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

Waheed Ur Rahman,Nela Klimova,Nataliya Balashova,Adriana Osickova,Jinery Lora,Radim Osicka

doi:10.3390/ijms21239092

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.

Highlights

The Gram-negative coccobacillus Kingella kingae is a member of the commensal oropharyngeal flora of young children and, until recently, it was believed to be a rare pathogen [1,2,3]
CyaA binding to CD11b/CD18-expressing J774A.1 macrophage-like cells, human neutrophils, and Chinese hamster ovary (CHO)-CD11b/CD18 cells was reduced by ~70 to 80%, when the cells were pretreated with neuraminidase that removes peripheral sialic acid residues from glycoproteins [26]
We have previously demonstrated that the RTX cytotoxin CyaA of B. pertussis interacts with N-linked oligosaccharide chains of the β2 integrin CD11b/CD18 located in the C-terminal part of the CD11b subunit [26,27]

Summary

Introduction

The Gram-negative coccobacillus Kingella kingae is a member of the commensal oropharyngeal flora of young children and, until recently, it was believed to be a rare pathogen [1,2,3]. RtxA belongs to the RTX (Repeats in ToXin) family of pore-forming cytotoxins that are produced by many Gram-negative bacterial pathogens, including the genera Actinobacillus, Aggregatibacter, Bordetella, Escherichia, Mannheimia, Moraxella, Morganella, Pasteurella, Proteus, and Vibrio [12]. Sequence homology with the RTX toxins revealed four functional segments in the 956 residues-long RtxA: (i) a pore-forming domain encompassing residues ~140 to 410, harboring four putative transmembrane α-helices; (ii) an acylated segment, where the RtxA protoxin (proRtxA) is posttranslationally activated; recently, we experimentally demonstrated that the acyltransferase RtxC activates proRtxA by fatty acyl modification on lysine residues 558 and 689, primarily with myristoyl and hydroxymyristoyl chains [9,13]; (iii) a typical calcium-binding RTX domain between residues ~730 to 810, harboring the conserved repetitions of a nonapeptide motif X-(L/I/F)-X-G-G-X-G-(N/D)-D (where X is any amino acid residue), which form calcium-binding sites and (iv) a C-terminal secretion signal. Upon binding to target cells that is facilitated by membrane cholesterol, RtxA inserts itself into the cell membrane and forms cation-selective membrane pores, which induce cation flux leading to cell death [9,14]

Methods

Results

Conclusion