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Epithelial Cell Infection Analyses with Shigella
Published on: February 9, 2024
Analysis of virulence and inflammatory potential of Shigella flexneri purine biosynthesis mutants
Antonella Cersini1, Maria Celeste Martino, Irene Martini
1Dipartimento di Biologia Cellulare e dello Sviluppo, Sezione di Scienze Microbiologiche, Università La Sapienza, 00185 Rome, Italy.
Abstract:
Several Shigella flexneri mutants with defects in aromatic amino acid and/or purine biosynthesis have been evaluated as vaccines in humans or in animal models. To be suitable as a vaccine, a mutant has to show virulence attenuation, minimal reactogenicity, and a good immunogenic potential in animal models. With this aim, we have constructed five S. flexneri 5 (wild-type strain M90T) mutants with inactivation of one or two of the loci purEK, purHD, and guaBA, governing early or late steps of purine biosynthesis. The mutants have been analyzed in vitro in cell cultures and in vivo in the Sereny test and in the murine pulmonary model of shigellosis. M90T guaBA, M90T guaBA purEK, M90T guaBA purHD, and M90T purHD purEK gave a negative result in the Sereny test. In contrast, in the murine pulmonary model all of the strains had the same 50% lethal dose as the wild type, except M90T guaBA purHD, which did not result in death of the animals. Nevertheless, bacterial counts in infected lungs, immunohistochemistry, and reverse transcription-PCR analysis of mRNAs for tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-1beta (IL-1beta), IL-6, IL-12, and inducible nitric oxide synthase (iNOS) revealed significant differences among the strains. At 72 h postinfection, M90T guaBA purHD still induced proinflammatory cytokines and factors such as IL-1beta, IL-6, TNF-alpha, and iNOS, along with cytokines such as IL-12 and IFN-gamma. Moreover, in the absence of evident lesions in murine tissues, this mutant highly stimulated major histocompatibility complex class II expression, showing a significant ability to activate the innate immunity of the host.
Insights
Shigella flexneri mutants targeting purine biosynthesis were tested as vaccines. The M90T guaBA purHD mutant showed promise, inducing innate immunity without causing lethal disease in mice.
Area of Science:
- Microbiology
- Vaccine Development
- Immunology
Background:
- Shigella flexneri is a pathogen requiring effective vaccine candidates.
- Mutants with defects in amino acid or purine biosynthesis are explored for vaccine potential.
- Vaccine suitability requires attenuation, low reactogenicity, and immunogenicity.
Purpose of the Study:
- To construct and evaluate S. flexneri 5 purine biosynthesis mutants as potential vaccines.
- To assess virulence, reactogenicity, and immunogenicity in vitro and in vivo.
- To identify a mutant with optimal vaccine characteristics.
Main Methods:
- Construction of five S. flexneri 5 mutants with targeted purine biosynthesis gene disruptions (purEK, purHD, guaBA).
- In vitro analysis in cell cultures.
- In vivo evaluation using the Sereny test and a murine pulmonary model of shigellosis.
- Analysis of cytokine and iNOS expression via RT-PCR and MHC class II expression.
Main Results:
- Four mutants (M90T guaBA, M90T guaBA purEK, M90T guaBA purHD, M90T purHD purEK) were Sereny test negative.
- M90T guaBA purHD mutant did not cause mortality in the murine pulmonary model, unlike other strains.
- Despite lack of lethality, M90T guaBA purHD induced proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha), iNOS, and adaptive immune markers (IL-12, IFN-gamma).
- This mutant significantly upregulated MHC class II expression, indicating innate immune activation without apparent tissue damage.
Conclusions:
- The M90T guaBA purHD S. flexneri mutant demonstrates significant promise as a live attenuated vaccine candidate.
- This mutant elicits a robust innate immune response, including cytokine production and MHC class II expression.
- Further studies are warranted to confirm its immunogenic potential and safety for human use.
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