Binding of Kingella kingae RtxA Toxin Depends on Cell Surface Oligosaccharides, but Not on β2 Integrins

Abstract

The Gram-negative coccobacillus Kingella kingae is increasingly recognized as an important invasive pediatric pathogen that causes mostly bacteremia and skeletal system infections. K. kingae secretes an RtxA toxin that belongs to a broad family of the RTX (Repeats in ToXin) cytotoxins produced by bacterial pathogens. Recently, we demonstrated that membrane cholesterol facilitates interaction of RtxA with target cells, but other cell surface structures potentially involved in toxin binding to cells remain unknown. We show that deglycosylation of cell surface structures by glycosidase treatment, or inhibition of protein N- and O-glycosylation by chemical inhibitors substantially reduces RtxA binding to target cells. Consequently, the deglycosylated cells were more resistant to cytotoxic activity of RtxA. Moreover, experiments on cells expressing or lacking cell surface integrins of the β2 family revealed that, unlike some other cytotoxins of the RTX family, K. kingae RtxA does not bind target cells via the β2 integrins. Our results, hence, show that RtxA binds cell surface oligosaccharides present on all mammalian cells but not the leukocyte-restricted β2 integrins. This explains the previously observed interaction of the toxin with a broad range of cell types of various mammalian species and reveals that RtxA belongs to the group of broadly cytolytic RTX hemolysins.

Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins

“…All these results showed that glycosylated structures on the cell surface and membrane cholesterol together act as the major membrane components used by RtxA to interact with target cells. Since glycosylated structures are present on the surface of all mammalian cells and cholesterol is a key structural component of all animal membranes, our results explain the previously observed promiscuity of RtxA binding to a wide spectrum of cells from various species and indicated that RtxA can be classified as a broadly cytolytic RTX hemolysin [37,262].…”

“…Based on the cited literature showing that several RTX toxins specifically interact with the β 2 integrins on the cell surface, we recently investigated whether the β 2 integrins could also be the potential receptors for the RTX cytolysin RtxA [ 262 ]. We examined the binding and cytotoxicity of RtxA on CHO cells that ectopically expressed three different human β 2 integrins, CD11a/CD18, CD11b/CD18, or CD11c/CD18.…”

“…Similarly, the viabilities of CD11a KO and CD11b KO macrophages treated with RtxA were reduced in the same time-dependent manner as the viability of the WT macrophages. Thus, the results showed that RtxA, unlike the RTX toxins described above, does not recognize the β 2 integrins as specific receptors on target cells [ 262 ].…”

“…Recently, we investigated whether the binding and cytotoxic activities of RtxA also depend on the recognition of glycosylated membrane structures, as previously shown for several other RTX toxins [ 262 ]. We showed that the binding and cytotoxic activity of RtxA could be significantly decreased when different cell types were preincubated with neuraminidase.…”

“…Furthermore, we demonstrated that both enzymatic (PNGase F, O-glycosidase) and inhibitor-mediated (tunicamycin, benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside) removal of N- or O-linked oligosaccharide chains from cell surface glycosylated structures resulted in a significant loss of RtxA binding, and deglycosylated cells were more resistant to the cytotoxic effect of RtxA than untreated cells. Thus, RtxA not only recognizes the sialic acid residues but also other saccharide units of the cell surface glycoproteins on the surface of target cells [ 262 ].…”