TolC facilitates the intracellular survival and immunomodulation of Salmonella Typhi in human host cells

Abstract

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a systemic infection that affects millions of people worldwide. S. Typhi can invade and survive within host cells, such as intestinal epithelial cells and macrophages, by modulating their immune responses. However, the immunomodulatory capability of S. Typhi in relation to TolC-facilitated efflux pump function remains unclear. The role of TolC, an outer membrane protein that facilitates efflux pump function, in the invasion and immunomodulation of S. Typhi, was studied in human intestinal epithelial cells and macrophages. The tolC deletion mutant of S. Typhi was compared with the wild-type and its complemented strain in terms of their ability to invade epithelial cells, survive and induce cytotoxicity in macrophages, and elicit proinflammatory cytokine production in macrophages. The tolC mutant, which has a defective outer membrane, was impaired in invading epithelial cells compared to the wild-type strain, but the intracellular presence of the tolC mutant exhibited greater cytotoxicity and induced higher levels of proinflammatory cytokines (IL-1β and IL-8) in macrophages compared to the wild-type strain. These effects were reversed by complementing the tolC mutant with a functional tolC gene. Our results suggest that TolC plays a role in S. Typhi to efficiently invade epithelial cells and suppress host immune responses during infection. TolC may be a potential target for the development of novel therapeutics against typhoid fever.

Keywords: Salmonella Typhi; immunomodulation of macrophages and epithelial cells; immunomodulatory ability of pathogens; invasion of bacteria into host cells; multidrug efflux pump AcrAB-TolC; mutant induce cytotoxic proinflammatory responses in host cell.

Graphical abstract

Figure 1.

The ST-ΔtolC mutant is impaired in invasion and induces proinflammatory chemokines in human macrophages.

Figure 2.

The ST-ΔtolC mutant induces cytotoxicity and pyroptosis in human THP-1 macrophages.

Figure 3.

S. Typhi ST-ΔtolC was hypercytotoxic to THP-1-derived human macrophages.

Figure 4.

S. Typhi ST-ΔtolC was not hypercytotoxic to human gut epithelial HT-29 cells.In the TEM Analysis of human gut epithelial HT-29 cells: TEM images were acquired 2 hours post-infection. (a) Control HT-29: An uninfected HT-29 epithelial cell with a prominent nucleus (Nu) and intact cellular structure throughout the 2-hour experiment, the epithelial cell remained healthy with an intact cell membrane. The cell displays a distinct nucleus (Nu), as indicated by the white arrow. The integrity of the epithelial cell membrane is highlighted by the black arrow. (b) HT-29 cell infected with ST-WT: HT-29 cells presented clear nuclear and minimal plasma membrane damage after 2 hours post-infection. The replication of ST-WT bacilli was evident from the visible bacterial septa, as indicated by the red arrows. The dense nucleus (Nu) is highlighted by the white arrow, while the black arrow points to the intact membrane of the HT-29 cells. (c) HT-29 cell infected with ST-ΔtolC: HT-29 cell showing relatively preserved cellular architecture with absence of any alterations. White arrows indicate the nucleus and black arrows indicate the intact membrane of epithelial cells. Significantly, the lack of ST-ΔtolC bacilli inside the HT-29 cells was apparent due to the complete absence of bacterial septa. This demonstrates that ST-ΔtolC was unsuccessful in invading the HT-29 cells. (d) HT-29 cell infected with ST-ΔtolC+: The HT-29 cell shown no significant cellular damage after infection. The cell membrane remained intact, showing no visible damage. The large nucleus (Nu) is easily identifiable, as pointed out by the white arrow. The black arrow indicates the undamaged membrane of the macrophages, while the presence of bacterial septa is marked by red arrows.

Figure 5.

The deletion of tolC decreases the integrity of the outer membrane, affecting the morphology of the cell surface. (a – c) Scanning electron micrographs reveal distinct changes in the cell surface appearance of the ST-ΔtolC, demonstrating phenotypic heterogeneity in comparison to the ST-WT and ST-ΔtolC+ strains. (a) The ST-WT strain, highlighted by the black square, shows a smooth surface. The presence of LPS patches is indicated by red arrows and a black dotted circle. (b) In contrast, the ST-ΔtolC strain, marked by red squares, displays a rough surface lacking visible LPS patches. Numerous dented areas are indicated by purple arrows. (c) The ST-∆tolC+ strain, denoted by the blue square, presents a smoother surface with LPS patches, as indicated by red arrows and a black dotted circle. This strain shows fewer dented areas, pointed out by black arrows. It’s important to note that the complementation of ST-ΔtolC did not completely reverse the effects of the tolC deletion. All cells were observed at a magnification of 100,000X, with scale bars representing 100 nm.