? We found it was not. The importance of this observation is that it identifies a protective niche for the growth of serum sensitive and nonsensitive Y. pestis strains.