Alteration of Salmonella enterica Virulence and Host Pathogenesis through Targeting sdiA by Using the CRISPR-Cas9 System

Abstract

<i>Salmonella enterica</i> 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 <i>S.</i><i>enterica</i>&nbsp;<i>sdiA</i> that encodes signal molecule receptor SdiA and responds to the quorum sensing (QS) signaling compounds N-acylhomoserine lactones (AHLs). For this purpose, <i>sdiA</i> was targeted in both <i>S.</i><i>enterica</i> wild type (WT) and the Δ<i>ssaV</i> mutant strain, where SsaV has been reported to be an essential component of SPI2-T3SS. The impact of <i>sdiA</i> mutation on <i>S.</i>&nbsp;<i>enterica</i> virulence was evaluated at both early invasion and later intracellular replication in both the presence and absence of AHL. Additionally, the influence of <i>sdiA</i> mutation on the pathogenesis <i>S.</i>&nbsp;<i>enterica</i> WT and mutants was investigated in vivo, using mice infection model. Finally, the minimum inhibitory concentrations (MICs) of various antibiotics against <i>S.</i>&nbsp;<i>enterica</i> strains were determined. Present findings show that mutation in <i>sdiA</i> significantly affects <i>S.</i><i>enterica</i> biofilm formation, cell adhesion and invasion. However, <i>sdiA</i> mutation did not affect bacterial intracellular survival. Moreover, in vivo bacterial pathogenesis was markedly lowered in <i>S.</i><i>enterica</i> Δ<i>sdiA</i> in comparison with the wild-type strain. Significantly, double-mutant <i>sdiA</i> and <i>ssaV</i> attenuated the <i>S. enterica</i> virulence and in vivo pathogenesis. Moreover, mutations in selected genes increased <i>Salmonella</i> 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 <i>S.</i><i>enterica</i> virulence. The present findings extend the understanding of virulence regulation and host pathogenesis of <i>Salmonella</i><i>enterica</i>.