A strategy to verify the absence of the pgm locus in Yersinia pestis strain candidates for select agent exemption

Jenkins, Amy; Worsham, Patricia L.; Welkos, Susan L.

doi:10.1016/j.mimet.2009.02.013

Cited by 10 publications

(13 citation statements)

References 12 publications

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“…Susan Welkos and Christopher Cote, USAMRIID, [47], [48] ) and virulent Y. pestis CO92 were used in these studies. For all macrophage infection studies, Y. pestis strains from frozen stocks were streaked onto sheep blood agar (SBA) plates and incubated at 35°C for 24 hr, unless otherwise noted.…”

Section: Methodsmentioning

confidence: 99%

Integrating High-Content Imaging and Chemical Genetics to Probe Host Cellular Pathways Critical for Yersinia Pestis Infection

et al. 2013

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The molecular machinery that regulates the entry and survival of Yersinia pestis in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent Y. pestis CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), E. coli LPS (a TLR4 agonist) or Y. pestis infection, while the PI3K and PKC inhibitors were selective only for Y. pestis infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to Y. pestis infection, and that Y. pestis entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics.

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Section: Methodsmentioning

confidence: 99%

Integrating High-Content Imaging and Chemical Genetics to Probe Host Cellular Pathways Critical for Yersinia Pestis Infection

et al. 2013

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“…Both Y. pestis CO92 and the C12 derivative are fully virulent tier-one select agents that require Biological Safety Level-3 (BSL-3) laboratory conditions. The Y. pestis VAX strain is an exempt strain and can safely be worked with at BSL-2 laboratory conditions [54]. Before use as either ELISA capture antigens or splenocyte stimulants, bacterial cells were subjected to approximately 21 kGy of γ-radiation using JL Shepherd irradiator model 109-68.…”

Section: Bacteriamentioning

confidence: 99%

“…The deletion mutation of the pgm locus and curing of the pPst plasmid resulted in significant attenuation [52,53]. The pgm locus encodes a pathogenicity island responsible for iron scavenging and utilization [54,[62][63][64]. The pPst plasmid encodes plasminogen activator, a protein critical for lethal infection [53,65,66].…”

Section: Clinical Observationsmentioning

confidence: 99%

The Use of Analgesics during Vaccination with a Live Attenuated Yersinia pestis Vaccine Alters the Resulting Immune Response in Mice

et al. 2019

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The administration of antipyretic analgesics prior to, in conjunction with, or due to sequelae associated with vaccination is a common yet somewhat controversial practice. In the context of human vaccination, it is unclear if even short-term analgesic regimens can significantly alter the resulting immune response, as literature exists to support several scenarios including substantial immune interference. In this report, we used a live attenuated Yersinia pestis vaccine to examine the impact of analgesic administration on the immune response elicited by a single dose of a live bacterial vaccine in mice. Mice were assessed by evaluating natural and provoked behavior, as well as food and water consumption. The resulting immune responses were assessed by determining antibody titers against multiple antigens and assaying cellular responses in stimulated splenocytes collected from vaccinated animals. We observed no substantial benefit to the mice associated with the analgesic administration. Splenocytes from both C57BL/6 and BALB/c vaccinated mice receiving acetaminophen have a significantly reduced interferon-gamma (IFN-γ) recall response. Additionally, there is a significantly lower immunoglobulin (Ig)G2a/IgG1 ratio in vaccinated BALB/c mice treated with either acetaminophen or meloxicam and a significantly lower IgG2c/IgG1 ratio in vaccinated C57BL/6 mice treated with acetaminophen. Taken together, our data indicate that the use of analgesics, while possibly ethically warranted, may hinder the accurate characterization and evaluation of novel vaccine strategies with little to no appreciable benefits to the vaccinated mice.

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“…Inclusivity was evaluated using genomic DNA extracted from 16 different Y. pestis strains containing the full pgm locus (pgmþ) to include a pgm þ KIM5 strain derived from pgm þ KIM6 [41]. Pgm status of assayed Y. pestis isolates was originally determined via multiple means including: observation of colony pigmentation on Congo red agar, Southern blot analysis, nucleotide sequencing, and/or detection of multiple pgm targets using conventional PCR assays previously established by Jenkins et al [39]. Given the non-pigmented phenotype is not exclusive to en bloc deletion of the entire 102-kb pgm locus nor absence of the entire 68-kb segment, observation of non-pigmented colonies on Congo red agar was not used exclusively to classify any Y. pestis isolate as pgm-prior to these experiments [16].…”

Section: Inclusivity For Real-time Pcr Assaysmentioning

confidence: 99%

“…In accordance with CDC rules and regulations that dictate the entirety of the pgm locus be absent for exemption, we previously adopted and used a set of conventional PCR assays developed by Jenkins et al as an integral part of our strategy to determine the pgm status of Y. pestis isolates in our laboratory [39]. However, because of the issues inherent to the use of conventional PCR (i.e.…”

Section: Introductionmentioning

confidence: 99%

Development of real-time PCR assays for specific detection of hmsH, hmsF, hmsR, and irp2 located within the 102-kb pgm locus of Yersinia pestis

Gaddy

Cuevas

Hartman

et al. 2014

Molecular and Cellular Probes

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A strategy to verify the absence of the pgm locus in Yersinia pestis strain candidates for select agent exemption

Cited by 10 publications

References 12 publications

Integrating High-Content Imaging and Chemical Genetics to Probe Host Cellular Pathways Critical for Yersinia Pestis Infection

Integrating High-Content Imaging and Chemical Genetics to Probe Host Cellular Pathways Critical for Yersinia Pestis Infection

The Use of Analgesics during Vaccination with a Live Attenuated Yersinia pestis Vaccine Alters the Resulting Immune Response in Mice

Development of real-time PCR assays for specific detection of hmsH, hmsF, hmsR, and irp2 located within the 102-kb pgm locus of Yersinia pestis

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