Construction and characterization of a Salmonella enterica serovar Typhi tolC deletion mutant

Abstract

IntroductionSalmonella enterica serovar Typhi (S.Typhi), the organism responsible for typhoid fever, is a human specific pathogen that is able to survive in the macrophage and form biofilm in the host. TolC belongs to a family of outer membrane proteins that are found in all Gramnegative pathogenic bacteria. Together with two other proteins, AcrA and AcrB, TolC forms a channel through the outer membrane. Besides its role in the efflux of various molecules from the cell, TolC has been hypothesized to also play a key role in bacterial colonisation, adhesion and invasion of macrophages. ObjectivesTo investigate the role of the outer membrane protein TolC of S.Typhi in macrophage infection and biofilm formation. Methods: The tolC deletion mutant (ΔTolC) strain of S.Typhi was constructed using a single-step gene knockout technique. S.Typhi cell invasion and biofilm formation ability of wild-type (WT) and ΔTolC strains were tested using an in vitro macrophage invasion assay and crystal violet biofilm assay, respectively. ResultsThe ΔTolC strain was compromised in their ability to infect macrophages and promote biofilm formation. The ΔTolC strain invaded THP-1 macrophage cells very poorly compared with the WT (counts for wild-type were mean 5.1×105cfu/ml [n=4] compared with mean 1.6×105cfu/ml [n=4] for ΔTolC; p<0.0001). Crystal violet biofilm assay revealed that WT strains were able to attach to polystyrene wells and form dense biofilm structures, whereas the ΔTolC strain were unable to do so (OD595 readings mean 3.43, SD ± 0.17 versus 0.30; S ± 0.08 p<0.0001). ConclusionsOur data demonstrated the role of the S. Typhi TolC protein in biofilm formation and invasion of human macrophage cells.