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...

Descripción completa

Detalles Bibliográficos
Autores principales: Tahir, Hamza, Basit, Abdul, Tariq, Hafsa, Haider, Zulquernain, Ullah, Asim, Hayat, Zafar, Rehman, Shafiq Ur
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