Salmonella

Genome Editing Service for Salmonella

1. Research Background

Salmonella is a common intestinal pathogen with significant clinical and food safety implications. Research into its multidrug resistance (MDR) and complex pathogenic mechanisms (e.g., pathogenicity islands, SPIs) represents a core challenge in the fields of microbiology and public health. Precise and efficient gene-editing technology provides a crucial tool for in-depth investigation of its functional genomics and for developing novel attenuated vaccines and control strategies.

In this context, GeneRulor offers microbial genome modification services using the CRISPR/Cas9 method. This enables customized gene knockout, large-fragment deletion, point mutation and gene knock-in (overexpression) in different Salmonellaserovars (such as S. Typhimurium, S. Enteritidis, etc.), with delivery of positive mutant strains. We are committed to providing professional and reliable customized Salmonellagenome editing solutions for global research and industrial clients.

2. Strain Characteristics and Biological Background

(1) Gram-staining property: Salmonella is a Gram-negative bacterium. Unlike Gram-positive bacteria, it has a relatively thin peptidoglycan layer but possesses a complex outer membrane structure and a lipopolysaccharide (LPS) layer.

(2) Clinical significance: It is a common pathogen in humans and animals, capable of causing food poisoning, enteritis, and severe diseases like typhoid fever. The emergence of multidrug-resistant strains has made the study of its pathogenic mechanisms a central challenge.

(3) Metabolism and applications: It exhibits strong environmental adaptability and serves as an ideal model for studying bacterial pathogenicity islands, biofilm formation, and host-pathogen interactions (e.g., epithelial cell invasion).

Figure 1. Microscopic Observation of Salmonella

3. Gene-Editing Strategies Reported in the Literature

To address the characteristics of the Salmonellaouter membrane barrier and its endogenous recombination systems, mainstream editing strategies in current literature and research practice include:

3.1 CRISPR/Cas9 System (Primary Solution):

(1) Principle: Utilizes the Cas9 protein (containing RuvC and HNH nuclease domains) to perform site-specific cleavage of target genomic DNA under the guidance of an sgRNA.

(2) Advantage: Induces repair via double-strand breaks (DSBs), enabling highly efficient gene knockout, large-fragment deletion, or gene knock-in (overexpression).

3.2 Homologous Recombination:

Often employed using suicide plasmids or shuttle plasmids, achieving precise base substitution or seamless editing through upstream and downstream homologous arms.

3.3 Site-Specific Recombination:

Utilizes recombinase systems (e.g., Flp/FRT) for gene insertion or removal of resistance markers at specific sites.

3.4 Transformation Efficiency Optimization:

It is typically necessary to optimize electroporation conditions to overcome the transformation barrier posed by the outer membrane, especially for clinical multidrug-resistant isolates.

4. Core Application Areas

(1) Functional Genomics: Precise deletion of target genes (e.g., virulence factors) to study their functions during infection.

(2) Drug Resistance Mechanism Analysis: Introduction of point mutations to mimic natural variants, analyzing the evolutionary patterns of antibiotic resistance.

(3) Synthetic Biology Engineering: Insertion of reporter genes (e.g., fluorescent proteins) into the genome for real-time monitoring of strain dynamics within the host.

(4) Attenuated Vaccine Construction: Sequential editing of multiple genes (e.g., knockout of key metabolic genes like aroA) to develop highly safe vaccine strains.

5. Project Workflow and Validation

We provide a one-stop service from design to delivery, ensuring the accuracy of editing outcomes:

(1) Design and Vector Construction: Designing knockout vectors for the target sites.

(2) Bacterial Transformation and Screening: Overcoming transformation challenges via optimized electroporation protocols.

(3) Comprehensive Validation: Ensuring positive mutations via PCR identification and Sanger sequencing.

Figure 2. Schematic Diagram of Project Workflow

6. Genome Editing Project Introduction

6.1 Core services include:

(1) Gene Knockout/Inactivation: Precise deletion or disruption of target genes for studying pathogenicity island function or constructing attenuated strains.

(2) Gene Knock-in/Overexpression: Insertion of reporter genes or exogenous genes at specific sites to construct fluorescent tracer strains.

(3) Point Mutation/Modification: Introduction of specific base mutations to study the relationship between key protein sites and resistance phenotypes.

(4) Multi-gene Editing: Achieving sequential or simultaneous editing of multiple virulence-related genes to construct complex attenuated models.

6.2 Technical Advantages:

(1) High Success Rate: Optimized protocols for transforming different serovars, including clinical drug-resistant strains.

(2) Customized Design: Designing the optimal strategy based on your research objectives (e.g., studying SPI-1 expression, antibiotic resistance).

(3) End-to-End Validation: Providing a complete closed-loop service from design to final genotype verification.

7. Case Study

Case: Targeted Modification of the Salmonella sdiAVirulence Gene Using the CRISPR-Cas9 System

Project Description: Investigated the impact of targeting the Salmonella sdiAgene (encoding a quorum-sensing receptor) via CRISPR-Cas9 technology on its virulence, host interaction, and antibiotic susceptibility.

(1) Research Background

Salmonella enterica is a common intestinal pathogen that can cause gastroenteritis, typhoid fever, and other diseases. Its virulence relies on Type III Secretion Systems (T3SS) encoded by pathogenicity islands (e.g., SPI1 and SPI2). SPI1-T3SS mediates bacterial invasion of host cells, while SPI2-T3SS promotes intracellular survival. SsaV is a key component of SPI2-T3SS, and its mutation can attenuate virulence. Salmonelladetects AHL signal molecules produced by other bacteria via the SdiA receptor, regulating virulence behaviors such as biofilm formation and adhesion, but does not synthesize AHL itself. Traditional gene editing methods (e.g., λ-Red recombination) are inefficient and time-consuming. The CRISPR-Cas9 technology provides an efficient and precise genomic editing tool for studying virulence gene function and developing attenuated vaccine vectors.

(2) Experimental Design

CRISPR-Cas9 System Construction: Designed two guide sequences (Oligo I/II and Oligo III/IV) targeting different regions of the sdiAgene. Utilized a two-plasmid system (pCas9 and pCRISPR) expressing the Cas9 protein and sdiA-targeting crRNA, respectively, introduced into wild-type (WT) Salmonellaand the ΔssaVmutant via electroporation. Positive clones were screened via colony PCR, and sdiAdeletion was confirmed by sequencing.

Experimental System: Bacterial adhesion ability was assessed using HeLa cells; biofilm formation was quantified in polystyrene plates; bacterial invasion and intracellular replication were analyzed via gentamicin protection assays; an SseJ-hSurvivin-HA reporter system was constructed to evaluate SPI2-T3SS function; and mouse infection experiments were conducted to assess mutant strain virulence.

(3) Experimental Conclusions

sdiAMutation Attenuates Virulence Phenotypes: AHL signals significantly enhanced adhesion and biofilm formation in WT and ΔssaVstrains, but adhesion capacity decreased by approximately 50-70% (p<0.001) in sdiAmutants (ΔsdiAand ΔssaVΔsdiA). The sdiAmutation did not affect bacterial invasion of HeLa cells, but intracellular replication was reduced in ΔssaVand the double mutant, indicating impaired SPI2 function. Both sdiAand ssaVsingle mutations reduced SPI2 effector translocation efficiency, with a more pronounced additive effect in the double mutant.

Attenuated Virulence In Vivo: In the mouse model, the lethality of ΔsdiAand the double mutant (ΔssaVΔsdiA) was significantly lower than that of WT, with the double mutant providing complete protection (p<0.001).

Enhanced Antibiotic Susceptibility: MIC and MBIC values for several antibiotics (e.g., ampicillin, ciprofloxacin) were lower in sdiAand ssaVmutants, indicating that attenuated virulence is associated with reduced drug resistance.

(4) Application Significance

Vaccine Development Prospects: The ΔssaVΔsdiAdouble mutant shows significantly attenuated virulence but may retain immunogenicity, holding promise as a safe vaccine vector for delivering heterologous antigens.

Anti-infection Strategy: Targeting the SdiA-mediated quorum-sensing pathway can inhibit biofilm formation, providing a new target for anti-virulence therapy (e.g., designing AHL analog inhibitors).

Technology Promotion Value: This study validates the efficient editing capability of CRISPR-Cas9 in Gram-negative bacteria, providing a template for virulence research in other pathogens.

Clinical Implications: Enhanced antibiotic susceptibility in the mutant strains suggests that combining gene editing with antibiotic therapy may improve the prognosis of infections caused by drug-resistant bacteria.

8. Reference

[1] Jajere, S. M. (2019). A review of Salmonella enterica with particular focus on the pathogenicity and virulence factors. Veterinary World.

[2] Datsenko, K. A., & Wanner, B. L. (2000). One step inactivation of chromosomal genes in Escherichia coli K12 and Salmonella using PCR products. PNAS.

[3] Jiang, W., et al. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology.

[4] Cui, L., & Bikard, D. (2016). Consequences of Cas9 cleavage in the chromosome of Escherichia coli and Salmonella. Nucleic Acids Research.

[5] Askoura M, et al. (2021). Alteration of Salmonella enterica Virulence and Host Pathogenesis through Targeting sdiA by Using the CRISPR-Cas9 System. Microorganisms.

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