Biothreats-Bacterial Warfare Agents

R, Arun Kumar; Nishanth, T

doi:10.4172/2157-2526.1000112

Cited by 2 publications

(2 citation statements)

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“…Overnight cultures of various Y. pestis strains were washed in PBS and normalized to the same absorbance by measuring the optical density at 600 nm (OD 600 ). These bacterial cultures were then inoculated separately (at approximately 1 ϫ 10 7 CFU) into 20 ml of modified M9 medium (1ϫ M9 salts [22 mM KH 2 PO 4 , 33.7 mM Na 2 HPO 4 , 8.55 mM NaCl, 9.35 mM NH 4 Cl], 1 mM MgSO 4 , 2.5 mM CaCl 2 , 0.001 mg/ml FeSO 4 , 0.0001% thiamine, 0.1% Casamino Acids) (all chemicals were obtained from Sigma-Aldrich, St. Louis, MO) contained in 125-ml polycarbonate Erlenmeyer flasks with HEPA-filtered tops. The medium either did not contain any sugar or was supplemented with 0.4% glucose or 0.4% ribose.…”

Section: Construction Of In-frame Deletion Mutants and Testing In Moumentioning

confidence: 99%

“…Although the disease is endemic in certain regions of the globe (2), the potential use of this organism as a biological warfare agent is a significant worldwide concern. In particular, aerosolized droplets charged with Y. pestis can lead to primary pneumonic plague and subsequent person-toperson spread, with a narrow window for antibiotic intervention (3)(4)(5). Consequently, an ideal strategy to combat the disease is to have a vaccine offering long-lasting immunity.…”

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confidence: 99%

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High-Throughput, Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection

et al. 2015

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The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a highthroughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD 50 ) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD 50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD 50 . Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD 50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD 50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the ⌬lpp single or the ⌬lpp ⌬msbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD 50 . The mice infected with the ⌬lpp ⌬msbB ⌬rbsA triple mutant at 50 LD 50 were 90% protected upon subsequent challenge with 12 LD 50 of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s). Yersinia pestis is a tier 1 select agent that leads to three pathodynamic manifestations in humans, namely, bubonic, septicemic, and pneumonic plague (1). Although the disease is endemic in certain regions of the globe (2), the potential use of this organism as a biological warfare agent is a significant worldwide concern. In particular, aerosolized droplets charged with Y. pestis can lead to primary pneumonic plague and subsequent person-toperson spread, with a narrow window for antibiotic intervention (3-5). Consequently, an ideal strategy to combat the disease is to have a vaccine offering long-lasting immunity.Until 1999, a heat-killed plague vaccine composed of Y. pestis 195/P strain was availab...

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Section: Construction Of In-frame Deletion Mutants and Testing In Moumentioning

confidence: 99%

mentioning

confidence: 99%