High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection

Duraisamy Ponnusamy, Eric C. Fitts, Jian Sha, Tatiana E. Erova, Elena V. Kozlova, Michelle L. Kirtley, Bethany L. Tiner, Jourdan A. Andersson, Ashok Chopra

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Abstract

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 high-throughput, 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<inf>50</inf>) 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<inf>50</inf> 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<inf>50</inf>. 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<inf>50</inf> 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<inf>50</inf> 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 Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD<inf>50</inf>. The mice infected with the Δlpp ΔmsbB ΔrbsA triple mutant at 50 LD<inf>50</inf> were 90% protected upon subsequent challenge with 12 LD<inf>50</inf> 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).

Original languageEnglish (US)
Pages (from-to)2065-2081
Number of pages17
JournalInfection and Immunity
Volume83
Issue number5
DOIs
StatePublished - 2015

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Yersinia pestis
Lethal Dose 50
Virulence Factors
Plague
Infection
Genes
Plague Vaccine
Acyltransferases
Attenuated Vaccines
Sequence Deletion
Gene Deletion
Lipoproteins
Virulence
Lipopolysaccharides
Carrier Proteins
Proteins
Vaccines
Spleen
Clone Cells
Adenosine Triphosphate

ASJC Scopus subject areas

  • Immunology
  • Microbiology
  • Parasitology
  • Infectious Diseases

Cite this

High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection. / Ponnusamy, Duraisamy; Fitts, Eric C.; Sha, Jian; Erova, Tatiana E.; Kozlova, Elena V.; Kirtley, Michelle L.; Tiner, Bethany L.; Andersson, Jourdan A.; Chopra, Ashok.

In: Infection and Immunity, Vol. 83, No. 5, 2015, p. 2065-2081.

Research output: Contribution to journalArticle

Ponnusamy, Duraisamy ; Fitts, Eric C. ; Sha, Jian ; Erova, Tatiana E. ; Kozlova, Elena V. ; Kirtley, Michelle L. ; Tiner, Bethany L. ; Andersson, Jourdan A. ; Chopra, Ashok. / High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection. In: Infection and Immunity. 2015 ; Vol. 83, No. 5. pp. 2065-2081.
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