Characterization of Actinobacillus actinomycetemcomitans leukotoxin pore formation in HL60 cells

Abstract

The mechanism of cell death induced by Actinobacillus actinomycetemcomitans leukotoxin (LTX) has been investigated with flow cytometry and patch electrode recording using cultured HL60 cells. The kinetics of propidium iodide (PI) positive staining of HL60 cells was measured as a function of LTX concentration at 37 degreesC. Results showed a concentration-dependent decrease in the tk times. Cell kill was slow at <1 microg/ml LTX concentrations with fewer than 50% of the cells killed after 1 h; at 1 microg/ml, the tk times ranged from approximately 15 to 30 min. At higher concentrations, the tk times decreased rapidly. The rate of cell kill was appreciably slowed at 20 degreesC. HL60 whole cell currents were recorded with patch electrodes. Immediately following exposure to high concentrations of LTX, large currents were recorded suggesting that the membrane potential of these cells had collapsed due to the large conductance increases. At low toxin concentrations, rapid conductance fluctuations were seen suggestive of a limited number of toxin-mediated events. Cells exposed to low concentrations of LTX exhibited these conductance fluctuations for up to 1 h, whereas toxin-insensitive cells were unaffected by long exposures to high concentrations of toxin. Our results are consistent with LTX-induced pores in susceptible cells which overwhelm the ability of the cell to maintain osmotic homeostasis causing cell death.

How Mannheimia haemolytica defeats host defence through a kiss of death mechanism

“…The authors interpreted this result by implying that the aqueous form of the toxin will not spontaneously incorporate into a bilayer, but if the toxin is partially unfolded, as likely happens at the lipid monolayer-water interface, insertion into the membrane occurs and channels are formed. These are also consistent with LTX being required to interact with a cell surface receptor in order to facilitate toxin activation [93,103]. It has been shown that this receptor is the human β2-integrin LFA-1, expressed on immune cell surfaces matching the profile of cytolytic targets [101].…”

“…Cells undergo morphological changes consistent with rapid cell death [93]. When adding LTX to the bathing solution of an artificial bilayer, no channel activity was seen.…”

Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and α-Hemolysin from Escherichia coli

“…S2 in the supplemental material). LtxA is an RTX toxin, and it is still debated whether it forms actual pores in biological membranes (50)(51)(52)(53). Peculiarly, we could demonstrate that LtxA releases ATP from phospholipid-1-palmitoyl-2-oleoyl-phosphatidyl choline (POPC) vesicles with kinetics quite similar to those of HlyA.…”

Intra- and Interspecies Regulation of Gene Expression byActinobacillus actinomycetemcomitansLuxS

“…Our results show that the expression of the A. actinomycetemcomitans leukotoxin is influenced by AI-2 and increases by several fold in early-log-phase cells after exposure to conditioned medium from recombinant E. coli cultures expressing luxS. The leukotoxin is an RTX pore-forming toxin that induces apoptosis (at low concentration) or cell lysis (at high concentration) in a defined set of human leukocytes (26,29,30). In addition, several recent studies have shown that A. actinomycetemcomitans strains which express high levels of leukotoxin are associated with severe forms of early-onset periodontal diseases (9,22,23), suggesting that the toxin is important for A. actinomycetemcomitans pathogenesis.…”