Heterologous expression of quorum sensing transcriptional regulator LitR and its function in virulence-related gene regulation in foodborne pathogen Aeromonas hydrophila.

Background: Aeromonas hydrophila is an important foodborne and zoonotic pathogen causing serious diseases. Hence, revealing the pathogenic mechanism of A. hydrophila will be of importance in the development of novel therapies. Aeromonas hydrophila litR was reported to be regulated by two quorum sensing (QS) pathways, indicating that it is involved in QS network regulation correlated with bacterial virulence. However, the function of LitR is currently not understood. Therefore, we aimed to reveal the potential regulatory mechanisms of LitR on virulence-related genes.
Methods and Results: In this study, amino acid sequences analysis of LitR was conducted, providing bioinformatics evidence for its function as a potential transcriptional regulator. LitR protein was heterologous expressed, purified and its in-vitro multimeric forms were observed with gel filtration chromatography. The correlation between intracellular LitR expression level and cell density was analyzed with immunoblots. Regulation mechanisms of LitR on several important virulence-related factors were investigated with qRT-PCR, EMSA, DNase I footprinting and microscale thermophoresis binding assays, etc. Results showed that recombinant LitR protein aggregated mainly as dimer and hexamer in vitro. Intracellular expression level of LitR was positively correlated with cell density of A. hydrophila. Furthermore, LitR exhibited complicated regulation modes on virulence-related genes; it could directly bind to promoter regions of the hemolysin, serine protease and T6SS effector protein VgrG encoded genes. The promoter region of the hemolysin gene showed high binding affinity and mainly two binding sites for LitR. Different dissociation constants were obtained for LitR interaction with the hemolysin gene binding motifs I and II. Assays focusing on physiological characteristics of A. hydrophila prove that LitR positively regulated hemolytic and total extracellular protease activities.
Conclusions: This study investigated the function of LitR as a quorum sensing transcriptional regulator in regulation of virulence-related genes, which will help reveal the mechanisms of A. hydrophila pathogenicity. LitR could serve as a potential target for development of new antimicrobial agents from the perspective of QS regulation.
(© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)