Structural basis of mammalian glycan targeting by Vibrio cholerae cytolysin and biofilm proteins

Abstract

Vibrio cholerae is an aquatic gram-negative microbe responsible for cholera, a pandemic disease causing life-threatening diarrheal outbreaks in populations with limited access to health care. Like most pathogenic bacteria, V. cholerae secretes virulence factors to assist colonization of human hosts, several of which bind carbohydrate receptors found on cell-surfaces. Understanding how pathogenic virulence proteins specifically target host cells is important for the development of treatment strategies to fight bacterial infections. Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin with a carboxy-terminal β-prism domain that targets complex N-glycans found on mammalian cell-surface proteins. To investigate glycan selectivity, we studied the VCC β-prism domain and two additional β-prism domains found within the V. cholerae biofilm matrix protein RbmC. We show that the two RbmC β-prism domains target a similar repertoire of complex N-glycan receptors as VCC and find through binding and modeling studies that a branched pentasaccharide core (GlcNAc2-Man3) represents the likely footprint interacting with these domains. To understand the structural basis of V. cholerae β-prism selectivity, we solved high-resolution crystal structures of fragments of the pentasaccharide core bound to one RbmC β-prism domain and conducted mutagenesis experiments on the VCC toxin. Our results highlight a common strategy for cell-targeting utilized by both toxin and biofilm matrix proteins in Vibrio cholerae and provide a structural framework for understanding the specificity for individual receptors. Our results suggest that a common strategy for disrupting carbohydrate interactions could affect multiple virulence factors produced by V. cholerae, as well as similar β-prism domains found in other vibrio pathogens.

The 1.9 Å crystal structure of the extracellular matrix protein Bap1 from Vibrio cholerae provides insights into bacterial biofilm adhesion

“…The structure of Bap1 ⌬57 reveals a two-domain arrangement consisting of an eight-bladed ␤-propeller with a ␤-prism domain inserted within blade 6 via a flexible linker. In addition, we show evidence of Bap1 ⌬57 binding to anionic polysaccharides in a manner that differs significantly from the lectin-binding activity displayed by the ␤-prism domains of RbmC (23). Our studies support a model of V. cholerae biofilm architecture that allows for varied roles of biofilm scaffolding and surface attachment in aquatic versus host environments.…”

“…However, we were successful in expressing a Bap1 construct in which the ␤-prism domain was genetically excised (Bap1 ␤-propeller ), suggesting that this domain contributes to the insolubility of the full-length Bap1 protein. Given that isolated RbmC and VCC ␤-prism domains express in a soluble form (23) and that the distinguishing feature of the Bap1 ␤-prism domain is the 57-amino acid insertion, we made Bap1 ⌬57 and ␤-prism ⌬57 domain constructs with this insertion removed. To aid in expression and screening, we additionally fused a protease-cleavable GFP UV domain to the N terminus of the Bap1 ⌬57 and ␤-prism ⌬57 constructs.…”

“…The Bap1 ⌬57 ␤-prism domain falls into the jacalin-related lectin (JRL) protein family, which consists of a pseudo-3-fold arrangement of Greek key motifs. Most JRLs bind carbohydrates in only one sugar site, located at the top of Greek key I, but three previously characterized ␤-prism I domains expressed by V. cholerae (two from RbmC and one from VCC) bind their carbohydrate ligands at a single site at the top of Greek key II (23,33).…”

“…The Bap1 ⌬57 ␤-prism domain adopts a canonical ␤-prism I architecture, made up of 12 ␤-strands arranged into three antiparallel ␤-sheets with Greek key folds. The surface of the Bap1 ⌬57 ␤-prism domain opposite its attachment to the ␤-propeller makes up the region that constitutes a carbohydrate-binding site in the other three ␤-prisms expressed by V. cholerae: RbmC ␤-prism 1 , RbmC ␤-prism 2 , and VCC ␤-prism (23,33). In the Bap1 ⌬57 structure, this end of the ␤-prism features a positivelycharged, lysine-rich surface with a central cavity not found in the other V. cholerae ␤-prism I lectin domains (Fig.…”

“…One additional ␤-prism is also found in the V. cholerae genome attached to the poreforming toxin V. cholerae cytolysin (VCC) with ϳ33% sequence identity to the Bap1 ␤-prism domain. The three ␤-prism domains from RbmC and VCC all exhibit low-nanomolar affinity for the central core of complex N-glycans found abundantly on eukaryotic cell surfaces (23). Sequence alignments indicate that Bap1 has a 57-amino acid insertion within the ␤-prism domain, not present in the RbmC or VCC ␤-prism domains (23).…”