Reversible Gene Expression Control in Yersinia pestis by Using an Optimized CRISPR Interference System.

Many genes in the bacterial pathogen Yersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system using Streptococcus pyogenes Cas9-derived dCas9 for gene knockdown in Y. pestis Multiple genes harbored on either the chromosome or plasmids of Y. pestis were efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of hmsH (responsible for biofilm formation) or cspB (encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance in in vitro phenotypic assays. Furthermore, silencing of the virulence-associated genes yscB or ail using this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported for yscB and ail null mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in Y. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening of Y. pestis genes with unknown functions.IMPORTANCEYersiniapestis is a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, many Y. pestis genes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported in Y. pestis Here, we systematically optimized the CRISPRi approach for use in Y. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected by in vitro assays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach in Y. pestis and highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.

Yersinia pestis YopK Inhibits Bacterial Adhesion to Host Cells by Binding to the Extracellular Matrix Adaptor Protein Matrilin-2.

Pathogenic yersiniae harbor a type III secretion system (T3SS) that injects Yersinia outer protein (Yop) into host cells. YopK has been shown to control Yop translocation and prevent inflammasome recognition of the T3SS by the innate immune system. Here, we demonstrate that YopK inhibits bacterial adherence to host cells by binding to the extracellular matrix adaptor protein matrilin-2 (MATN2). YopK binds to MATN2, and deleting amino acids 91 to 124 disrupts binding of YopK to MATN2. A yopK null mutant exhibits a hyperadhesive phenotype, which could be responsible for the established Yop hypertranslocation phenotype of yopK mutants. Expression of YopK, but not YopKΔ91-124, in a yopK mutant restored the wild-type phenotypes of adhesion and Yop translocation, suggesting that binding to MATN2 might be essential for YopK to inhibit bacterial adhesion and negatively regulate Yop translocation. A green fluorescent protein (GFP)-YopK fusion specifically binds to the endogenous MATN2 on the surface of HeLa cells, whereas GFP-YopKΔ91-124 cannot. Addition of purified YopK protein during infection decreased adhesion of Y. pestis to HeLa cells, while YopKΔ91-124 protein showed no effect. Taking these results together, we propose a model that the T3SS-secreted YopK hinders bacterial adhesion to HeLa cells by binding to MATN2, which is ubiquitously exposed on eukaryotic cells.

Taxonomy of Yersinia pestis.

This chapter summarized the taxonomy and typing works of Yersinia pestis since it's firstly identified in Hong Kong in 1894. Phenotyping methods that based on phenotypic characteristics, including biotyping, serotyping, antibiogram analysis, bacteriocin typing, phage typing, and plasmid typing, were firstly applied in classification of Y. pestis in subspecies level. And then, with the advancement of molecular biological technology, the methods based on outer membrane protein profiles, fatty acid composition, and bacterial mass fingerprinting were also used to identify the populations within Y. pestis. However, Y. pestis is a highly homogenous species; therefore, the above typing methods could only provide low resolution, e.g., only one serotype and one phage type were observed for the whole species. Since the 1990s, molecular typing based on DNA variations, including single-nucleotide polymorphism, gene gain/loss, variable-number tandem repeats, clustered regularly interspaced short palindromic repeat, etc., was introduced and improved the resolution and robust of typing result. Especially in recent years, genotyping-based whole-genome-wide variations were successfully employed in Y. pestis, which built the "gold standard" of typing scheme of the species and could distinguish the samples under the strain level. The taxonomy and typing works leaved us enormous polymorphism data; therefore, a comprehensive fingerprint database of Y. pestis was needed to collect and standardize these data, for facilitating future works on evolution, plague surveillance and control, anti-bioterrorism, and microbial forensic researches.

Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis.

The genetic diversity of Yersinia pestis, the etiologic agent of plague, is extremely limited because of its recent origin coupled with a slow clock rate. Here we identified 2,326 SNPs from 133 genomes of Y. pestis strains that were isolated in China and elsewhere. These SNPs define the genealogy of Y. pestis since its most recent common ancestor. All but 28 of these SNPs represented mutations that happened only once within the genealogy, and they were distributed essentially at random among individual genes. Only seven genes contained a significant excess of nonsynonymous SNP, suggesting that the fixation of SNPs mainly arises via neutral processes, such as genetic drift, rather than Darwinian selection. However, the rate of fixation varies dramatically over the genealogy: the number of SNPs accumulated by different lineages was highly variable and the genealogy contains multiple polytomies, one of which resulted in four branches near the time of the Black Death. We suggest that demographic changes can affect the speed of evolution in epidemic pathogens even in the absence of natural selection, and hypothesize that neutral SNPs are fixed rapidly during intermittent epidemics and outbreaks.

Analysis of Himalayan marmot distribution and plague risk in Qinghai province of China using the "3S" technology.

To provide guidance for plague surveillance and a reliable basis for plague prevention and control, we analyzed the habitat characteristics of Himalayan marmots, developed Himalayan marmot information collection system V3.0 based on global navigation satellite system (GNSS), remote sensing, and geographic information system ("3S") technology, and drew a predictive spatial distribution map of Himalayan marmots in Qinghai Province. Field survey data of 352 marmot plague sites in Qinghai Province were collected in 2014, and the data from 80 sample sites were included. The Himalayan marmot habitat characteristics were analyzed based on "3S" technology using five environment variables (elevation, slope, aspect, vegetation cover, and grass type) and the geographical coordinates. Himalayan marmot information collection system V3.0, which has been approved by the National Copyright Administration of the People's Republic of China (No.00764743), was used to draw a predictive spatial distribution map of Himalayan marmots in Qinghai province. Moreover, from 2015 to 2017, positioning data of the plague-foci and plague-free areas in Qinghai Province were collected using GNSS receptor for field validations to verify the accuracy of the marmot predictive spatial distribution map. Elevation, slope, vegetation cover, and grassland type were identified as important environmental factors that determine the spatial distribution of Himalayan marmots. The suitable range of environmental features was 3400-4600 m elevation, 5°-20° slope, 0.60-1.00 vegetation cover, and alpine meadows. The Himalayan marmot predictive spatial distribution map in Qinghai Province based on "3S" technology and marmot information collection system V3.0 had a spatial resolution of 30 m. Field validation in areas of Qinghai Province revealed a prediction accuracy and mean absolute error of 0.8669 and 0.1331, respectively, which indicated excellent prediction accuracy. This study greatly improved the work efficiency of plague surveillance and effectively reduced the work intensity of researchers. Application of "3S" technology and marmot information collection system V3.0 has improved the data collection efficiency, provided new technical means for plague investigation and research, and provided a reference for development of plague surveillance programs. The research results will play a positive role in promoting the improvement and perfection of plague prevention and control strategies in Qinghai province and even in China.

Characterization of Mu-Like Yersinia Phages Exhibiting Temperature Dependent Infection.

Yersinia pestis is the etiological agent of plague. Marmota himalayana of the Qinghai-Tibetan plateau is the primary host of flea-borne Y. pestis. This study is the report of isolation of Mu-like bacteriophages of Y. pestis from M. himalayana. The isolation and characterization of four Mu-like phages of Y. pestis were reported, which were named as vB_YpM_3, vB_YpM_5, vB_YpM_6, and vB_YpM_23 according to their morphology. Comparative genome analysis revealed that vB_YpM_3, vB_YpM_5, vB_YpM_6, and vB_YpM_23 are phylogenetically closest to Escherichia coli phages Mu, D108 and Shigella flexneri phage SfMu. The role of LPS core structure of Y. pestis in the phages' receptor was pinpointed. All the phages exhibit "temperature dependent infection," which is independent of the growth temperature of the host bacteria and dependent of the temperature of phage infection. The phages lyse the host bacteria at 37°C, but enter the lysogenic cycle and become prophages in the chromosome of the host bacteria at 26°C. IMPORTANCE Mu-like bacteriophages of Y. pestis were isolated from M. himalayana of the Qinghai-Tibetan plateau in China. These bacteriophages have a unique temperature dependent life cycle, follow a lytic cycle at the temperature of warm-blooded mammals (37°Ð¡), and enter the lysogenic cycle at the temperature of its flea-vector (26°Ð¡). A switch from the lysogenic to the lytic cycle occurred when lysogenic bacteria were incubated from lower temperature to higher temperature (initially incubating at 26°C and shifting to 37°C). It is speculated that the temperature dependent lifestyle of bacteriophages may affect the population dynamics and pathogenicity of Y. pestis.

Interplays of mutations in waaA, cmk, and ail contribute to phage resistance in Yersinia pestis.

Plague caused by Yersinia pestis remains a public health threat worldwide. Because multidrug-resistant Y. pestis strains have been found in both humans and animals, phage therapy has attracted increasing attention as an alternative strategy against plague. However, phage resistance is a potential drawback of phage therapies, and the mechanism of phage resistance in Y. pestis is yet to be investigated. In this study, we obtained a bacteriophage-resistant strain of Y. pestis (S56) by continuously challenging Y. pestis 614F with the bacteriophage Yep-phi. Genome analysis identified three mutations in strain S56: waaA* (9-bp in-frame deletion 249GTCATCGTG257), cmk* (10-bp frameshift deletion 15CCGGTGATAA24), and ail* (1-bp frameshift deletion A538). WaaA (3-deoxy-D-manno-octulosonic acid transferase) is a key enzyme in lipopolysaccharide biosynthesis. The waaA* mutation leads to decreased phage adsorption because of the failure to synthesize the lipopolysaccharide core. The mutation in cmk (encoding cytidine monophosphate kinase) increased phage resistance, independent of phage adsorption, and caused in vitro growth defects in Y. pestis. The mutation in ail inhibited phage adsorption while restoring the growth of the waaA null mutant and accelerating the growth of the cmk null mutant. Our results confirmed that mutations in the WaaA-Cmk-Ail cascade in Y. pestis contribute to resistance against bacteriophage. Our findings help in understanding the interactions between Y. pestis and its phages.

Attenuation of Yersinia pestis fyuA Mutants Caused by Iron Uptake Inhibition and Decreased Survivability in Macrophages.

Yersinia pestis is the etiological agent of plague, a deadly infectious disease that has caused millions of deaths throughout history. Obtaining iron from the host is very important for bacterial pathogenicity. Y. pestis possesses many iron uptake systems. Yersiniabactin (Ybt) plays a major role in iron uptake in vivo and in vitro, and in virulence toward mice as well. FyuA, a ß-barrel TonB-dependent outer membrane protein, serves as the receptor for Ybt. In this study, we examined the role of the fyuA gene in Y. pestis virulence using different challenging ways and explored the underlying mechanisms. The BALB/c mouse infection assay showed that the virulence of the mutant strains (ΔfyuA and ΔfyuAGCAdel) was lower when compared with that of the wild-type (WT) strain 201. Furthermore, the attenuation of virulence of the mutant strains via subcutaneous and intraperitoneal challenges was far greater than that via intravenous injection. Iron supplementation restored lethality during subcutaneous challenge with the two mutants. Thus, we speculated that the attenuated virulence of the mutant strains toward the mice may be caused by dysfunctional iron uptake. Moreover, ΔfyuA and ΔfyuAGCAdel strains exhibited lower survival rates in murine RAW264.7 macrophages, which might be another reason for the attenuation. We further explored the transcriptomic differences between the WT and mutant strains at different temperatures and found that the expressions of genes related to Ybt synthesis and its regulation were significantly downregulated in the mutant strains. This finding indicates that fyuA might exert a regulatory effect on Ybt. Additionally, the expressions of the components of the type III secretion system were unexpectedly upregulated in the mutants, which is inconsistent with the conventional view that the upregulation of the virulence genes enhances the virulence of the pathogens.

Identification and characterization of P2-like bacteriophages of Yersinia pestis.

Yersinia pestis is the cause of plague, historically known as the "Black Death". Marmota himalayana in the Qinghai-Tibet Plateau (QTP) natural plague focus is the primary host in China. Although several phages originating from Y. pestis have been characterized. This is the first report of isolation of P2-like phages of Y. pestis from M. himalayana. In this study, the isolation and characterization of three P2-like phages of Y. pestis were reported, which were named as vB_YpM_22, vB_YpM_46 and vB_YpM_50. Comparative genome analysis revealed that vB_YpM_22, vB_YpM_46 and vB_YpM_50 are members of the nonlambdoid P2 family, and are highly similar and collinear with enterobacteriophage P2, plague diagnostic phage L-413C and enterobacteriophage fiAA91-ss. The role of LPS core structure of Y. pestis in the phages' receptor was pinpointed. The findings of this study contribute an advance in our current knowledge of Y. pestis phages and will also play a key role in understanding the evolution of Y. pestis phages.

A dog-associated primary pneumonic plague in Qinghai Province, China.

BACKGROUND: Primary pneumonic plague (PPP) caused by Yersinia pestis is the most threatening clinical form of plague. An outbreak was reported in July 2009 in Qinghai Province, China. METHODS: This outbreak was investigated by clinical, epidemiological, bacteriological, and immunological methods. Multilocus variable number tandem repeat analysis (MLVA) was used to track the source of the outbreak. RESULTS: The index case, a patient with PPP, contaminated 11 close contacts. All the 12 cases, including the index patient, experienced sudden onset of fever, headache, and productive coughing with bloody sputum. Three of them died. Nevertheless, another 61 direct and 256 indirect contacts were not infected during the 2-week quarantine. Antibodies to F1 antigen were detected in 9 survival cases, with a 4-fold increase in titers in serum samples collected at different periods. Seven strains of Y. pestis were isolated from dogs and patients. Field investigation and MLVA of the isolated strains revealed that this outbreak was started by a deceased dog. CONCLUSION: Dogs are believed to be an indicator animal for plague surveillance, but their association with PPP is rare. Our results provide evidence for this possibility, which suggests the public health significance of dogs as a source of plague.

The complete genome sequence and proteomics of Yersinia pestis phage Yep-phi.

Yep-phi, a lytic phage of Yersinia pestis, was isolated in China and is routinely used as a diagnostic phage for the identification of the plague pathogen. Yep-phi has an isometric hexagonal head containing dsDNA and a short non-contractile conical tail. In this study, we sequenced the Yep-phi genome (GenBank accession no. HQ333270) and performed proteomics analysis. The genome consists of 38 ,616 bp of DNA, including direct terminal repeats of 222 bp, and is predicted to contain 45 ORFs. Most structural proteins were identified by proteomics analysis. Compared with the three available genome sequences of lytic phages for Y. pestis, the phages could be divided into two subgroups. Yep-phi displays marked homology to the bacteriophages Berlin (GenBank accession no. AM183667) and Yepe2 (GenBank accession no. EU734170), and these comprise one subgroup. The other subgroup is represented by bacteriophage ΦA1122 (GenBank accession no. AY247822). Potential recombination was detected among the Yep-phi subgroup.

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 Co(2+) 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.

Tackling COVID-19: Insights from the Qinghai Province plague prevention and control (PPC) model.

Plague, caused by Yersinia pestis, is a natural focus infectious disease. In China, plague is classified as category A, with the highest risk and hazard among the infectious diseases. Qinghai used to be considered as one of the most serious areas of plague in China. In recent years, thank to the measures in eight aspects summarized as the "Qinghai model" which were adopted to prevent and control the human plague in Qinghai, Qinghai has not experienced any plague case reported for eight years. In early 2020, coronavirus disease 2019 (COVID-19) outbroke in China. The Qinghai model on plague was employed to deal with the COVID-19 emergency in Qinghai Province. The Qinghai Center for Disease Control and Prevention (Qinghai CDC) and hospitals, along with the departments of public security, animal husbandry and other departments, quickly tracked and treated the patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and started surveillance programs on close contacts timely. At present, the cure rate of patients has reached 100%, and close contacts have been effectively quarantined and tested to avoid the spread of COVID-19. The findings from the study suggest that the prevention and control measures undertaken in Qinghai Province might be effective in dealing with the category A infectious diseases such as COVID-19 and other diseases.

Evolutionary selection of biofilm-mediated extended phenotypes in Yersinia pestis in response to a fluctuating environment.

Yersinia pestis is transmitted from fleas to rodents when the bacterium develops an extensive biofilm in the foregut of a flea, starving it into a feeding frenzy, or, alternatively, during a brief period directly after feeding on a bacteremic host. These two transmission modes are in a trade-off regulated by the amount of biofilm produced by the bacterium. Here by investigating 446 global isolated Y. pestis genomes, including 78 newly sequenced isolates sampled over 40 years from a plague focus in China, we provide evidence for strong selection pressures on the RNA polymerase ω-subunit encoding gene rpoZ. We demonstrate that rpoZ variants have an increased rate of biofilm production in vitro, and that they evolve in the ecosystem during colder and drier periods. Our results support the notion that the bacterium is constantly adapting-through extended phenotype changes in the fleas-in response to climate-driven changes in the niche.

[Evaluation of immunization protection efficacy of plague subunit vaccine].

OBJECTIVE: To evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study. METHODS: Groups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization. RESULTS: The immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively. CONCLUSION: BALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.

Human Macrophages Clear the Biovar Microtus Strain of Yersinia pestis More Efficiently Than Murine Macrophages.

Yersinia pestis is the etiological agent of the notorious plague that has claimed millions of deaths in history. Of the four known Y. pestis biovars (Antiqua, Medievalis, Orientalis, and Microtus), Microtus strains are unique for being highly virulent in mice but avirulent in humans. Here, human peripheral lymphocytes were infected with the fully virulent 141 strain or the Microtus strain 201, and their transcriptomes were determined and compared. The most notable finding was that robust responses in the pathways for cytokine-cytokine receptor interaction, chemokine signaling pathway, Toll-like receptor signaling and Jak-STAT signaling were induced at 2 h post infection (hpi) in the 201- but not the 141-infected lymphocytes, suggesting that human lymphocytes might be able to constrain infections caused by strain 201 but not 141. Consistent with the transcriptome results, much higher IFN-γ and IL-1ß were present in the supernatants from the 201-infected lymphocytes, while inflammatory inhibitory IL-10 levels were higher in the 141-infected lymphocytes. The expressions of CSTD and SLC11A1, both of which are functional components of the lysosome, increased in the 201-infected human macrophage-like U937 cells. Further assessment of the survival rate of the 201 bacilli in the U937 cells and murine macrophage RAW 264.7 cells revealed no viable bacteria in the U937 cells at 32 hpi.; however, about 5-10% of the bacteria were still alive in the RAW264.7 cells. Our results indicate that human macrophages can clear the intracellular Y. pestis 201 bacilli more efficiently than murine macrophages, probably by interfering with critical host immune responses, and this could partially account for the host-specific pathogenicity of Y. pestis Microtus strains.

A new purification strategy for fraction 1 capsular antigen and its efficacy against Yersinia pestis virulent strain challenge.

F1 antigen is an attractive candidate for the development of a subunit vaccine against plague. In previous study, the extraction of this antigen from Yersinia pestis is characterized by using organic solvents. In this work, a new purification strategy that produced high-purity F1 antigen from Y. pestis EV76 was developed by the substitution of physical disruption for organic solvent one, followed by a combination of ammonium sulfate fractionation and Sephacryl S-200HR column filtration chromatography. As revealed in this study, this purification procedure is simple and effective, and avoids potential adverse effect on the antigen by organic solvents. Highly purified F1 that adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to F1 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10(4) CFU of Y. pestis virulent strain 141.

Genetic diversity and spatial-temporal distribution of Yersinia pestis in Qinghai Plateau, China.

BACKGROUND: Plague, caused by the bacterium Yersinia pestis, is a highly infectious, zoonotic disease. Hundreds of human plague cases are reported across the world annually. Qinghai Plateau is one of the most severely affected plague regions in China, with more than 240 fatal cases of Y. pestis in the last 60 years. Conventional epidemiologic analysis has effectively guided the prevention and control of local plague transmission; however, molecular genetic analysis is more effective for investigating population diversity and transmission. In this report, we employed different genetic markers to analyze the population structure of Y. pestis in Qinghai Plateau. METHODOLOGY/PRINCIPAL FINDING: We employed a two-step hierarchical strategy to analyze the phylogeny of 102 Qinghai Plateau isolates of Y. pestis, collected between 1954 and 2011. First, we defined the genealogy of Y. pestis by constructed minimum spanning tree based on 25 key SNPs. Seven groups were identified, with group 1.IN2 being identified as the dominant population. Second, two methods, MLVA and CRISPR, were applied to examine the phylogenetic detail of group 1.IN2, which was further divided into three subgroups. Subgroups of 1.IN2 revealed a clear geographic cluster, possibly associated with interaction between bacteriophage and Y. pestis. More recently, Y. pestis populations appear to have shifted from the east toward the center and west of Qinghai Plateau. This shift could be related to destruction of the local niche of the original plague focus through human activities. Additionally, we found that the abundance and relative proportion of 1.IN2 subgroups varied by decade and might be responsible for the fluctuations of plague epidemics in Qinghai Plateau. CONCLUSION/SIGNIFICANCE: Molecular genotyping methods provided us with detailed information on population diversity and the spatial-temporal distribution of dominant populations of Y. pestis, which will facilitate future surveillance, prevention, and control of plague in Qinghai Plateau.

Phenotypic characteristics of wild-type plague phage growth in different experimental environments / 中国热带医学

@#Abstract: Objective To observe the phenotypic characteristics of 3 wild-type plague phages under different experimental environments, providing scientific evidence for the identification of phage biological characteristics and the study of their interaction with host bacteria in the future. Methods The sensitivity of 3 wild-type plague phages were detected by using liquid culture method, emisolid medium method and micro-liquid culture method based on OmniLog TM microbial identification system. Results The growth result based on LB liquid medium showed that the growth of plague phage 476 for 20-24 hours at both 28 ℃ and 37 ℃was better than that of plague phages 087 and 072204 at 37 ℃, and the growth of plague phages 087 was better than that of plague phages 072204 at 37 ℃. With the attenuated plague bacterium EV76 as the host bacterium, phage 476 was able to form visible plaque on double-layer agar medium for 20-20 hours at both 28 ℃ and 37 ℃, phages 087 and 072204 were only able to form opaque plaque on double-layer agar medium for 20-24 hours at 37 ℃. The growth results based on OmniLogTM system showed that when plague phage was lysed in EV76 strain at 33 ℃, the first row appeared as a straight line with a peak of no more than 100 in the 96-well microplate curve chart. As the phage quantity decreased, the dilution plate appeared with growth curve similar to EV76 strain in turn, and the color of tetrazolium dyes in the experimental wells gradually deepened as the phage number decreased and the host bacteria number increased. Therefore, it indicates that phage 476 was sensitively at both 28 ℃ and 37 ℃, while phage 087 and 072204 were temperature-dependent only at 37 ℃ to attenuated plague bacterium EV76. Conclusions The lysing ability of 3 wild-type plague phages are temperature-dependent, and the growth results are consistent under the three experimental conditions.

Isolation and molecular characterisation of Achromobacter phage phiAxp-3, an N4-like bacteriophage.

Achromobacter xylosoxidans, an opportunistic pathogen, is responsible for various nosocomial and community-acquired infections. We isolated phiAxp-3, an N4-like bacteriophage that infects A. xylosoxidans, from hospital waste and studied its genomic and biological properties. Transmission electron microscopy revealed that, with a 67-nm diameter icosahedral head and a 20-nm non-contractile tail, phiAxp-3 has features characteristic of Podoviridae bacteriophages (order Caudovirales). With a burst size of 9000 plaque-forming units and a latent period of 80 min, phiAxp-3 had a host range limited to only four A. xylosoxidans strains of the 35 strains that were tested. The 72,825 bp phiAxp-3 DNA genome, with 416-bp terminal redundant ends, contains 80 predicted open reading frames, none of which are related to virulence or drug resistance. Genome sequence comparisons place phiAxp-3 more closely with JWAlpha and JWDelta Achromobacter phages than with other N4 viruses. Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles. Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3. Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.