Cargando…
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
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 Path...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9776377/ https://www.ncbi.nlm.nih.gov/pubmed/36551784 http://dx.doi.org/10.3390/biomedicines10123028 |
_version_ | 1784855850589880320 |
---|---|
author | Tahir, Hamza Basit, Abdul Tariq, Hafsa Haider, Zulquernain Ullah, Asim Hayat, Zafar Rehman, Shafiq Ur |
author_facet | Tahir, Hamza Basit, Abdul Tariq, Hafsa Haider, Zulquernain Ullah, Asim Hayat, Zafar Rehman, Shafiq Ur |
author_sort | Tahir, Hamza |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9776377 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-97763772022-12-23 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 Tahir, Hamza Basit, Abdul Tariq, Hafsa Haider, Zulquernain Ullah, Asim Hayat, Zafar Rehman, Shafiq Ur Biomedicines Article 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. MDPI 2022-11-24 /pmc/articles/PMC9776377/ /pubmed/36551784 http://dx.doi.org/10.3390/biomedicines10123028 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Tahir, Hamza Basit, Abdul Tariq, Hafsa Haider, Zulquernain Ullah, Asim Hayat, Zafar Rehman, Shafiq Ur 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 |
title | 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 |
title_full | 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 |
title_fullStr | 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 |
title_full_unstemmed | 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 |
title_short | 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 |
title_sort | 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 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9776377/ https://www.ncbi.nlm.nih.gov/pubmed/36551784 http://dx.doi.org/10.3390/biomedicines10123028 |
work_keys_str_mv | AT tahirhamza couplingcrisprcas9andlambdaredrecombineeringsystemforgenomeeditingofsalmonellagallinarumandtheeffectofssauknockoutmutantonthevirulenceofbacteria AT basitabdul couplingcrisprcas9andlambdaredrecombineeringsystemforgenomeeditingofsalmonellagallinarumandtheeffectofssauknockoutmutantonthevirulenceofbacteria AT tariqhafsa couplingcrisprcas9andlambdaredrecombineeringsystemforgenomeeditingofsalmonellagallinarumandtheeffectofssauknockoutmutantonthevirulenceofbacteria AT haiderzulquernain couplingcrisprcas9andlambdaredrecombineeringsystemforgenomeeditingofsalmonellagallinarumandtheeffectofssauknockoutmutantonthevirulenceofbacteria AT ullahasim couplingcrisprcas9andlambdaredrecombineeringsystemforgenomeeditingofsalmonellagallinarumandtheeffectofssauknockoutmutantonthevirulenceofbacteria AT hayatzafar couplingcrisprcas9andlambdaredrecombineeringsystemforgenomeeditingofsalmonellagallinarumandtheeffectofssauknockoutmutantonthevirulenceofbacteria AT rehmanshafiqur couplingcrisprcas9andlambdaredrecombineeringsystemforgenomeeditingofsalmonellagallinarumandtheeffectofssauknockoutmutantonthevirulenceofbacteria |