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Bioluminescent tracing of a Yersinia pestis pCD1+-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice
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Microbes and Infection xx (2017) 1e10 www.elsevier.com/locate/micinf
Original article
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Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice Yazhou Zhou, Jiyuan Zhou, Yuxin Ji, Lu Li, Yafang Tan, Guang Tian, Ruifu Yang, Xiaoyi Wang* Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China Received 22 July 2017; accepted 7 November 2017 Available online ▪ ▪ ▪
Abstract Yersinia pestis has evolved from Yersinia pseudotuberculosis serotype O:1b. A typical Y. pestis contains three plasmids: pCD1, pMT1 and pPCP1. However, some isolates only harbor pCD1 (pCD1þ-mutant). Y. pestis and Y. pseudotuberculosis share a common plasmid (pCD1 or pYV), but little is known about whether Y. pseudotuberculosis exhibited plague-inducing potential before it was evolved into Y. pestis. Here, the luxCDABE::Tn5::kan was integrated into the chromosome of the pCD1þ-mutant, Y. pseudotuberculosis or Escherichia coli K12 to construct stable bioluminescent strains for investigation of their dissemination in mice by bioluminescence imaging technology. After subcutaneous infection, the pCD1þ-mutant entered the lymph nodes, followed by the liver and spleen, and, subsequently, the lungs, causing pathological changes in these organs. Y. pseudotuberculosis entered the lymph nodes, but not the liver, spleen and lungs. It also resided in the lymph nodes for several days, but did not cause lymphadenitis or pathological lesions. By contrast, E. coli K12-lux was not isolatable from mouse lymph nodes, liver, spleen and lungs. These results indicate that the pCD1þ-mutant can cause typical bubonic and pneumonic plague-like diseases, and Y. pestis has inherited lymphoid tissue tropism from its ancestor rather than acquiring these properties independently. © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Plague; Yersinia pestis; Yersinia pseudotuberculosis; In vivo bioluminescence imaging
1. Introduction Of the 17 Yersinia genus species, Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica all cause invasive diseases in humans and other mammals. Y. pestis is genetically very closely related to the enteric pathogen Y. pseudotuberculosis, and both of them share 90% DNA sequence homology. Despite the highly distinct diseases they cause and their different transmission modes, it is generally accepted that Y. pestis evolved from the Y. pseudotuberculosis O:1 b serotype as a clone about 5000 to 7000 years ago [1,2]. All three pathogenic species share a related plasmid (called pCD1 in Y. pestis and pYV in Y. pseudotuberculosis and Y.
* Corresponding author. Fax: þ86 10 63815689. E-mail address: [email protected] (X. Wang).
enterocolitica), which is associated with virulence and in vitro Ca2þ-dependent growth at 37 C [3]. Y. pestis causes plague, a zoonotic disease occasionally transmitted to humans from Y. pestis-infected rodents via the bite of infected fleas [4]. Historically, Y. pestis has caused three major plague pandemics, leading to millions of human deaths. Recently, an increase in the annual number of global plague cases was reported to the World Health Organization (WHO), and plague is now considered by them to be a re-emerging infectious disease [5,6]. Plague has also attracted much attention because of its potential for use in classical biological warfare or bioterrorism agents [7,8]. A typical Y. pestis strain usually contains three virulence plasmids: pCD1, pMT1 and pPCP1. pCD1 was inherited from its Y. pseudotuberculosis ancestor, whereas pMT1 and pPCP1 were acquired by horizontal transfer from unknown pathogens. The pCD1 and pYV plasmids encode the type three secretion
https://doi.org/10.1016/j.micinf.2017.11.005 1286-4579/© 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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system (T3SS) [9], which is responsible for injecting into host cells a number of Yersinia outer proteins (Yops) that inhibit bacterial phagocytosis, the respiratory burst, the host innate immune response, and trigger apoptosis [10,11]. The newlyacquired pPCP1 encodes the plasminogen activator (Pla), a surface proteinase responsible for increasing the pathogen's invasive ability in bubonic plague and enabling rapid replication of Y. pestis in the lungs [12,13]. Another newly acquired plasmid (pMT1) encodes the most important protective antigens in Y. pestis, the F1 antigen, along with the murine toxin (Ymt), which is required for Y. pestis survival in the flea midgut [14]. We recently analyzed 133 natural isolates of Y. pestis, including 107 strains representing the geographical and genetic diversity of >5000 Chinese isolates, 11 isolates from Mongolia, the former Soviet Union, Myanmar, and Madagascar, and 15 previously sequenced genomes through sequence alignment analyses. We found several Y. pestis plasmid deletion mutants that only contain the pCD1 plasmid (pCD1þ-mutant). These pCD1þ-mutants were isolated from rodents, but whether these mutants can cause typical plague disease requires investigation. Y. pestis and Y. pseudotuberculosis share a common virulence plasmid (pCD1 or pYV), but no study has investigated whether Y. pseudotuberculosis has the potential to induce plague disease via the subcutaneous route before it evolved into Y. pestis. To answer this question, the pCD1þ-mutant, Y. pseudotuberculosis O:1b and Escherichia coli K12 were used to study their dissemination routes during primary bubonic plague in a mouse experiment infection model using a bioluminescence imaging (BLI) technology. E. coli K12 does not contain pCD1 or pYV plasmids, and therefore, will not induce bubonic plague after subcutaneous infection with it. In this study, Y. pseudotuberculosis was compared with E. coli K12 to assess its ability to induce bubonic plague in mice. 2. Material and methods 2.1. Bacterial strains and mice The pCD1þ-mutant (containing only the pCD1 plasmid) was constructed from the Y. pestis subsp. microtus 201 strain using the plasmid incompatibility principle described in our previous study [15]. Y. pseudotuberculosis Pa3606 (serotype O:1b) was isolated from humans in Japan. E. coli K12 was purchased from NEB (New England Biolabs). The pXENluxCDABE (pXEN-18) bioluminescent plasmid was a generous gift from Dr. Tao Zhou (National Center of Biomedical Analysis, Beijing, China). The plasmid is under the control of a native promoter, confers resistances to ampicillin and kanamycin, and contains Tn5 and sacB transposon genes. All the Male BALB/c mice (6e8 weeks old) used in this study were provided with food and fresh water ad libitum throughout the study. All the animal experiments were conducted in accordance with the Guidelines for the Welfare and Ethics of Laboratory Animals of China and were approved by
the Committee of the Welfare and Ethics of Laboratory Animals. 2.2. Bioluminescent reporter strain construction The pCD1þ-mutant, Y. pseudotuberculosis Pa3606 and E. coli K12 were transformed with pXEN-18 by electroporation, and then grown on Hottinger agar plates containing kanamycin (50 mg/ml). Bioluminescent bacterial strains were selected on the NightOWL II LB983 imaging system, and then grown on 10% (W/V) sucrose LuriaeBertani (LB) plates containing kanamycin (50 mg/ml) to enable chromosomal integration of the luxCDABE::Tn5::kan transposon. Each bioluminescent clone isolated via sucrose plate selection was replated on LB plates containing kanamycin (50 mg/ml) and ampicillin (100 mg/ml), and the clones with kanamycin resistance but not ampicillin resistance were considered positive. The positive clones that were selected were screened for the position of luxCDABE on the chromosome. The resultant bioluminescent strains were designated as pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux. 2.3. Identification of the chromosomal position of lux CDABE The chromosomal integration position of lux CDABE was determined using a genome walking kit (Takara GW) and DNA sequencing. Briefly, three forward primers based on the kanamycin gene sequence were designed (designated SP1_SP3), and four reverse primers (AP1_AP4) were provided by the genome walking kit. The SP1-SP3 primer sequences are 50 -CGTAATGGCTGGCCTGTTGAACAAG-30 (SP1), 50 -CACCGGATTCAGTCGTCACTCATGGT-30 (SP2) and 50 -CGATACCAGGATCTTGCCATCCTATG-30 (SP3). The DNA samples from the selected clones were used as templates for the first round of nested PCR with SP1 combined with AP1, AP2, AP3 or AP4 in four separate PCR tubes. After the first round of nested PCR, 1 ml of PCR product was removed from each of the four tubes, and used individually as the template for the second round of nested PCR with SP2 combined with AP1, AP2, AP3 or AP4 in four separate test tubes. After the second round of nested PCR reactions, 1 ml of PCR product was removed from each of the four tubes, and then used as the template for the third round of nested PCR with SP3 combined with AP1, AP2, AP3 or AP4 in the four PCR tubes. Finally, the PCR products from four test tubes were analyzed by 1% agarose gel electrophoresis, and sequenced with the SP3 primer. The position of luxCDABE::Tn5::kan on the chromosome was identified by BLAST analysis of the National Center for Biotechnology Infection NCBI website. 2.4. Growth curves for the bioluminescent strains A single colony of the lux-expressing strain or its parent strain was inoculated into 5 ml of LB medium, and then grown to the mid-log phase at 26 C (pCD1þ-mutant-lux, Y.
Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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2.5. Bacterial virulence determination
Fig. 1. Chromosomal position of the luxCDABE::Tn5::kan transposon in the pCD1þ-mutant, Y. pseudotuberculosis Pa3606, and E. coli K12.
The pCD1þ-mutant and pCD1þ-mutant-lux were inoculated from overnight cultures into fresh LB medium and grown to an OD of 0.6 at 620 nm at 26 C, and then diluted 10-fold into the same medium and cultivated at 26 C to the mid-log phase. Bacterial cells were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS), diluted in PBS and the cell density was adjusted to the desired concentration for the challenge experiments. A groups of ten male BALB/c mice were subcutaneously injected with the pCD1þmutant (1.61 107 colony forming units, CFU) or pCD1þmutant-lux (1.56 107 CFU). The time of death for each animal was recorded during the observation intervals, and a percentage survival analysis was performed using GraphPad Prism. 2.6. BLI and microbiological examination
pseudotuberculosis Pa3606-lux, plus their parents) or at 37 C (E. coli K12-lux and its parent). The bacterial suspension (900 ml) was transferred to 18 ml of fresh LB medium and grown to an OD600 of 1.0 (determined by an ultraviolet spectrophotometer UV-5100B, METASH, China), after which 900 ml (OD600 1.0) of it was transferred to 18 ml of fresh LB medium for cultivation. During the cultivation process, 300 ml of bacterial suspension was collected at different time points, 30 ml of 40% formalin was added, and the OD600 value was determined. Graphs of the OD600 values versus time were plotted.
Three bioluminescent bacterial strains of pCD1þ-mutantlux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux were individually grown to an OD 600 of 1.0 in LB medium, harvested by centrifugation, and then resuspended in PBS to prepare serial 10-fold bacterial suspension dilutions. Individual groups of six male BALB/c mice were subcutaneously injected at the end of the tail with the pCD1þ-mutantlux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux. The infected mice were anesthetized with 3% isoflurane for 5 min, and imaged with the NightOWL II LB983 imaging system
Fig. 2. Growth curves, virulence and bacterial loads. (A) The growth curve of the pCD1þ-mutant and pCD1þ-mutant-lux. (B) The growth curve of Y. pseudotuberculosis Pa3606 and Y. pseudotuberculosis Pa3606-lux. (C) The growth curve of E. coli K12 and E. coli K12-lux. (D) Comparison of virulence between the pCD1þ-mutant and pCD1þ-mutant-lux in mice after infection via the subcutaneous route. (E) Bacterial loads in the groin lymph nodes from the mice subcutaneously infected with Y. pseudotuberculosis Pa3606-lux. Bacterial numbers are reported in CFU/lymph node. Data points are expressed as the means (±standard deviations) of the measurements taken for three mice. Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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(Berthold Technologies, Bad Wildbad, Germany) at various post-infection (pi) time point. The bioluminescence signals analyzed with IndiGO software (Berthold Technologies, Germany) were recorded as counts per second, with monitoring taking place at regular time intervals. The bacterial load or degree of colonization in each animal tissue was determined after pentobarbital sodium anesthesia during the post-mortem examinations. The groin lymph nodes, liver, spleen and lung tissues of the infected mice were harvested, homogenized, and then subjected to agar plate counts to confirm whether any bacteria were present in these organs. 2.7. Pathological observations The mice infected with pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux were killed by CO2 inhalation followed by cervical dislocation on the fifth day of infection. Tissues were collected and placed in 10% neutralbuffered formalin, dehydrated through a serial alcohol gradient (70%, 80%, 90%, 95%, and 100%), cleared with xylene, infiltrated with wax, and then embedded in paraffin [16]. The issue sections were stained with hematoxylin and eosin (HE) for histopathological examination under light microscopy. 2.8. Statistical analyses Statistical analyses were performed using GraphPad Prism version 5.0. The bacterial loads in the groin lymph nodes from the pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux were determined by viable plate counts. The bacterial load difference between two strains was investigated by the ManneWhitney t test with a two-tailed nonparametric analysis. The survival rates in the different infection groups were compared by a log-rank test. A probability value of <0.05 was considered statistically significant. 3. Results 3.1. Identification of bioluminescent strains The bioluminescent reporter strains pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux were constructed by combining each of them with the pXEN-18 plasmid containing the luxCDABE::Tn5::kan transposon and sucrose selection, thereby facilitating transposon integration into the chromosome of each strain. The chromosomal position of luxCDABE::Tn5::kan was mapped by genome walking, DNA sequencing and BLAST analysis of the NCBI website (Fig. 1). In the pCD1þ-mutant-lux bioluminescent strain, luxCDABE::Tn5::kan was inserted into a non-coding region between the YP_3609 and YP_3610 genes. In the E.
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coli K12-lux bioluminescent strain, luxCDABE::Tn5::kan was inserted inside the NEB5A_03800 c-di-GMP phosphodiesterase encoding gene. In the Y. pseudotuberculosis Pa3606-lux strain, luxCDABE::Tn5::kan was inserted into the gene BZ19_1372 encoding a ROK family protein. 3.2. Growth curves and bacterial strain virulence To investigate whether transpose integration into the chromosomes of the wild-type strains affected their growth, the growth rate of each lux-expressing strain was compared with that of the corresponding parental strain in vitro. The results showed no obvious growth difference between the luxexpressing strains and their parent strains (Fig. 2AeC). In investigating whether a virulence difference existed between the wild-type strains and their lux-expressing counterparts, our preliminary experiments found that Y. pseudotuberculosis Pa3606 and E. coli K12 had extremely low virulent profiles in mice by the subcutaneous route. Even when the mice received a high dose of Y. pseudotuberculosis Pa3606 or E. coli K12 (>108 CFU), all the animals still survived, but the pCD1þmutant still possessed high virulence in the mouse model of bubonic plague. Here, we only compared the virulence of the pCD1þ-mutant (1.61 107 CFU) with that of its luxexpressing strain (1.56 107 CFU). Statistical analysis showed no significant virulence difference between the pCD1þ-mutant and its lux-expressing counterpart (Fig. 2D). 3.3. Bacterial tissue colonization To further verify the BLI results, bacterial colonization of the groin lymph nodes, livers, spleens and lungs from the pCD1þ-mutant-lux-infected mice (4.5 106 CFU), the Y. pseudotuberculosis Pa3606-lux-infected mice (8.6 107 CFU) or E. coli K12-lux-infected mice (8.2 107 CFU) was assessed after subcutaneous infection administered by the end of the tail route. A small number of the pCD1þ-mutant-lux group had infected groin lymph nodes, livers and spleens on day 1 pi, but a large number of bacteria were isolated from these organs on day 3 pi. A large number of bacteria were observed in the infected lungs until day five pi. Microbiological examination also showed that Y. pseudotuberculosis Pa3606-lux was transferred to the groin lymph nodes one day after the infection commenced, but E. coli K12lux was not transferred to the groin lymph nodes during the whole eleven-day observation period. However, no Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux was isolated from the liver, spleen and lungs of each infected animal. To evaluate the colonization of Y. pseudotuberculosis Pa3606-lux in the infected groin lymph nodes, the bacterial loads were assessed during the eleven-day observation period (Fig. 2E). Small numbers of Y. pseudotuberculosis Pa3606-lux were
Fig. 3. In vivo imaging of mice infected with pCD1þ-mutant-lux. Six mice were subcutaneously infected at the end of the tail with pCD1þ-mutant-lux, and the bioluminescence signal emitted from each animal was determined by the NightOWL II LB983 imaging system. The numbers “1e6” represent six different mice, whereas “P and S” denote prone and supine positional imaging, respectively, of the same animal. “Х” represents the dead animal. Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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observed in the infected groin lymph nodes on day 1 pi, reached a peak on day five pi, and the bacteria were completely eliminated from the lymph nodes on day 11 pi. 3.4. In vivo bacterial BLI Three groups of six mice were subcutaneously infected with pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux, and then monitored for the bioluminescence emitted from each animal. The bioluminescent scale provided in the figures ranged from most intense (red) to least intense (violet). After subcutaneous administration of 2.0 106 CFU of pCD1þ-mutant-lux to the end of each mouse tail, the bioluminescence emitted from each animal was observed immediately using the NightOWL II LB983 imaging system at the time point designated as day 0, after which the mice were imaged on day 1, 3, 5, 7, 9, 11 and 13 (Fig. 3). On day 0 pi, the bioluminescence signal intensity was concentrated similarly at the injection site, indicating that each animal received the same dose of the bacterial suspension. The bioluminescence signal was observed in the region corresponding to the groin lymph node on day 1 pi. By day 3 of the infection, the bacteria had entered the mouse livers and spleens, on day 7 the mice started to die, and all were dead on day 14 pi. After each mouse received 6.2 107 CFU of Y. pseudotuberculosis Pa3606-lux by administration to the tail end, BLI of each animal was performed on days 0, 1, 3, 5 and 7 (Fig. 4A). The bioluminescence signal intensity was similarly observed at the injection site on day 0 after infection, indicating that each mouse received approximately the same dose of bacterial suspension. From days 0e5 pi, the bioluminescence signal attenuated gradually, disappearing by day 7 pi. All the animals survived the 14-days observation period. After the mice were received 8.6 107 CFU of E. coli K12-lux via their tail ends, BLI of each animal was undertaken on days 0, 1, 3 and 5 (Fig. 4B). Similar bioluminescence signal intensities were observed at the injection site on day 0 pi, indicating that each mouse received approximately the same dose of bacterial suspension. From days 0e3 pi, the bioluminescence signal gradually weakened, disappearing completely on day 5 pi. All the animals survived the 14-day of observation period. 3.5. Pathological changes in the tissues After subcutaneous injection of pCD1þ-lux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux on the fifth day, lymph nodes, livers, spleens and lungs were collected from each animal group and one untreated mouse, and then fixed in 10% neutral-buffered formalin for paraffin block preparation. The tissues were trimmed, paraffin embedded, sectioned and stained with HE for histopathological examination. No obvious histopathological changes were observed in the
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tissues examined from the animals infected with Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux or in the control animal (Fig. 5aed, panel C, D and A). By contrast, the animals infected with pCD1þ-mutant-lux had obvious lesions in the same tissues (Fig. 5aed, panel B). Congestion, necrosis, inflammatory cell infiltration and reduced lymphocyte numbers were observed in the lymph node tissues (Fig. 5a, panel B). Vascular engorgement and cellular degeneration were seen in the liver tissues (Fig. 5b, panel B). Congestion, necrosis and inflammatory cell infiltration were also seen in the spleen tissues (Fig. 5c, panel B). Vascular engorgement and slight partial edema were observed in the lung tissues (Fig. 5d, panel B). 4. Discussion BLI technology, which was developed over the past decade, allows for the noninvasive study of ongoing biological processes in small mammals [17]. This technology has been used successfully to visualize the dissemination of viruses, bacteria, fungus and parasites in vivo [18e28]. In our previous study, the pCD1þ-mutant used here was constructed by applying a method based on plasmid incompatibility to cure pMT1 and pPCP1 from the Y. pestis subsp. microtus 201 strain [15]. We have also previously developed a plasmidbased luxCDABE bioreporter in the pCD1þ-mutant, and investigated its dissemination by BLI during primary septicemic plague in a mouse model. However, this plasmid-based luxCDABE bioreporter was unstable in vivo, which limits its application for in vivo imaging [28]. Consequently, in the present study, the pCD1þ-mutant, Y. pseudotuberculosis Pa3606, and E. coli K12 were transformed with pXEN-luxCDABE (pXEN-18), and grown on the sucrose LB plates to facilitate chromosomal integration of the luxCDABE::Tn5::kan transposon, thereby yielding pCD1þ-mutantlux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux bioluminescent strains. The genome walking results showed that the luxCDABE::Tn5::kan transposon had integrated randomly into a non-encoding region of the pCD1þ-mutant's genome, and as expected, this did not affect bacterial growth and virulence in the host strain. However, the transposon was inserted into BZ19_1372, a ROK family protein encoding gene in Y. pseudotuberculosis Pa3606, and into the c-di-GMP phosphodiesterase-encoding NEB5A_03800 gene in E. coli K12. Bacterial growth curve analysis showed that inactivation of these two genes did not decrease the growth rates of Y. pseudotuberculosis Pa3606 and E. coli K12. Therefore, it appears likely that replacement of the pCD1þ-mutant, Y. pseudotuberculosis Pa3606 or E. coli K12 with its bioluminescent counterpart strain did not affect the experimental results. The BLI results indicated that the lux-strains have high bioluminescent stability in vivo.
Fig. 4. In vivo imaging of mice infected with Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux. Two groups of six mice were subcutaneously infected with Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux at the end of the tail, and the bioluminescence signal emitted from each animal was determined by the NightOWL II LB983 imaging system. (A) In vivo imaging of mice infected with Y. pseudotuberculosis Pa3606-lux. (B) In vivo imaging of mice infected with E. coli K12-lux. The numbers “1e6” represent six different mice, whereas “P and S” denote prone and supine positional imaging, respectively, of the same animal. Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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Fig. 5. Histopathological observation of the tissues from mice infected with pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux. (A) Tissue sections from one uninfected control animal. (B) Tissue sections from the animals infected with pCD1þ-mutant-lux. (C) Tissue sections from the animals infected with Y. pseudotuberculosis Pa3606-lux. (D) Tissue sections from the animals infected with E. coli K12-lux.
In a previous study, we found that many natural strains from rodents have lost their pPCP1 and pMT1 plasmids. The reason why these natural strains have lost two newly acquired plasmids is not known, but whether a strain containing only pCD1 is able to cause typical bubonic or pneumonic plague merits investigation. In vivo imaging technology has the great advantage of real-time in vivo detection of bacterial dissemination in an infected animal at different time points. In the present study, three groups of six mice were subcutaneously infected with the pCD1þmutant-lux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux, and each animal was observed at different time points using BLI technology. One day after the commencement of infection with the pCD1þ-mutant-lux in mice, a bioluminescent signal was observed in the region corresponding to the groin lymph nodes. Microbiological examination also found the bacteria in the groin lymph nodes, livers and spleens at the same time point in the mice. In contrast, the pCD1þ-mutant-lux could not be isolated from the lungs of the infected mice until the fifth day pi.
These results are consistent with the previous description on plague whereby bacteria multiply initially at the site of infection, enter the regional lymph nodes, and finally spread to other organs such as the spleen, liver and lungs [29]. It was reported that the loss of the pla gene or pPCP1 plasmid decreases the establishment of pneumonic infection in mice [12,30]. However, bacteria can be isolated from the lungs of mice infected with the Y. pestis strain that lacks the pPCP1 plasmid (pPCP1e mutant) at 1 h pi. Moreover, the bacterial load in the lungs at 48 h pi starts to fall in when compared with the 1 h pi time point [30]. After the infection with the pCD1þ-mutant in mice, bacteria were not isolatable from the lungs until the fifth day pi. These results indicate that the loss of both pMT1 and pPCP1 plasmids delayed the infection in the mouse lungs compared with the pPCP1e mutant. Histopathological observation also showed that pCD1þmutant-lux caused different degrees of pathological changes to the lymph nodes, livers, spleens and lungs of the mice, indicating that invasion of the pCD1þ-mutant had indeed caused bubonic and pneumonic disease.
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The Y. pestis infection route is primarily via the bites of infected fleas, whereas Y. pseudotuberculosis is transmitted by the fecal-oral route [31]. Both pathogens therefore utilize different means of transmission and cause distinct diseases, but both pathogens display a pronounced tropism for lymphoid tissue. Y. pestis often disseminate from the injection site to the regional draining lymph nodes after infection from an infected flea commences, and this causes lymphadenitis (the enlarged lymph node referred to as a bubo) [32], whereas Y. pseudotuberculosis usually enters the mesenteric lymph nodes (the nodes around the intestine) and multiplies there after infection by the oral route, causing mesenteric lymphadenitis [33]. Buboes typically occur in the inguinal and femoral regions, but sometimes arise in other regional lymph node sites, including the popliteal, axillary, supraclavicular, cervical, post-auricular, pharyngeal and deeper nodes, such as the intraabdominal or intrathoracic nodes [34]. It was reported that nonpathogenic E. coli could enter the mesenteric lymph nodes at 6 h post-inoculation [35]. In one study, Y. pseudotuberculosis lacking pYV was able to multiply in the lymphatic organs of mice for several days, but its sustained replication required pYV [36]. Although both Y. pestis and Y. pseudotuberculosis harbor a common ~70-kb virulence plasmid, it was not known whether Y. pseudotuberculosis could cause lymphadenitis by the subcutaneous route. In this study, a strain of O:1 b serotype of Y. pseudotuberculosis and E. coli k12 were used to infect mice subcutaneously at the end of the tail, respectively. The results showed that both pathogens were not transferred to the liver, spleen and lungs from the injection site, whereas Y. pseudotuberculosis entered the groin lymph nodes one day after infection commenced, remained there for a short time, and did not cause lymphadenitis. In comparison with E. coli k12, before it evolved into Y. pestis, pseudotuberculosis possessed the potential for lymph node invasion. Therefore, it is possible that Y. pestis inherited lymphoid tissue tropism from a Y. pseudotuberculosis ancestor rather than acquiring this property independently. Acknowledgements Financial support for this study came from Natural Science foundation Committee of China (contract no. 31430006). We thank Sandra Cheesman, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. All the authors have read this MS and approved of its submission.
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References [1] Cui Y, Yu C, Yan Y, Li D, Li Y, Jombart T, et al. Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis. Proc Natl Acad Sci U S A 2013;110:577e82. [2] Achtman M, Zurth K, Morelli G, Torrea G, Guiyoule A, Carniel E. Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A 1999;96: 14043e8.
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[3] Portnoy DA, Wolf-Watz H, Bolin I, Beeder AB, Falkow S. Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins. Infect Immun 1984;43: 108e14. [4] Perry RD, Fetherston JD. Yersinia pestis-etiologic agent of plague. Clin Microbiol Rev 1997;10:35e66. [5] Human plague: review of regional morbidity and mortality, 2004e2009. Wkly Epidemiol Rec 2009;85:40e5. [6] Stenseth NC, Atshabar BB, Begon M, Belmain SR, Bertherat E, Carniel E, et al. Plague: past, present, and future. PLoS Med 2008; 5:e3. [7] Riedel S. Plague: from natural disease to bioterrorism. Proc (Bayl Univ Med Cent) 2005;18:116e24. [8] Inglesby TV, Dennis DT, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA 2000;283: 2281e90. [9] Cornelis GR, Van Gijsegem F. Assembly and function of type III secretory systems. Annu Rev Microbiol 2000;54:735e74. [10] Brubaker RR. Interleukin-10 and inhibition of innate immunity to Yersiniae: roles of Yops and LcrV (V antigen). Infect Immun 2003;71: 3673e81. [11] Cornelis GR. The Yersinia Ysc-Yop ‘type III’ weaponry. Nat Rev Mol Cell Biol 2002;3:742e52. [12] Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. Science 2007;315:509e13. [13] Zimbler DL, Schroeder JA, Eddy JL, Lathem WW. Early emergence of Yersinia pestis as a severe respiratory pathogen. Nat Commun 2015;6: 7487. [14] Felek S, Muszynski A, Carlson RW, Tsang TM, Hinnebusch BJ, Krukonis ES. Phosphoglucomutase of Yersinia pestis is required for autoaggregation and polymyxin B resistance. Infect Immun 2010;78: 1163e75. [15] Ni B, Du Z, Guo Z, Zhang Y, Yang R. Curing of four different plasmids in Yersinia pestis using plasmid incompatibility. Lett Appl Microbiol 2008;47:235e40. [16] Rohr LR, Layfield LJ, Wallin D, Hardy D. A comparison of routine and rapid microwave tissue processing in a surgical pathology laboratory. Am J Clin Pathol 2001;115:703e8. [17] Massoud TF, Gambhir SS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 2003;17: 545e80. [18] Kong Y, Subbian S, Cirillo SLG, Cirillo JD. Application of optical imaging to study of extrapulmonary spread by tuberculosis. Tuberc (Edinb) 2009;89:S15e7. [19] Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 2008;16:1073e80. [20] Hyland KV, Asfaw SH, Olson CL, Daniels MD, Engman DM. Bioluminescent imaging of Trypanosoma cruzi infection. Int J Parasitol 2008; 38:1391e400. [21] Hutchens M, Luker GD. Applications of bioluminescence imaging to the study of infectious diseases. Cell Microbiol 2007;9:2315e22. [22] Francis KP, Yu J, Bellinger-Kawahara C, Joh D, Hawkinson MJ, Xiao G, et al. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Infect Immun 2001;69:3350e8. [23] Lane MC, Alteri CJ, Smith SN, Mobley HLT. Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract. Proc Natl Acad Sci U S A 2007;104:16669e74. [24] Brock M, Jouvion G, Droin-Bergere S, Dussurget O, Nicola MA, Ibrahim-Granet O. Bioluminescent Aspergillus fumigatus, a new tool for drug efficiency testing and in vivo monitoring of invasive aspergillosis. Appl Environ Microbiol 2008;74:7023e35. [25] Cook SH, Griffin DE. Luciferase imaging of a neurotropic viral infection in intact animals. J Virol 2003;77:5333e8.
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[26] Glomski IJ, Piris-Gimenez A, Huerre M, Mock Ml, Goossens PL. Primary involvement of pharynx and Peyer's patch in inhalational and intestinal anthrax. PLoS Pathog 2007;3:e76. [27] Bina XR, Miller MA, Bina JE. Construction of a bioluminescence reporter plasmid for Francisella tularensis. Plasmid 2010;64:156e61. [28] Zhou J, Bi Y, Xu X, Qiu Y, Wang Q, Feng N, et al. Bioluminescent tracking of colonization and clearance dynamics of plasmid-deficient Yersinia pestis strains in a mouse model of septicemic plague. Microbes Infect 2014;16:214e24. [29] Pujol C, Bliska JB. The ability to replicate in macrophages is conserved between Yersinia pestis and Yersinia pseudotuberculosis. Infect Immun 2003;71:5892e9. [30] Stacy LA, Jian S, Wallace BB, Tatiana EE, Sheri MF, Giovanni S, et al. Deletion of Braun lipoprotein gene (lpp) and curing of plasmid pPCP1 dramatically alter the virulence of Yersinia pestis CO92 in a mouse model of pneumonic plague. Microbiology 2009;155:3247e59.
[31] Brubaker RR. Factors promoting acute and chronic diseases caused by yersiniae. Clin Microbiol Rev 1991;4:309e24. [32] Putzker M, Sauer H, Sobe D. Plague and other human infections caused by Yersinia species. Clin Lab 2001;47:453e66. [33] Smego RA, Frean J, Koornhof HJ, Yersiniosis I. Microbiological and clinicoepidemiological aspects of plague and non-plague Yersinia infections. Eur J Clin Microbiol Infect Dis 1999;18: 1e15. [34] Anisimov AP, Amoako KK. Treatment of plague: promising alternatives to antibiotics. J Med Microbiol 2006;55:1461e75. [35] Balada-Llasat JM, Mecsas J. Yersinia has a tropism for B and T cell zones of lymph nodes that is independent of the type III secretion system. PLoS Pathog 2006;2:e86. [36] Simonet M, Richard S, Berche P. Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudotuberculosis harboring the pYV plasmid. Infect Immun 1990;58:841e5.
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Microbes and Infection xx (2017) 1e10 www.elsevier.com/locate/micinf
Original article
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Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice Yazhou Zhou, Jiyuan Zhou, Yuxin Ji, Lu Li, Yafang Tan, Guang Tian, Ruifu Yang, Xiaoyi Wang* Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China Received 22 July 2017; accepted 7 November 2017 Available online ▪ ▪ ▪
Abstract Yersinia pestis has evolved from Yersinia pseudotuberculosis serotype O:1b. A typical Y. pestis contains three plasmids: pCD1, pMT1 and pPCP1. However, some isolates only harbor pCD1 (pCD1þ-mutant). Y. pestis and Y. pseudotuberculosis share a common plasmid (pCD1 or pYV), but little is known about whether Y. pseudotuberculosis exhibited plague-inducing potential before it was evolved into Y. pestis. Here, the luxCDABE::Tn5::kan was integrated into the chromosome of the pCD1þ-mutant, Y. pseudotuberculosis or Escherichia coli K12 to construct stable bioluminescent strains for investigation of their dissemination in mice by bioluminescence imaging technology. After subcutaneous infection, the pCD1þ-mutant entered the lymph nodes, followed by the liver and spleen, and, subsequently, the lungs, causing pathological changes in these organs. Y. pseudotuberculosis entered the lymph nodes, but not the liver, spleen and lungs. It also resided in the lymph nodes for several days, but did not cause lymphadenitis or pathological lesions. By contrast, E. coli K12-lux was not isolatable from mouse lymph nodes, liver, spleen and lungs. These results indicate that the pCD1þ-mutant can cause typical bubonic and pneumonic plague-like diseases, and Y. pestis has inherited lymphoid tissue tropism from its ancestor rather than acquiring these properties independently. © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Plague; Yersinia pestis; Yersinia pseudotuberculosis; In vivo bioluminescence imaging
1. Introduction Of the 17 Yersinia genus species, Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica all cause invasive diseases in humans and other mammals. Y. pestis is genetically very closely related to the enteric pathogen Y. pseudotuberculosis, and both of them share 90% DNA sequence homology. Despite the highly distinct diseases they cause and their different transmission modes, it is generally accepted that Y. pestis evolved from the Y. pseudotuberculosis O:1 b serotype as a clone about 5000 to 7000 years ago [1,2]. All three pathogenic species share a related plasmid (called pCD1 in Y. pestis and pYV in Y. pseudotuberculosis and Y.
* Corresponding author. Fax: þ86 10 63815689. E-mail address: [email protected] (X. Wang).
enterocolitica), which is associated with virulence and in vitro Ca2þ-dependent growth at 37 C [3]. Y. pestis causes plague, a zoonotic disease occasionally transmitted to humans from Y. pestis-infected rodents via the bite of infected fleas [4]. Historically, Y. pestis has caused three major plague pandemics, leading to millions of human deaths. Recently, an increase in the annual number of global plague cases was reported to the World Health Organization (WHO), and plague is now considered by them to be a re-emerging infectious disease [5,6]. Plague has also attracted much attention because of its potential for use in classical biological warfare or bioterrorism agents [7,8]. A typical Y. pestis strain usually contains three virulence plasmids: pCD1, pMT1 and pPCP1. pCD1 was inherited from its Y. pseudotuberculosis ancestor, whereas pMT1 and pPCP1 were acquired by horizontal transfer from unknown pathogens. The pCD1 and pYV plasmids encode the type three secretion
https://doi.org/10.1016/j.micinf.2017.11.005 1286-4579/© 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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system (T3SS) [9], which is responsible for injecting into host cells a number of Yersinia outer proteins (Yops) that inhibit bacterial phagocytosis, the respiratory burst, the host innate immune response, and trigger apoptosis [10,11]. The newlyacquired pPCP1 encodes the plasminogen activator (Pla), a surface proteinase responsible for increasing the pathogen's invasive ability in bubonic plague and enabling rapid replication of Y. pestis in the lungs [12,13]. Another newly acquired plasmid (pMT1) encodes the most important protective antigens in Y. pestis, the F1 antigen, along with the murine toxin (Ymt), which is required for Y. pestis survival in the flea midgut [14]. We recently analyzed 133 natural isolates of Y. pestis, including 107 strains representing the geographical and genetic diversity of >5000 Chinese isolates, 11 isolates from Mongolia, the former Soviet Union, Myanmar, and Madagascar, and 15 previously sequenced genomes through sequence alignment analyses. We found several Y. pestis plasmid deletion mutants that only contain the pCD1 plasmid (pCD1þ-mutant). These pCD1þ-mutants were isolated from rodents, but whether these mutants can cause typical plague disease requires investigation. Y. pestis and Y. pseudotuberculosis share a common virulence plasmid (pCD1 or pYV), but no study has investigated whether Y. pseudotuberculosis has the potential to induce plague disease via the subcutaneous route before it evolved into Y. pestis. To answer this question, the pCD1þ-mutant, Y. pseudotuberculosis O:1b and Escherichia coli K12 were used to study their dissemination routes during primary bubonic plague in a mouse experiment infection model using a bioluminescence imaging (BLI) technology. E. coli K12 does not contain pCD1 or pYV plasmids, and therefore, will not induce bubonic plague after subcutaneous infection with it. In this study, Y. pseudotuberculosis was compared with E. coli K12 to assess its ability to induce bubonic plague in mice. 2. Material and methods 2.1. Bacterial strains and mice The pCD1þ-mutant (containing only the pCD1 plasmid) was constructed from the Y. pestis subsp. microtus 201 strain using the plasmid incompatibility principle described in our previous study [15]. Y. pseudotuberculosis Pa3606 (serotype O:1b) was isolated from humans in Japan. E. coli K12 was purchased from NEB (New England Biolabs). The pXENluxCDABE (pXEN-18) bioluminescent plasmid was a generous gift from Dr. Tao Zhou (National Center of Biomedical Analysis, Beijing, China). The plasmid is under the control of a native promoter, confers resistances to ampicillin and kanamycin, and contains Tn5 and sacB transposon genes. All the Male BALB/c mice (6e8 weeks old) used in this study were provided with food and fresh water ad libitum throughout the study. All the animal experiments were conducted in accordance with the Guidelines for the Welfare and Ethics of Laboratory Animals of China and were approved by
the Committee of the Welfare and Ethics of Laboratory Animals. 2.2. Bioluminescent reporter strain construction The pCD1þ-mutant, Y. pseudotuberculosis Pa3606 and E. coli K12 were transformed with pXEN-18 by electroporation, and then grown on Hottinger agar plates containing kanamycin (50 mg/ml). Bioluminescent bacterial strains were selected on the NightOWL II LB983 imaging system, and then grown on 10% (W/V) sucrose LuriaeBertani (LB) plates containing kanamycin (50 mg/ml) to enable chromosomal integration of the luxCDABE::Tn5::kan transposon. Each bioluminescent clone isolated via sucrose plate selection was replated on LB plates containing kanamycin (50 mg/ml) and ampicillin (100 mg/ml), and the clones with kanamycin resistance but not ampicillin resistance were considered positive. The positive clones that were selected were screened for the position of luxCDABE on the chromosome. The resultant bioluminescent strains were designated as pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux. 2.3. Identification of the chromosomal position of lux CDABE The chromosomal integration position of lux CDABE was determined using a genome walking kit (Takara GW) and DNA sequencing. Briefly, three forward primers based on the kanamycin gene sequence were designed (designated SP1_SP3), and four reverse primers (AP1_AP4) were provided by the genome walking kit. The SP1-SP3 primer sequences are 50 -CGTAATGGCTGGCCTGTTGAACAAG-30 (SP1), 50 -CACCGGATTCAGTCGTCACTCATGGT-30 (SP2) and 50 -CGATACCAGGATCTTGCCATCCTATG-30 (SP3). The DNA samples from the selected clones were used as templates for the first round of nested PCR with SP1 combined with AP1, AP2, AP3 or AP4 in four separate PCR tubes. After the first round of nested PCR, 1 ml of PCR product was removed from each of the four tubes, and used individually as the template for the second round of nested PCR with SP2 combined with AP1, AP2, AP3 or AP4 in four separate test tubes. After the second round of nested PCR reactions, 1 ml of PCR product was removed from each of the four tubes, and then used as the template for the third round of nested PCR with SP3 combined with AP1, AP2, AP3 or AP4 in the four PCR tubes. Finally, the PCR products from four test tubes were analyzed by 1% agarose gel electrophoresis, and sequenced with the SP3 primer. The position of luxCDABE::Tn5::kan on the chromosome was identified by BLAST analysis of the National Center for Biotechnology Infection NCBI website. 2.4. Growth curves for the bioluminescent strains A single colony of the lux-expressing strain or its parent strain was inoculated into 5 ml of LB medium, and then grown to the mid-log phase at 26 C (pCD1þ-mutant-lux, Y.
Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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2.5. Bacterial virulence determination
Fig. 1. Chromosomal position of the luxCDABE::Tn5::kan transposon in the pCD1þ-mutant, Y. pseudotuberculosis Pa3606, and E. coli K12.
The pCD1þ-mutant and pCD1þ-mutant-lux were inoculated from overnight cultures into fresh LB medium and grown to an OD of 0.6 at 620 nm at 26 C, and then diluted 10-fold into the same medium and cultivated at 26 C to the mid-log phase. Bacterial cells were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS), diluted in PBS and the cell density was adjusted to the desired concentration for the challenge experiments. A groups of ten male BALB/c mice were subcutaneously injected with the pCD1þmutant (1.61 107 colony forming units, CFU) or pCD1þmutant-lux (1.56 107 CFU). The time of death for each animal was recorded during the observation intervals, and a percentage survival analysis was performed using GraphPad Prism. 2.6. BLI and microbiological examination
pseudotuberculosis Pa3606-lux, plus their parents) or at 37 C (E. coli K12-lux and its parent). The bacterial suspension (900 ml) was transferred to 18 ml of fresh LB medium and grown to an OD600 of 1.0 (determined by an ultraviolet spectrophotometer UV-5100B, METASH, China), after which 900 ml (OD600 1.0) of it was transferred to 18 ml of fresh LB medium for cultivation. During the cultivation process, 300 ml of bacterial suspension was collected at different time points, 30 ml of 40% formalin was added, and the OD600 value was determined. Graphs of the OD600 values versus time were plotted.
Three bioluminescent bacterial strains of pCD1þ-mutantlux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux were individually grown to an OD 600 of 1.0 in LB medium, harvested by centrifugation, and then resuspended in PBS to prepare serial 10-fold bacterial suspension dilutions. Individual groups of six male BALB/c mice were subcutaneously injected at the end of the tail with the pCD1þ-mutantlux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux. The infected mice were anesthetized with 3% isoflurane for 5 min, and imaged with the NightOWL II LB983 imaging system
Fig. 2. Growth curves, virulence and bacterial loads. (A) The growth curve of the pCD1þ-mutant and pCD1þ-mutant-lux. (B) The growth curve of Y. pseudotuberculosis Pa3606 and Y. pseudotuberculosis Pa3606-lux. (C) The growth curve of E. coli K12 and E. coli K12-lux. (D) Comparison of virulence between the pCD1þ-mutant and pCD1þ-mutant-lux in mice after infection via the subcutaneous route. (E) Bacterial loads in the groin lymph nodes from the mice subcutaneously infected with Y. pseudotuberculosis Pa3606-lux. Bacterial numbers are reported in CFU/lymph node. Data points are expressed as the means (±standard deviations) of the measurements taken for three mice. Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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(Berthold Technologies, Bad Wildbad, Germany) at various post-infection (pi) time point. The bioluminescence signals analyzed with IndiGO software (Berthold Technologies, Germany) were recorded as counts per second, with monitoring taking place at regular time intervals. The bacterial load or degree of colonization in each animal tissue was determined after pentobarbital sodium anesthesia during the post-mortem examinations. The groin lymph nodes, liver, spleen and lung tissues of the infected mice were harvested, homogenized, and then subjected to agar plate counts to confirm whether any bacteria were present in these organs. 2.7. Pathological observations The mice infected with pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux were killed by CO2 inhalation followed by cervical dislocation on the fifth day of infection. Tissues were collected and placed in 10% neutralbuffered formalin, dehydrated through a serial alcohol gradient (70%, 80%, 90%, 95%, and 100%), cleared with xylene, infiltrated with wax, and then embedded in paraffin [16]. The issue sections were stained with hematoxylin and eosin (HE) for histopathological examination under light microscopy. 2.8. Statistical analyses Statistical analyses were performed using GraphPad Prism version 5.0. The bacterial loads in the groin lymph nodes from the pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux were determined by viable plate counts. The bacterial load difference between two strains was investigated by the ManneWhitney t test with a two-tailed nonparametric analysis. The survival rates in the different infection groups were compared by a log-rank test. A probability value of <0.05 was considered statistically significant. 3. Results 3.1. Identification of bioluminescent strains The bioluminescent reporter strains pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux were constructed by combining each of them with the pXEN-18 plasmid containing the luxCDABE::Tn5::kan transposon and sucrose selection, thereby facilitating transposon integration into the chromosome of each strain. The chromosomal position of luxCDABE::Tn5::kan was mapped by genome walking, DNA sequencing and BLAST analysis of the NCBI website (Fig. 1). In the pCD1þ-mutant-lux bioluminescent strain, luxCDABE::Tn5::kan was inserted into a non-coding region between the YP_3609 and YP_3610 genes. In the E.
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coli K12-lux bioluminescent strain, luxCDABE::Tn5::kan was inserted inside the NEB5A_03800 c-di-GMP phosphodiesterase encoding gene. In the Y. pseudotuberculosis Pa3606-lux strain, luxCDABE::Tn5::kan was inserted into the gene BZ19_1372 encoding a ROK family protein. 3.2. Growth curves and bacterial strain virulence To investigate whether transpose integration into the chromosomes of the wild-type strains affected their growth, the growth rate of each lux-expressing strain was compared with that of the corresponding parental strain in vitro. The results showed no obvious growth difference between the luxexpressing strains and their parent strains (Fig. 2AeC). In investigating whether a virulence difference existed between the wild-type strains and their lux-expressing counterparts, our preliminary experiments found that Y. pseudotuberculosis Pa3606 and E. coli K12 had extremely low virulent profiles in mice by the subcutaneous route. Even when the mice received a high dose of Y. pseudotuberculosis Pa3606 or E. coli K12 (>108 CFU), all the animals still survived, but the pCD1þmutant still possessed high virulence in the mouse model of bubonic plague. Here, we only compared the virulence of the pCD1þ-mutant (1.61 107 CFU) with that of its luxexpressing strain (1.56 107 CFU). Statistical analysis showed no significant virulence difference between the pCD1þ-mutant and its lux-expressing counterpart (Fig. 2D). 3.3. Bacterial tissue colonization To further verify the BLI results, bacterial colonization of the groin lymph nodes, livers, spleens and lungs from the pCD1þ-mutant-lux-infected mice (4.5 106 CFU), the Y. pseudotuberculosis Pa3606-lux-infected mice (8.6 107 CFU) or E. coli K12-lux-infected mice (8.2 107 CFU) was assessed after subcutaneous infection administered by the end of the tail route. A small number of the pCD1þ-mutant-lux group had infected groin lymph nodes, livers and spleens on day 1 pi, but a large number of bacteria were isolated from these organs on day 3 pi. A large number of bacteria were observed in the infected lungs until day five pi. Microbiological examination also showed that Y. pseudotuberculosis Pa3606-lux was transferred to the groin lymph nodes one day after the infection commenced, but E. coli K12lux was not transferred to the groin lymph nodes during the whole eleven-day observation period. However, no Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux was isolated from the liver, spleen and lungs of each infected animal. To evaluate the colonization of Y. pseudotuberculosis Pa3606-lux in the infected groin lymph nodes, the bacterial loads were assessed during the eleven-day observation period (Fig. 2E). Small numbers of Y. pseudotuberculosis Pa3606-lux were
Fig. 3. In vivo imaging of mice infected with pCD1þ-mutant-lux. Six mice were subcutaneously infected at the end of the tail with pCD1þ-mutant-lux, and the bioluminescence signal emitted from each animal was determined by the NightOWL II LB983 imaging system. The numbers “1e6” represent six different mice, whereas “P and S” denote prone and supine positional imaging, respectively, of the same animal. “Х” represents the dead animal. Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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observed in the infected groin lymph nodes on day 1 pi, reached a peak on day five pi, and the bacteria were completely eliminated from the lymph nodes on day 11 pi. 3.4. In vivo bacterial BLI Three groups of six mice were subcutaneously infected with pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux, and then monitored for the bioluminescence emitted from each animal. The bioluminescent scale provided in the figures ranged from most intense (red) to least intense (violet). After subcutaneous administration of 2.0 106 CFU of pCD1þ-mutant-lux to the end of each mouse tail, the bioluminescence emitted from each animal was observed immediately using the NightOWL II LB983 imaging system at the time point designated as day 0, after which the mice were imaged on day 1, 3, 5, 7, 9, 11 and 13 (Fig. 3). On day 0 pi, the bioluminescence signal intensity was concentrated similarly at the injection site, indicating that each animal received the same dose of the bacterial suspension. The bioluminescence signal was observed in the region corresponding to the groin lymph node on day 1 pi. By day 3 of the infection, the bacteria had entered the mouse livers and spleens, on day 7 the mice started to die, and all were dead on day 14 pi. After each mouse received 6.2 107 CFU of Y. pseudotuberculosis Pa3606-lux by administration to the tail end, BLI of each animal was performed on days 0, 1, 3, 5 and 7 (Fig. 4A). The bioluminescence signal intensity was similarly observed at the injection site on day 0 after infection, indicating that each mouse received approximately the same dose of bacterial suspension. From days 0e5 pi, the bioluminescence signal attenuated gradually, disappearing by day 7 pi. All the animals survived the 14-days observation period. After the mice were received 8.6 107 CFU of E. coli K12-lux via their tail ends, BLI of each animal was undertaken on days 0, 1, 3 and 5 (Fig. 4B). Similar bioluminescence signal intensities were observed at the injection site on day 0 pi, indicating that each mouse received approximately the same dose of bacterial suspension. From days 0e3 pi, the bioluminescence signal gradually weakened, disappearing completely on day 5 pi. All the animals survived the 14-day of observation period. 3.5. Pathological changes in the tissues After subcutaneous injection of pCD1þ-lux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux on the fifth day, lymph nodes, livers, spleens and lungs were collected from each animal group and one untreated mouse, and then fixed in 10% neutral-buffered formalin for paraffin block preparation. The tissues were trimmed, paraffin embedded, sectioned and stained with HE for histopathological examination. No obvious histopathological changes were observed in the
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tissues examined from the animals infected with Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux or in the control animal (Fig. 5aed, panel C, D and A). By contrast, the animals infected with pCD1þ-mutant-lux had obvious lesions in the same tissues (Fig. 5aed, panel B). Congestion, necrosis, inflammatory cell infiltration and reduced lymphocyte numbers were observed in the lymph node tissues (Fig. 5a, panel B). Vascular engorgement and cellular degeneration were seen in the liver tissues (Fig. 5b, panel B). Congestion, necrosis and inflammatory cell infiltration were also seen in the spleen tissues (Fig. 5c, panel B). Vascular engorgement and slight partial edema were observed in the lung tissues (Fig. 5d, panel B). 4. Discussion BLI technology, which was developed over the past decade, allows for the noninvasive study of ongoing biological processes in small mammals [17]. This technology has been used successfully to visualize the dissemination of viruses, bacteria, fungus and parasites in vivo [18e28]. In our previous study, the pCD1þ-mutant used here was constructed by applying a method based on plasmid incompatibility to cure pMT1 and pPCP1 from the Y. pestis subsp. microtus 201 strain [15]. We have also previously developed a plasmidbased luxCDABE bioreporter in the pCD1þ-mutant, and investigated its dissemination by BLI during primary septicemic plague in a mouse model. However, this plasmid-based luxCDABE bioreporter was unstable in vivo, which limits its application for in vivo imaging [28]. Consequently, in the present study, the pCD1þ-mutant, Y. pseudotuberculosis Pa3606, and E. coli K12 were transformed with pXEN-luxCDABE (pXEN-18), and grown on the sucrose LB plates to facilitate chromosomal integration of the luxCDABE::Tn5::kan transposon, thereby yielding pCD1þ-mutantlux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux bioluminescent strains. The genome walking results showed that the luxCDABE::Tn5::kan transposon had integrated randomly into a non-encoding region of the pCD1þ-mutant's genome, and as expected, this did not affect bacterial growth and virulence in the host strain. However, the transposon was inserted into BZ19_1372, a ROK family protein encoding gene in Y. pseudotuberculosis Pa3606, and into the c-di-GMP phosphodiesterase-encoding NEB5A_03800 gene in E. coli K12. Bacterial growth curve analysis showed that inactivation of these two genes did not decrease the growth rates of Y. pseudotuberculosis Pa3606 and E. coli K12. Therefore, it appears likely that replacement of the pCD1þ-mutant, Y. pseudotuberculosis Pa3606 or E. coli K12 with its bioluminescent counterpart strain did not affect the experimental results. The BLI results indicated that the lux-strains have high bioluminescent stability in vivo.
Fig. 4. In vivo imaging of mice infected with Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux. Two groups of six mice were subcutaneously infected with Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux at the end of the tail, and the bioluminescence signal emitted from each animal was determined by the NightOWL II LB983 imaging system. (A) In vivo imaging of mice infected with Y. pseudotuberculosis Pa3606-lux. (B) In vivo imaging of mice infected with E. coli K12-lux. The numbers “1e6” represent six different mice, whereas “P and S” denote prone and supine positional imaging, respectively, of the same animal. Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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Fig. 5. Histopathological observation of the tissues from mice infected with pCD1þ-mutant-lux, Y. pseudotuberculosis Pa3606-lux or E. coli K12-lux. (A) Tissue sections from one uninfected control animal. (B) Tissue sections from the animals infected with pCD1þ-mutant-lux. (C) Tissue sections from the animals infected with Y. pseudotuberculosis Pa3606-lux. (D) Tissue sections from the animals infected with E. coli K12-lux.
In a previous study, we found that many natural strains from rodents have lost their pPCP1 and pMT1 plasmids. The reason why these natural strains have lost two newly acquired plasmids is not known, but whether a strain containing only pCD1 is able to cause typical bubonic or pneumonic plague merits investigation. In vivo imaging technology has the great advantage of real-time in vivo detection of bacterial dissemination in an infected animal at different time points. In the present study, three groups of six mice were subcutaneously infected with the pCD1þmutant-lux, Y. pseudotuberculosis Pa3606-lux and E. coli K12-lux, and each animal was observed at different time points using BLI technology. One day after the commencement of infection with the pCD1þ-mutant-lux in mice, a bioluminescent signal was observed in the region corresponding to the groin lymph nodes. Microbiological examination also found the bacteria in the groin lymph nodes, livers and spleens at the same time point in the mice. In contrast, the pCD1þ-mutant-lux could not be isolated from the lungs of the infected mice until the fifth day pi.
These results are consistent with the previous description on plague whereby bacteria multiply initially at the site of infection, enter the regional lymph nodes, and finally spread to other organs such as the spleen, liver and lungs [29]. It was reported that the loss of the pla gene or pPCP1 plasmid decreases the establishment of pneumonic infection in mice [12,30]. However, bacteria can be isolated from the lungs of mice infected with the Y. pestis strain that lacks the pPCP1 plasmid (pPCP1e mutant) at 1 h pi. Moreover, the bacterial load in the lungs at 48 h pi starts to fall in when compared with the 1 h pi time point [30]. After the infection with the pCD1þ-mutant in mice, bacteria were not isolatable from the lungs until the fifth day pi. These results indicate that the loss of both pMT1 and pPCP1 plasmids delayed the infection in the mouse lungs compared with the pPCP1e mutant. Histopathological observation also showed that pCD1þmutant-lux caused different degrees of pathological changes to the lymph nodes, livers, spleens and lungs of the mice, indicating that invasion of the pCD1þ-mutant had indeed caused bubonic and pneumonic disease.
Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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Q1
The Y. pestis infection route is primarily via the bites of infected fleas, whereas Y. pseudotuberculosis is transmitted by the fecal-oral route [31]. Both pathogens therefore utilize different means of transmission and cause distinct diseases, but both pathogens display a pronounced tropism for lymphoid tissue. Y. pestis often disseminate from the injection site to the regional draining lymph nodes after infection from an infected flea commences, and this causes lymphadenitis (the enlarged lymph node referred to as a bubo) [32], whereas Y. pseudotuberculosis usually enters the mesenteric lymph nodes (the nodes around the intestine) and multiplies there after infection by the oral route, causing mesenteric lymphadenitis [33]. Buboes typically occur in the inguinal and femoral regions, but sometimes arise in other regional lymph node sites, including the popliteal, axillary, supraclavicular, cervical, post-auricular, pharyngeal and deeper nodes, such as the intraabdominal or intrathoracic nodes [34]. It was reported that nonpathogenic E. coli could enter the mesenteric lymph nodes at 6 h post-inoculation [35]. In one study, Y. pseudotuberculosis lacking pYV was able to multiply in the lymphatic organs of mice for several days, but its sustained replication required pYV [36]. Although both Y. pestis and Y. pseudotuberculosis harbor a common ~70-kb virulence plasmid, it was not known whether Y. pseudotuberculosis could cause lymphadenitis by the subcutaneous route. In this study, a strain of O:1 b serotype of Y. pseudotuberculosis and E. coli k12 were used to infect mice subcutaneously at the end of the tail, respectively. The results showed that both pathogens were not transferred to the liver, spleen and lungs from the injection site, whereas Y. pseudotuberculosis entered the groin lymph nodes one day after infection commenced, remained there for a short time, and did not cause lymphadenitis. In comparison with E. coli k12, before it evolved into Y. pestis, pseudotuberculosis possessed the potential for lymph node invasion. Therefore, it is possible that Y. pestis inherited lymphoid tissue tropism from a Y. pseudotuberculosis ancestor rather than acquiring this property independently. Acknowledgements Financial support for this study came from Natural Science foundation Committee of China (contract no. 31430006). We thank Sandra Cheesman, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. All the authors have read this MS and approved of its submission.
Q2
References [1] Cui Y, Yu C, Yan Y, Li D, Li Y, Jombart T, et al. Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis. Proc Natl Acad Sci U S A 2013;110:577e82. [2] Achtman M, Zurth K, Morelli G, Torrea G, Guiyoule A, Carniel E. Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A 1999;96: 14043e8.
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[3] Portnoy DA, Wolf-Watz H, Bolin I, Beeder AB, Falkow S. Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins. Infect Immun 1984;43: 108e14. [4] Perry RD, Fetherston JD. Yersinia pestis-etiologic agent of plague. Clin Microbiol Rev 1997;10:35e66. [5] Human plague: review of regional morbidity and mortality, 2004e2009. Wkly Epidemiol Rec 2009;85:40e5. [6] Stenseth NC, Atshabar BB, Begon M, Belmain SR, Bertherat E, Carniel E, et al. Plague: past, present, and future. PLoS Med 2008; 5:e3. [7] Riedel S. Plague: from natural disease to bioterrorism. Proc (Bayl Univ Med Cent) 2005;18:116e24. [8] Inglesby TV, Dennis DT, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA 2000;283: 2281e90. [9] Cornelis GR, Van Gijsegem F. Assembly and function of type III secretory systems. Annu Rev Microbiol 2000;54:735e74. [10] Brubaker RR. Interleukin-10 and inhibition of innate immunity to Yersiniae: roles of Yops and LcrV (V antigen). Infect Immun 2003;71: 3673e81. [11] Cornelis GR. The Yersinia Ysc-Yop ‘type III’ weaponry. Nat Rev Mol Cell Biol 2002;3:742e52. [12] Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. Science 2007;315:509e13. [13] Zimbler DL, Schroeder JA, Eddy JL, Lathem WW. Early emergence of Yersinia pestis as a severe respiratory pathogen. Nat Commun 2015;6: 7487. [14] Felek S, Muszynski A, Carlson RW, Tsang TM, Hinnebusch BJ, Krukonis ES. Phosphoglucomutase of Yersinia pestis is required for autoaggregation and polymyxin B resistance. Infect Immun 2010;78: 1163e75. [15] Ni B, Du Z, Guo Z, Zhang Y, Yang R. Curing of four different plasmids in Yersinia pestis using plasmid incompatibility. Lett Appl Microbiol 2008;47:235e40. [16] Rohr LR, Layfield LJ, Wallin D, Hardy D. A comparison of routine and rapid microwave tissue processing in a surgical pathology laboratory. Am J Clin Pathol 2001;115:703e8. [17] Massoud TF, Gambhir SS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 2003;17: 545e80. [18] Kong Y, Subbian S, Cirillo SLG, Cirillo JD. Application of optical imaging to study of extrapulmonary spread by tuberculosis. Tuberc (Edinb) 2009;89:S15e7. [19] Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 2008;16:1073e80. [20] Hyland KV, Asfaw SH, Olson CL, Daniels MD, Engman DM. Bioluminescent imaging of Trypanosoma cruzi infection. Int J Parasitol 2008; 38:1391e400. [21] Hutchens M, Luker GD. Applications of bioluminescence imaging to the study of infectious diseases. Cell Microbiol 2007;9:2315e22. [22] Francis KP, Yu J, Bellinger-Kawahara C, Joh D, Hawkinson MJ, Xiao G, et al. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Infect Immun 2001;69:3350e8. [23] Lane MC, Alteri CJ, Smith SN, Mobley HLT. Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract. Proc Natl Acad Sci U S A 2007;104:16669e74. [24] Brock M, Jouvion G, Droin-Bergere S, Dussurget O, Nicola MA, Ibrahim-Granet O. Bioluminescent Aspergillus fumigatus, a new tool for drug efficiency testing and in vivo monitoring of invasive aspergillosis. Appl Environ Microbiol 2008;74:7023e35. [25] Cook SH, Griffin DE. Luciferase imaging of a neurotropic viral infection in intact animals. J Virol 2003;77:5333e8.
Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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[26] Glomski IJ, Piris-Gimenez A, Huerre M, Mock Ml, Goossens PL. Primary involvement of pharynx and Peyer's patch in inhalational and intestinal anthrax. PLoS Pathog 2007;3:e76. [27] Bina XR, Miller MA, Bina JE. Construction of a bioluminescence reporter plasmid for Francisella tularensis. Plasmid 2010;64:156e61. [28] Zhou J, Bi Y, Xu X, Qiu Y, Wang Q, Feng N, et al. Bioluminescent tracking of colonization and clearance dynamics of plasmid-deficient Yersinia pestis strains in a mouse model of septicemic plague. Microbes Infect 2014;16:214e24. [29] Pujol C, Bliska JB. The ability to replicate in macrophages is conserved between Yersinia pestis and Yersinia pseudotuberculosis. Infect Immun 2003;71:5892e9. [30] Stacy LA, Jian S, Wallace BB, Tatiana EE, Sheri MF, Giovanni S, et al. Deletion of Braun lipoprotein gene (lpp) and curing of plasmid pPCP1 dramatically alter the virulence of Yersinia pestis CO92 in a mouse model of pneumonic plague. Microbiology 2009;155:3247e59.
[31] Brubaker RR. Factors promoting acute and chronic diseases caused by yersiniae. Clin Microbiol Rev 1991;4:309e24. [32] Putzker M, Sauer H, Sobe D. Plague and other human infections caused by Yersinia species. Clin Lab 2001;47:453e66. [33] Smego RA, Frean J, Koornhof HJ, Yersiniosis I. Microbiological and clinicoepidemiological aspects of plague and non-plague Yersinia infections. Eur J Clin Microbiol Infect Dis 1999;18: 1e15. [34] Anisimov AP, Amoako KK. Treatment of plague: promising alternatives to antibiotics. J Med Microbiol 2006;55:1461e75. [35] Balada-Llasat JM, Mecsas J. Yersinia has a tropism for B and T cell zones of lymph nodes that is independent of the type III secretion system. PLoS Pathog 2006;2:e86. [36] Simonet M, Richard S, Berche P. Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudotuberculosis harboring the pYV plasmid. Infect Immun 1990;58:841e5.
Please cite this article in press as: Zhou Y, et al., Bioluminescent tracing of a Yersinia pestis pCD1þ-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice, Microbes and Infection (2017), https://doi.org/10.1016/j.micinf.2017.11.005
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