Coupling CRISPR/Cas9 and lambda red recombineering system for genome editing of Salmonella Gallinarum and the effect of ssaU knock-out mutant on the virulence of bacteria

Hamza Tahir, Abdul Basit, Hafsa Tariq, Zulquernain Haider, Asim Ullah, Zafar Hayat, Shafiq Ur Rehman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

The poultry industry in developing countries still faces a significant threat from fowl typhoid, a disease caused by Salmonella Gallinarum that has been well contained in more economically developed countries. In addition to the virulence exhibited by large virulence plasmid (85 kb), Salmonella Pathogenicity Island 2 in S. Gallinarum plays a key role in mediating disease through its type III secretion systems (TTSS). The TTSS secrete effector protein across the Salmonella containing vacuoles and mediate the internalization of bacteria by modulating vesicular passage. In this study, candidate virulent ssaU gene (~1 kb) encoding type III secretion system was successfully deleted from indigenously isolated S. Gallinarum genome through homology-directed repair using CRISPR/Cas9 and lambda recombination systems. CRISPR/Cas9-based genome editing of poultry-derived Salmonella Gallinarum has not been previously reported, which might be linked to a lack of efficiency in its genetic tools. This is the first study which demonstrates a complete CRISPR/Cas9-based gene deletion from this bacterial genome. More importantly, a poultry experimental model was employed to assess the virulence potential of this mutant strain (ΔssaU_SG18) which was unable to produce any mortality in the experimentally challenged birds as compared to the wild type strain. No effect on weight gain was observed whereas bacteria were unable to colonize the intestine and liver in our challenge model. This in vivo loss of virulence in mutant strain provides an excellent functionality of this system to be useful in live vaccine development against this resistant and patho genic bacteria.
Original languageEnglish
Article number3028
Number of pages13
JournalBiomedicines
Volume10
Issue number12
DOIs
Publication statusPublished - 24 Nov 2022

Keywords

  • CRISPR/Cas9
  • Lambda recombineering
  • Genome editing
  • Type three secretion system
  • Virulent genes
  • Salmonella Gallinarum
  • Poultry experimental model

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