Salmonella enterica is a common cause of many enteric infections worldwide and is successfully engineered to deliver heterologous antigens to be used as vaccines. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 endonuclease is a promising genome editing tool. In the current study, a CRISPR-Cas9 system was used to target S.enterica sdiA that encodes signal molecule receptor SdiA and responds to the quorum sensing (QS) signaling compounds N-acylhomoserine lactones (AHLs). For this purpose, sdiA was targeted in both S.enterica wild type (WT) and the ΔssaV mutant strain, where SsaV has been reported to be an essential component of SPI2-T3SS. The impact of sdiA mutation on S. enterica virulence was evaluated at both early invasion and later intracellular replication in both the presence and absence of AHL. Additionally, the influence of sdiA mutation on the pathogenesis S. enterica WT and mutants was investigated in vivo, using mice infection model. Finally, the minimum inhibitory concentrations (MICs) of various antibiotics against S. enterica strains were determined. Present findings show that mutation in sdiA significantly affects S.enterica biofilm formation, cell adhesion and invasion. However, sdiA mutation did not affect bacterial intracellular survival. Moreover, in vivo bacterial pathogenesis was markedly lowered in S.enterica ΔsdiA in comparison with the wild-type strain. Significantly, double-mutant sdiA and ssaV attenuated the S. enterica virulence and in vivo pathogenesis. Moreover, mutations in selected genes increased Salmonella susceptibility to tested antibiotics, as revealed by determining the MICs and MBICs of these antibiotics. Altogether, current results clearly highlight the importance of the CRISPR-Cas9 system as a bacterial genome editing tool and the valuable role of SdiA in S.enterica virulence. The present findings extend the understanding of virulence regulation and host pathogenesis of Salmonellaenterica.

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