Different Strategies for Preparation of Non-tagged rV270 Protein and Its Efficacy against Yersinia Pestis Challenge

Objective LcrV is an important component for the development of a subunit vaccine against plague.To reduce immunosuppressive activity of LcrV,a recombinant LcrV variant lacking amino acids 271 to 326 （rV270） was prepared by different methods in this study.Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a,or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a.After Co2＋ affinity chromatography,a purification strategy was developed by cleavage of His tag on column,following Sephacryl S-200HR column filtration chromatography.Results Removal of His tag by thrombin,enterokinase and factor Xa displayed a yield of 99.5%,32.4% and 15.3%,respectively.Following Sephacryl S-200HR column filtration chromatography,above 97% purity of rV270 protein was obtained.Purified rV270 that was adsorbed to 25% （v/v） Al（OH）3 adjuvant in phosphate-buffered saline （PBS） induced very high titers of antibody to rV270 in BALB/c mice and protected them （100% survival） against subcutaneous challenge with 106 CFU of Y.pestis virulent strain 141.Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa,but they exhibited extremely low cleavage activity to the corresponding recognition site.Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy.The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

Comparative Genomic Analysis of Gene Variations of Two Chinese Yersinia pestis Isolates from Vaccine Strain EV76

Objective To investigate genomic variations of two Chinese Yersinia pestis isolates that were isolated from different plague foci obtained from vaccine strain EV76 from the Yunnan province of China. Methods A microarray containing 12 000 probes covering the entire genome of seven Yersinie pestis and two Yersinia pseudotuberculosis strains, was used. PCR assays were performed to confirm microarray results. Results The gene variations detected included the absence of five genes related to the synthesis of betaine in both EV76 and another sequenced attenuated strain, KIM D27. Several genes related to phage-related membrane proteins were found to be absent in the Antiqua biovar Yunnan strain, 485, which was isolated from a rodent plague foci. Conclusion These findings provide initial insight into the distinct strains isolated from natural foci, within their genomic context, including Yunnan Y. pestis strains. This information will be used therefore to establish subsequent comparisons of these sequences with published complete genomes of other strains.

Use of Rich BHI Medium Instead of Synthetic TMH Medium for Gene Regulation Study in Yersinia pestis

Objective This study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis. Methods The transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 ~C, and with temperature shifting from 26 to 37 ~C, the wild-type （WT） strain or its phoP or crp null mutant （AphoP or Acrp, respectively） was subject to RNA isolation, and then the promoter activity of rovA or plo in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and AphoP to measure the promoter activity of rovA in these two strains with the ^-Galactosidase enzyme assay system. Results When Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that ofpla was stimulated by CRP. Conclusion The rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions （which are essential to triggering relevant gene regulatory cascades）, can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.

Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis,and Roles of RyhBs in Biofilm Formation

Objective To investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis(Y.pestis),as well as the roles of RyhBs in biofilm formation.Methods Regulatory relationships were assessed by a combination of colony morphology assay,primer extension,electrophoretic mobility shift assay and DNase I footprinting.Results Fur bound to the promoter-proximal DNA regions of ryhB1 and ryhB2 to repress their transcription,while both RyhB1 and RyhB2 repressed the expression of Fur at the post-transcriptional level.In addition,both RyhB1 and RyhB2 positively regulated Y.pestis biofilm exopolysaccharide(EPS)production and the expression of hmsHFRS and hmsT.Conclusion Fur and the two RyhB homologs exert negative reciprocal regulation,and RyhB homologs have a positive regulatory effect on biofilm formation in Y.pestis.

Genotyping of strains of Yersinia pestis from Marmota baibacina-Spermophilus undulates Plague Focus of Tianshan Mountains in China

The aim of this study is to carry out genotyping of 61 Y. pestts strmns tsolated trom lwarmota baibacina-Spermophilus undulates Plague Focus of Tianshan Mountains in China. Primer pairs targeting the 22 DFRs were designed for detecting the genotypes of 61 strains. As a result, 61 strains of Y. pestis were divided into four genotypes 1, 2, 3, and 4. Genotypes 1, 2 and 3 were found from west part in Northern Tianshan Mountains Plague Focus, but type 1 was only from Nileke county. The type of strains from Aheqi was different from those of Atushi counties in Southern Tianshan Mountains and similar to strains in Northern Tianshan Mountains Plague Focus. The type 4 distributed over Atushi county, which was identical with that of strains from Marmota caudae Plague Focus of the Pamiri Plateau. It is concluded that geonotyping is identical with ecotyping made by Shuli Ji. Tianshan Mountains Plague Focus and Marmota caudae Plague Focus of the Pamiri Plateau have a cross spreading profile.

Yersinia pestis: examining wildlife plague surveillance in China and the USA

Plague is a zoonotic disease caused by the bacterium Yersinia pestis Lehmann and Neumann,1896.Although it is essentially a disease of rodents,plague can also be transmitted to people.Historically,plague has caused mas-sive morbidity and mortality events in human populations,and has recently been classified as a reemerging dis-ease in many parts of the world.This public health threat has led many countries to set up wild and domestic an-imal surveillance programs in an attempt to monitor plague activity that could potentially spill over into human populations.Both China and the USA have plague surveillance programs in place,but the disease dynamics dif-fer in each country.We present data on plague seroprevalence in wildlife and review different approaches for plague surveillance in the 2 countries.The need to better comprehend plague dynamics,combined with the fact that there are still several thousand human plague cases per year,make well-designed wildlife surveillance pro-grams a critical part of both understanding plague risks to humans and preventing disease outbreaks in the future.

Yersinia pestis, a problem of the past and a re-emerging threat

Yersinia pestis is the bacteria that causes plague,one of the deadliest diseases in human history.Three major plague pandemics(the Justinian Plague,the Black Death and the Modern Plague)have been recorded.Each caused massive fatalities and has become defining events in the time periods in places that were affected.The presence of natural plague foci in rodents across the world is one of the risk factors for human plague.While plague is a relatively rare problem for most countries,more than 90%of plague cases in the world still occur in Africa.This article discusses the threat of Yersinia pestis in the modern world by considering its prevalence and severity of illness it causes,transmission,antibiotic resistance,and its potential as a bioweapon.

Transcriptomic Response to Yersinia pestis:RIG-I Like Receptor Signaling Response Is Detrimental to the Host against Plague

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-13 production, higher Thl-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 E pestis also altered the expression of the RLR pathway, suggesting that the response of RLR pathway to bacterial infection is a universal mechanism.

Formation and regulation of Yersinia biofilms

Flea-borne transmission is a recent evolutionary adaptation that distinguishes the deadly Yersinia pestis from its progenitor Y.pseudotuberculosis,a mild pathogen transmitted via the food-borne route.Y.pestis synthesizes biofilms in the flea gut,which is important for fleaborne transmission.Yersinia biofilms are bacterial colonies surrounded by extracellular matrix primarily containing a homopolymer of N-acetyl-D-glucosamine that are synthesized by a set of specific enzymes.Yersinia biofilm production is tightly regulated at both transcriptional and post-transcriptional levels.All the known structural genes responsible for biofilm production are harbored in both Y.pseudotuberculosis and Y.pestis,but Y.pestis has evolved changes in the regulation of biofilm development,thereby acquiring efficient arthropod-borne transmission.

Protection against lethal subcutaneous challenge of virulent Y. pestis strain 141 using an F1-V subunit vaccine

In this study,we designed and engineered a two-component recombinant fusionprotein antigen as a vaccine candidate against the possible biological threat of Yersinia pestis.Therecombinant F1-V protein was formulated with Alhydrogel.A four-time injection with a dosage of10,20 and 50 ug/mouse in about two months was adopted for vaccination.Serum antibodies and subclassof T helper cells were measured and analyzed.After the final vaccination,the mice were challenged by141 strain with 25- 600 LD50.In conclusion,the recombinant vaccine was capable of inducingprotective immunity against subcutaneous challenge.The level of serum IgG was supposed to be a mainfactor that affected the final protection of challenge.20 ug recombinant protein could induce anendpoint titre of serum IgG as high as 51200,which was enough to afford 100% protection against 400LD50 virulent 141 challenge.The antibody isotype analysis showed that the vaccine inducedpredominantly an lgG1 rather than lgG2a response.Flow cytometric analysis revealed that Alhydrogelsignificantly helped induce a stronger humoral immunity instead of CTL cellular response.Thesefindings suggested that the plague F1-V subunit vaccine is promising for the next plague vaccine.

A historical evaluation of Chinese tongue diagnosis in the treatment of septicemic plague in the pre-antibiotic era, and as a new direction for revolutionary clinical research applications

Chinese tongue diagnosis was initially developed to quickly and efficiently diagnose and prescribe medicine, while at the same time allowing the doctor to have minimal contact with the patient. At the time of its compiling, the spread of Yersinia pestis, often causing septicaemia and gangrene of the extremities,may have discouraged doctors to come in direct contact with their patients and take the pulse.However, in recent decades, modern developments in the field of traditional Chinese medicine, as well as the spread of antibiotics in conjunction with the advancements of microbiology, have overshadowed the original purpose of this methodology. Nevertheless, the fast approaching post-antibiotic era and the development of artificial intelligence may hold new applications for tongue diagnosis. This article focuses on the historical development of what is the world＇s earliest tongue diagnosis monograph, and discusses the directions that such knowledge may be used in future clinical research.

Interrelationship of soil moisture and temperature to sylvatic plague cycle among prairie dogs in the Western United States

Plague,caused by Yersinia pestis,is a flea-borne disease that is endemic in areas throughout the world due to its successful maintenance in a sylvatic cycle,mainly in areas with temperate climates.Burrowing rodents are thought to play a key role in the enzootic maintenance as well as epizootic outbreaks of plague.In the United States,prairie dogs(Cynomys),rodents(Muridae),and ground squirrels(Spermophilus)are susceptible to infection and are parasitized by fleas that transmit plague.In particular,prairie dogs can experience outbreaks that rapidly spread,which can lead to extirpation of colonies.A number of ecological parameters,including climate,are associated with these epizootics.In this study,we asked whether soil parameters,primarily moisture and temperature,are associated with outbreaks of plague in black-tailed prairie dogs and Gunnison’s prairie dogs in the Western United States,and at what depth these associations were apparent.We collected publicly available county-level information on the occurrence of population declines or colony extirpation,while historical soil data was collected from SCAN and USCRN stations in counties and states where prairie dogs have been located.The analysis suggests that soil moisture at lower depths correlates with colony die-offs,in addition to temperature near the surface,with key differences within the landscape ecology that impact the occurrence of plague.Overall,the model suggests that the burrow environment may play a significant role in the epizootic spread of disease amongst black-tailed and Gunnison’s prairie dogs.

Field assessment of dog as sentinel animal for plague in endemic foci of Madagascar

The epidemiology of Yersinia pestis,the causative agent of plague,involves vectors and reservoirs in its transmission cycle.The passive plague surveillance in Madagascar targets mainly rodent and fleas.However,carnivores are routinely surveyed as sentinels of local plague activity in some countries.The aim of this study is to assess the use of domestic dog(Canis familiaris)as sentinel animal for field surveillance of plague in a highly endemic area in Madagascar.Cross-sectional surveys of plague antibody prevalence in C.familiaris were conducted in endemic areas with contrasting histories of plague cases in humans,as well as a plague free area.Rodent capture was done in parallel to evaluate evidence for Y.pestis circulation in the primary reservoirs.In 2 sites,dogs were later re-sampled to examine evidence of seroconversion and antibody persistence.Biological samplings were performed between March 2008 and February 2009.Plague antibody detection was assessed using anti-F1 ELISA.Our study showed a significant difference in dog prevalence rates between plague-endemic and plague-free areas,with no seropositive dogs detected in the plague free area.No correlation was found between rodents and dog prevalence rates,with an absence of seropositive rodents in some area where plague circulation was indicated by seropositive dogs.This is consistent with high mortality rates in rodents following infection.Re-sampling dogs identified individuals seropositive on both occasions,indicating high rates of re-exposure and/or persistence of plague antibodies for at least 9 months.Seroconversion or seropositive juvenile dogs indicated recent local plague circulation.In Madagascar,dog surveillance for plague antibody could be useful to identify plague circulation in new areas or quiescent areas within endemic zones.Within active endemic areas,monitoring of dog populations for seroconversion(negative to positive)or seropositive juvenile dogs could be useful for identifying areas at greatest risk of human outbreaks.

One Health: navigating plague in Madagascar amidst COVID-19

Background Africa sees the surge of plague cases in recent decades,with hotspots in the Democratic Republic of Congo,Madagascar,and Peru.A rodent-borne scourge,the bacterial infection known as plague is transmitted to humans via the sneaky bites of fleas,caused by Yersinia pestis.Bubonic plague has a case fatality rate of 20.8%with treatment,but in places such as Madagascar the mortality rate can increase to 40–70%without treatment.Main text Tragedy strikes in the Ambohidratrimo district as three lives are claimed by the plague outbreak and three more fight for survival in the hospitals,including one man in critical condition,from the Ambohimiadana,Antsaharasty,and Ampanotokana communes,bringing the total plague victims in the area to a grim to five.Presently,the biggest concern is the potential plague spread among humans during the ongoing COVID-19 pandemic.Effective disease control can be achieved through training and empowering local leaders and healthcare providers in rural areas,implementing strategies to reduce human–rodent interactions,promoting water,sanitation and hygiene practices(WASH)practices,and carrying out robust vector,reservoir and pest control,diversified animal surveillance along with human surveillance should be done to more extensively to fill the lacunae of knowledge regarding the animal to human transmission.The lack of diagnostic laboratories equipped represents a major hurdle in the early detection of plague in rural areas.To effectively combat plague,these tests must be made more widely available.Additionally,raising awareness among the general population through various means such as campaigns,posters and social media about the signs,symptoms,prevention,and infection control during funerals would greatly decrease the number of cases.Furthermore,healthcare professionals should be trained on the latest methods of identifying cases,controlling infections and protecting themselves from the disease.Conclusions Despite being endemic to Madagascar,the outbreak’s pace is unparalleled,and it may spread to non-endemic areas.The utilization of a One Health strategy that encompasses various disciplines is crucial for minimizing catastrophe risk,antibiotic resistance,and outbreak readiness.Collaboration across sectors and proper planning ensures efficient and consistent communication,risk management,and credibility during disease outbreaks.

No evidence for enzootic plague within black-tailed prairie dog(Cynomys ludovicianus)populations

Yersinia pestis,causative agent of plague,occurs throughout the western United States in rodent populations and periodically causes epizootics in susceptible species,including black-tailed prairie dogs(Cynomys ludovicianus).How Y.pestis persists long-term in the environment between these epizootics is poorly understood but multiple mechanisms have been proposed,including,among others,a separate enzootic transmission cycle that maintains Y.pestis without involvement of epizootic hosts and persistence of Y.pestis within epizootic host populations without causing high mortality within those populations.We live-trapped and collected fleas from black-tailed prairie dogs and other mammal species from sites with and without black-tailed prairie dogs in 2004 and 2005 and tested all fleas for presence of Y.pestis.Y.pestis was not detected in 2126 fleas collected in 2004 but was detected in 294 fleas collected from multiple sites in 2005,before and during a widespread epizootic that drastically reduced black-tailed prairie dog populations in the affected colonies.Temporal and spatial patterns of Y.pestis occurrence in fleas and genotyping of Y.pestis present in some infected fleas suggest Y.pestis was introduced multiple times from sources outside the study area and once introduced,was dispersed between several sites.We conclude Y.pestis likely was not present in these black-tailed prairie dog colonies prior to epizootic activity in these colonies.Although we did not identify likely enzootic hosts,we found evidence that deer mice(Peromyscus maniculatus)may serve as bridging hosts for Y.pestis between unknown enzootic hosts and black-tailed prairie dogs.

Identifying the spatiotemporal clusters of plague occurrences in China during the Third P Identifying the spatiotemporal clusters of plague occurrences in China during the Third Pandemic andemic

Plague,a devastating infectious disease caused by Yersinia pestis,has killed millions of people in the past and is still active in the natural foci of the world today.Understanding the spatiotemporal patterns of plague outbreaks in history is critically important,as it may help to facilitate prevention and control of potential future outbreaks.In this study,we explored spatiotemporal clusters of human plague occurrences in China using a machine-learning clustering method and reconstructed the potential transmission pattern during the Third Pandemic(1772-1964).We succeeded in identifying 6 clusters in the space domain(2D)and 13 clusters in the spatiotemporal domain(3D).Our results suggest that there were several temporal outbreaks and transmissions of plague in different spatial clusters.Together with the spatiotemporal nearest neighbor approach(ST-NNA),this method could allow us to have a clearer look at the spatiotemporal patterns of plague.

Single-cell transcriptomics of immune cells in lymph nodes reveals their composition and alterations in functional dynamics during the early stages of bubonic plague

Bubonic plague caused by Yersinia pestis is highly infectious and often fatal.Characterization of the host immune response and its subsequent suppression by Y.pestis is critical to understanding the pathogenesis of Y.pestis.Here,we utilized single-cell RNA sequencing to systematically profile the transcriptomes of immune cells in draining lymph nodes(d LNs)during the early stage of Y.pestis infection.Dendritic cells responded to Y.pestis within 2 h post-infection(hpi),followed by the activation of macrophages/monocytes(Mφs/Mons)and recruitment of polymorphonuclear neutrophils(PMNs)to d LNs at 24 hpi.Analysis of cell-to-cell communication suggests that PMNs may be recruited to lymph nodes following the secretion of CCL9 by Mφs/Mons stimulated through CCR1-CCL9 interaction.Significant functional suppression of all the three innate immune cell types occurred during the early stage of infection.In summary,we present a dynamic immune landscape,at single-cell resolution,of murine d LNs involved in the response to Y.pestis infection,which may facilitate the understanding of the plague pathogenesis of during the early stage of infection.