Alteration in apyrase enzyme attenuated virulence of Shigella flexneri

Area of Science:

• Microbiology and Immunology
• Molecular Biology
• Vaccine Development

Background:

• Shigella virulence is linked to genes on its chromosome and megaplasmid.
• The apy gene, located on the megaplasmid, encodes apyrase, an enzyme contributing to pathogenesis by damaging host cells.
• Apyrase's role in mitochondrial damage and host cell death highlights its significance in Shigella pathogenesis.

Purpose of the Study:

• To construct and characterize an apyrase (apy) mutant of Shigella flexneri.
• To evaluate the protective efficacy of the constructed apy mutant strain in a relevant animal model.
• To explore the potential of the apy mutant as a vaccine target for shigellosis.

Main Methods:

• Construction of the apy mutant using insertional activation with a kanamycin resistance gene cassette (aphA).
• PCR amplification, cloning, and mutation of the wild-type apy gene.
• Introduction of the mutated construct into Shigella flexneri 2a via conjugation and homologous recombination, followed by sucrose selection for gene deletion.
• Confirmation of mutation via DNA sequencing and assessment of apyrase enzyme activity using a colorimetric test.
• Evaluation of protective efficacy in the guinea pig keratoconjunctivitis model.

Main Results:

• Successful construction of a Shigella flexneri apy mutant with non-functional apyrase enzyme activity.
• The apy mutant strain was Sereny negative, indicating reduced keratoconjunctivitis-inducing potential.
• The apy mutant elicited significant protection against challenge with wild-type Shigella flexneri in the guinea pig model.

Conclusions:

• The developed apy mutant strain of Shigella flexneri is attenuated and immunogenic.
• This apy mutant demonstrates significant protective efficacy, suggesting its potential as a live attenuated vaccine candidate.
• The study provides a foundation for developing novel vaccine strategies against shigellosis.