Role of the Lipopolysaccharide-CD14 Complex for the Activity of Hemolysin from Uropathogenic Escherichia coli

Abstract

Bacterial pathogens produce a variety of exotoxins, which often become associated with the bacterial outer membrane component lipopolysaccharide (LPS) during their secretion. LPS is a potent proinflammatory mediator; however, it is not known whether LPS contributes to cell signaling induced by those microbial components to which it is attached. This is partly due to the common view that LPS present in bacterial component preparations is an experimental artifact. The Escherichia coli exotoxin hemolysin (Hly) is a known inducer of proinflammatory signaling in epithelial cells, and the signal transduction pathway involves fluctuation of the intracellular-Ca 2؉ concentration. Since LPS is known to interact with Hly, we investigated whether it is required as a cofactor for the activity of Hly. We found that the LPS/Hly complex exploits the CD14/LPSbinding protein recognition system to bring Hly to the cell membrane, where intracellular-Ca 2؉ signaling is initiated via specific activation of the small GTPase RhoA. Hly-induced Ca 2؉ signaling was found to occur independently of the LPS receptor TLR4, suggesting that the role of LPS/CD14 is to deliver Hly to the cell membrane. In contrast, the cytolytic effect triggered by exposure of cells to high Hly concentrations occurs independently of LPS/CD14. Collectively, our data reveal a novel molecular mechanism for toxin delivery in bacterial pathogenesis, where LPS-associated microbial compounds are targeted to the host cell membrane as a consequence of their association with LPS.The exotoxin hemolysin (Hly) produced by uropathogenic Escherichia coli (UPEC) is an important virulence factor in extraintestinal infections, such as urinary tract infections, newborn meningitis, bacteremia, and septicemia (33). Hly exerts a cytolytic effect when present in high concentrations, while low, sublytic concentrations have been demonstrated to induce intracellular-Ca 2ϩ signaling in epithelial cells, leading to proinflammatory responses, such as production of interleukin-6 (IL-6) and . Hly is secreted from bacteria via the type I secretion pathway (26) and is found associated with bacterial outer membranes, in supernatants, and in outer membrane vesicles (5, 34). Hly forms high-molecular-weight complexes with lipopolysaccharide (LPS), and this interaction has been suggested to prevent aggregation and degradation of Hly as well as to promote an active conformation of the toxin (6, 12, 15). Whether there is also a molecular function of LPS in the Ca 2ϩ signaling and cytolytic activities of Hly remains unknown. Detailed information regarding the signal transduction pathway elicited by LPS does exist, however. LPS is a large amphipathic molecule composed of the acylated, hydrophobic lipid A component, the LPS core, and the O-antigen polysaccharide. Host recognition of LPS is a multistep process initiated by the binding of LPS by the LPS-binding protein (LBP). This interaction enhances the binding of LPS to the glycosylphosphatidylinositol-linked receptor CD14, whose protein structure conta...

E. coli Alpha Hemolysin and Properties

“…In addition, sublytic concentrations of HlyA have been recently found to potently stimulate the inactivation of the serine/threonine protein kinase B (PKB), which enzyme plays a central role in host cellcycle progression, metabolism, vesicular trafficking, survival, and inflammatory-signaling pathways . These findings may help to explain previously published results implicating sublytic concentrations of HlyA in the inhibition of chemotaxis and in bacterial killing by phagocytes in addition to the HlyA-mediated stimulation of host apoptotic and inflammatory pathways (Cavalieri & Snyder, 1982), (Koschinski et al, 2006), (Mansson et al, 2007), (Tran Van Nhieu et al,2004), (Uhlen et al, 2000).…”

Inactivation of Host Akt/Protein Kinase B Signaling by Bacterial Pore-forming Toxins

“…Specifically, the treatment of bladder cells with the K ϩ ionophore valinomycin (1-40 M for 2 h) did not alter the phosphorylation status of Akt and neither chelation of intracellular Ca 2ϩ pools nor the use of 5 mM KCl in the cell culture medium (to prevent K ϩ release during infection with UPEC) had any inhibitory effect on HlyAmediated dephosphorylation of Akt ( Figure 4B; unpublished data). Moreover, the mechanism by which HlyA induces Ca 2ϩ oscillations has recently been shown to be dependent on the small GTPase RhoA (Mansson et al, 2007a). By using a cell-permeable variant of the RhoA inhibitor C3 transferase (4 g/ml), which effectively inactivates RhoA in 5637 bladder cells, we again observed no abrogation of HlyA-mediated dephosphorylation of Akt (unpublished data).…”

“…Furthermore, we found that disruption of LPS (lipopolysaccharide) biosynthetic genes within the rfa and rfb operons, which were recently shown to contribute to UPEC-mediated suppression of host cytokine responses (Hunstad et al, 2005), did not prevent UTI89-mediated dephosphorylation of Akt ( Figure 2E). LPS promotes hemolysin interactions with host cells and mutations within the rfa operon, which affect the generation of the LPS core polysaccharide, result in decreased levels of hemolysin secretion (Bauer and Welch, 1997;Mansson et al, 2007a). Interestingly, in our experiments the rfa mutant was reproducibly less effective than wild-type UTI89 at inducing Akt dephosphorylation ( Figure 2E), possibly reflecting either decreased HlyA secretion by the rfa mutant and/or impaired LPS-modulated HlyA interactions with the target host cells.…”

Structure and function of RTX toxins

“…Interestingly, mutations in either TolC or HlyD also result in secreted HlyA with reduced hemolytic activity, suggesting a tight coupling between the secretion process and the folding of the exported protein [113,137]. Other studies have suggested a role of the outer membrane lipopolysaccharide (LPS) in the secretion, folding, and activity of several RTX toxins [138][139][140][141].…”