CheV enhances the virulence of Salmonella Enteritidis, and the Chev-deleted Salmonella vaccine provides immunity in mice

Abstract

Background Salmonella enteritidis (SE) is a major zoonotic pathogen and causes infections in a variety of hosts. The development of novel vaccines for SE is necessary to eradicate this pathogen. Genetically engineered attenuated live vaccines are more immunogenic and safer. Thus, to develop a live attenuated Salmonella vaccine, we constructed a cheV gene deletion strain of SE (named ΔcheV) and investigated the role of cheV in the virulence of SE. First, the ability to resist environmental stress in vitro, biofilm formation capacity, drug resistance and motility of ΔcheV were analyzed. Secondly, the bacterial adhesion, invasion, intracellular survival assays were performed by cell model. Using a mouse infection model, an in vivo virulence assessment was conducted. To further evaluate the mechanisms implicated by the reduced virulence, qPCR analysis was utilized to examine the expression of the strain’s major virulence genes. Finally, the immune protection rate of ΔcheV was evaluated using a mouse model. Results Compared to C50336, the ΔcheV had significantly reduced survival ability under acidic, alkaline and thermal stress conditions, but there was no significant difference in survival under oxidative stress conditions. There was also no significant change in biofilm formation ability, drug resistance and motility. It was found that the adhesion ability of ΔcheV to Caco-2 cells remained unchanged, but the invasion ability and survival rate in RAW264.7 cells were significantly reduced. The challenge assay results showed that the LD50 values of C50336 and ΔcheV were 6.3 × 105 CFU and 1.25 × 107 CFU, respectively. After the deletion of the cheV gene, the expression levels of fimD, flgG, csgA, csgD, hflK, lrp, sipA, sipB, pipB, invH, mgtC, sodC, rfbH, xthA and mrr1 genes were significantly reduced. The live attenuated ΔcheV provided 100% protection in mice against SE infection. Conclusion All the results confirmed that the deletion of the cheV gene reduces the virulence of SE and provides significant immune protection in mice, indicating that ΔcheV could be potential candidates to be explored as live-attenuated vaccines.

The peptidoglycan-associated lipoprotein gene mutant elicits robust immunological defense in mice against Salmonella enteritidis

“…RNA from strain C50336 and Δ pal was extracted using a bacterial RNA extraction kit (Aidlab, China), with DNA removal followed by reverse transcription into cDNA. Primers can be found in the references ( Pande et al, 2016 ; Gupta et al, 2020 ; Zhang et al, 2024 ). The expression levels of biofilm-associated genes fimD , csgD , bcsA , ompR , and rpoS in Δ pal were detected using dye-based quantitative PCR (qPCR), Primer sequences and gene functions can be found in Table 3 .…”

“…According to the method described in the reference ( Zhang et al, 2024 ), one milliliter of logarithmic phase bacterial culture of C50336 and Δ pal was centrifuged, washed, and resuspended in an equal volume for tenfold serial dilutions, followed by colony counting using the spread plate method (recorded as A). For acid and alkaline stress, C50336 and Δ pal cultures were added to physiological saline at pH 3.5 and pH 10.0, respectively, and incubated at 37°C for 1 h. For oxidative stress, the cultures were added to physiological saline containing H 2 O 2 (10 mmol/L) and incubated at 37°C for 10 min.…”

“…RNA from strain C50336 and Δ pal was extracted using a bacterial RNA extraction kit (Aidlab, China), according to the manufacturer’s instructions, with subsequent DNA removal and reverse transcription into cDNA. Primers can be found in the references ( Upadhyaya et al, 2013 ; Gupta et al, 2020 ; Zhang et al, 2024 ). The expression levels of virulence genes including mgtC , invH , spvB , sipA , sipB , ssaV , csgA , and pipB in strain C50336 and Δ pal were quantified using quantitative PCR (qPCR).…”