We show here that RBC lysis by A. actinomycetemcomitans
LtxA is efficiently blocked by soluble gangliosides. Gangliosides also abrogate binding of LtxA-FITC to ganglioside-rich glioma cells. Numerous bacterial toxins utilize gangliosides as cellular receptors on various cell types. These toxins are highly selective for specific gangliosides. For example, cholera toxin and E. coli
heat-labile toxin bind to GM1, botulinum toxin to GT1b and GQ1b, and pertussis toxin to GD1a [26
]. In contrast, based on the work presented here, LtxA does not show a preference for any specific ganglioside. To our knowledge, only one other bacterial toxin, Clostridium perfringens
δ toxin, has been shown to interact with a ganglioside on RBCs, namely GM2 [33
While gangliosides effectively prevented LtxA-mediated lysis of RBCs, they were much less effective at protecting WBCs from LtxA. LFA-1, the WBC receptor for LtxA, is a heavily glycosylated membrane protein [15
]. Asada et al.
] reported that ~60% of the oligosaccharide moieties that modify LFA-1 have the terminal structure: sialic acid→galactose→N-acetylglucosamine (). Interestingly, this terminal sugar structure found on LFA-1 is strikingly similar to the gangliosides that block LtxA (). In addition, Morova et al.
] recently reported that RTX toxins can recognize β2
integrin receptors through their N-linked oligosaccharide chains and suggest that interaction between toxin and oligosaccharide represents the initial binding step. They showed that treatment of Jurkat T-cells with glycosidases rendered these cells more resistant to killing by RTX toxins, including LtxA. However, LtxA was still able to kill glycosidase-treated cells, but less efficiently than untreated cells. This result suggests that the deglycosylation was not complete or that LtxA can still interact with deglycosylated LFA-1, albeit less efficiently. WBCs have been reported to also express gangliosides on their surfaces [35
]. However, cell lines that do not express LFA-1 (CD11a, CD18, or both) are completely resistant to LtxA-mediated toxicity [14
], indicating that LtxA does not function through gangliosides on WBCs. This result is similar to the effect of LtxA on C6 glioma cells, which are recognized by LtxA but not killed. Thus, it appears that gangliosides do not act as potential functional receptors on cells except RBCs.
Based on several lines of evidence, we hypothesize that LtxA originally evolved as a toxin to target the immune system by interacting with LFA-1, but because of the similarities between the N-linked oligosaccharides of LFA-1 and gangliosides on the surface of RBCs, the toxin “gained” the ability to recognize and lyse RBCs. First, all of the sialidated gangliosides we tested were able to block LtxA-mediated hemolysis equally well with no apparent preference. All other bacterial toxins that interact with ganglioside receptors show strong preference for one or two gangliosides [26
]. Second, gangliosides were only partially effective at blocking LtxA-mediated killing of WBCs (THP-1 cells) at low doses of LtxA and short incubation times. At high LtxA doses, blocking was not apparent. Third, when LtxA bound to C6 glioma cells via gangliosides, there was no subsequent toxicity or cellular changes, even at high doses after 24 hours. Interaction between other toxins and gangliosides always leads to noticeable downstream effects, especially, cell death.
Interaction between RTX toxins and cellular receptors, such as LFA-1, leads to membrane disruption [37
] and cellular signaling that ultimately results in cell death [40
]. However, because RBCs lack LFA-1 and there is unlikely to be an intracellular signaling cascade activated by LtxA in RBCs, we suggest that interaction between LtxA and RBC gangliosides results in disruption of the membrane, which leads to cell lysis. Following interaction with LFA-1 on WBCs, LtxA is proposed to undergo significant conformational changes that result in membrane insertion of the toxin [39
]. Thus, contact between LtxA and gangliosides may result in a similar change in conformation that allows LtxA to insert into and disrupt RBC membranes. Further biochemical studies will be required to test our hypothesis.
In conclusion, we demonstrate that gangliosides can block hemolysis by a toxin from an important oral pathogen. Our data and knowledge of other bacterial toxins suggest that gangliosides may act as a RBC receptor for LtxA. For individuals with HACEK-causing IE, the most common route of infection is through the oral cavity since the HACEK bacteria are part of the normal oro-pharyngeal flora. A. actinomycetemcomitans
can be found on infected heart tissue where it exists as vegetative growths [9
]. This indicates that A. actinomycetemcomitans
has the ability to enter the blood stream, evade host immune defenses, colonize heart tissue, and persist in this environment. Furthermore, one of the clinical symptoms of IE is marked anemia, and hemolysis is considered to be an important mechanism leading to this anemia [44
]. Thus, targeting of host RBCs by A. actinomycetemcomitans
may be a crucial step in the pathogenic process and understanding this interaction could lead to novel therapeutic modalities.