Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification

Abstract

A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Galp-(1→3)-β-d-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Galp-(1→3)-β-d-Galp-(1→] or O2c [→3)-β-d-GlcpNAc-(1→5)-β-d-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp–dependent GT-A galactosyltransferase adding β-(1→3)–linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)–linked d-Galp. These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf–dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria.

Diversity of mucoid to non-mucoid switch among carbapenemase-producing Klebsiella pneumoniae

“…In-depth analysis of CNR152A1 genome compared to isolates from the same STs retrieved from the National Center for Biotechnology (NCBI) revealed an IS 903b at codon 264 disrupting wbbM, belonging to the antigen O biosynthesis gene cluster. It encodes a glycosyltransferase and its disruption has been shown to induce defects in LPS biosynthesis [ 37 ], increasing the electronegative charge of the bacterial cell surface. The IS insertion was also present in the CNR152A1-NM2 and in the other NM variants included in our analysis.…”

K-PAM: a unified platform to distinguish Klebsiella species K- and O-antigen types, model antigen structures and identify hypervirulent strains

“…Sequence identity cutoff criteria used for K-typing are followed in O-typing also. As the Wzm and Wzt sequences of O1, O2 and O2ac are highly similar (> 98%), WbbY and WbbZ sequences corresponding to O1 and O2ac have been used to distinguish them from each other as well as from O2 as suggested in the earlier studies 23,35 . Similarly, WbdA and WbdD sequences have been added to the reference database to identify the sub-types of O3 serotype (O3/O3a & O3b) 36 .…”

“…In fact, the O-antigen biosynthesis locus arrangement is also identical for these serotypes. Thus, wbbY and wbbZ genes that are located outside the O-antigen biosynthesis locus are additionally used to distinguish O1, O2 and O2ac serotypes 23,35 . Similarly, wbdA and wbdD genes are used to distinguish O3 variants.…”

K-PAM: A unified platform to distinguishKlebsiellaspecies K- and O-antigen types, model antigen structures and identify hypervirulent strains

“…WbbY and WbbZ sequences corresponding to O1 and O2ac have been used to distinguish them from each other as well as from O2 as suggested in the earlier studies [25,34].…”