The regulatory network of Salmonella biofilm formation: the role of CsgD on autoinducer-2 activity and expression of quorum-sensing-related genes in Salmonella Typhimurium

Abstract

Salmonella biofilms are mainly formed by cellulose and curli fimbriae under the control of CsgD. Biofilm regulation also involves small RNAs and LuxS-mediated AI-2 quorum sensing. This study aimed to investigate the relationship between CsgD and quorum-sensing regulation in Salmonella Typhimurium. By modulating csgD expression, the impact on the expression levels of MicA, CsrB, CsrC, and RyeE sRNAs—sRNAs implicated in QS-linked regulatory pathways—was assessed. Quantitative real-time PCR (qRT-PCR) analyses demonstrated that the expression of these sRNAs was increased following perturbation of CsgD levels. Furthermore, the influence of CsgD levels on AI-2 activity was evaluated, revealing that strains overexpressing csgD displayed a 277% increase in relative AI-2 activity compared with the wild-type strain. Additionally, the effects of csgD on the expression of lsrA, lsrK, and lsrR genes, which are part of the Lsr transport system for AI-2 uptake, were investigated. In the strain with elevated AI-2 activity, a marked increase in lsrK expression was observed. Conversely, loss of csgD resulted in the upregulation of lsrA, lsrK, and lsrR, while AI-2 activity remained largely unchanged. This pattern suggests that the inhibitory effect of LsrR on QS signaling may be counterbalanced by increased expression of lsrA and lsrK. Collectively, our findings indicate that changes in CsgD levels are associated with shifts in QS-linked sRNA expression and AI-2 signaling dynamics. In addition, deletion of csgD resulted in reduced biofilm-associated phenotypes, including curli- and cellulose-related traits and biofilm biomass, whereas partial restoration of these phenotypes was observed in the complemented strain. Results suggest that CsgD may act as an integrative regulatory node linking transcriptional control of biofilm formation with quorum-sensing signaling pathways in Salmonella. Proposed model of CsgD-associated regulatory interactions linking quorum sensing and biofilm-related pathways in Salmonella Typhimurium. Red dashed arrows indicate experimentally supported findings obtained in the present study, whereas black interactions represent previously reported regulatory relationships derived from the literature; specifically, the CsgD-to-biofilm (curli/cellulose) regulatory links shown in black are established from prior reports and were not re-tested here, whereas the CsgD–sRNA–AI-2/Lsr connections shown as red dashed arrows are those investigated in this work. Altered csgD expression modulates quorum-sensing–associated sRNAs (MicA, CsrB/C, and RyeE) and influences AI-2 dynamics through gene-specific effects on the Lsr transport system.