Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining
Bethany L Tiner1, Jian Sha2, Michelle L Kirtley1
1Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
Abstract:
Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection.
Insights
Deleting the ail gene from Yersinia pestis CO92, alongside Lpp and MsbB deletions, severely weakened the bacteria. This triple mutant protected mice against pneumonic plague and subsequent challenge, offering a potential vaccine candidate.
Area of Science:
- Microbiology and Immunology
- Infectious Diseases
- Vaccine Development
Background:
- Yersinia pestis causes plague; previous studies attenuated it by deleting Braun lipoprotein (Lpp) and MsbB genes.
- Lpp activates Toll-like receptor 2, while MsbB modifies lipopolysaccharide, both key bacterial components.
Purpose of the Study:
- To investigate the effect of deleting the attachment-invasion locus (ail) gene from Yersinia pestis CO92, individually and in combination with Lpp and MsbB deletions.
- To evaluate the resulting mutant's attenuation, immunogenicity, and protective potential against pneumonic plague in a mouse model.
Main Methods:
- Genetic manipulation of Yersinia pestis CO92 to create single, double, and triple mutants (Δail, Δlpp, ΔmsbB, Δlpp Δail, Δlpp ΔmsbB Δail).
- Assessment of bacterial attenuation using lethal dose (LD50) determination in a mouse pneumonic plague model.
- Evaluation of immune responses (antibody isotyping, cytokine production) and bacterial clearance via in vivo imaging and organ burden analysis.
- In vitro assays examining bacterial adherence, invasion, and intracellular survival in epithelial and macrophage cell lines.
- Assessment of protection against subsequent challenge with wild-type Yersinia pestis CO92 in surviving mice.
Main Results:
- The Δlpp ΔmsbB Δail triple mutant was severely attenuated, with an LD50 equivalent to 6,800 LD50s of wild-type CO92.
- Triple mutant-infected mice showed balanced TH1/TH2 immune responses, rapid bacterial clearance, reduced cytokine production, and fewer lesions.
- Surviving mice from triple mutant infection were protected against a subsequent challenge with wild-type Yersinia pestis CO92.
- The triple mutant exhibited decreased adherence, invasion, and intracellular survival in relevant cell lines, and sensitivity to human serum bactericidal activity.
Conclusions:
- Deletion of the ail gene from the Δlpp ΔmsbB double mutant significantly attenuates Yersinia pestis CO92, rendering it avirulent in a pneumonic plague model.
- The severely attenuated triple mutant elicits protective immunity against subsequent wild-type Yersinia pestis CO92 challenge, indicating its potential as a live vaccine candidate.
- The combined genetic modifications enhance bacterial clearance through reduced virulence and improved host immune responses.
More Related Videos
06:30A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis
Published on: January 27, 2021
11:32Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria
Published on: February 23, 2014
