Targeting the Sugary Armor of Klebsiella Species

Abstract

The emergence of multidrug-resistant strains of Gram-negative Klebsiella species is an urgent global threat. The World Health Organization has listed Klebsiella pneumoniae as one of the global priority pathogens in critical need of next-generation antibiotics. Compared to other Gram-negative pathogens, K. pneumoniae accumulates a greater diversity of antimicrobial-resistant genes at a higher frequency. The evolution of a hypervirulent phenotype of K. pneumoniae is yet another concern. It has a broad ecological distribution affecting humans, agricultural animals, plants, and aquatic animals. Extracellular polysaccharides of Klebsiella, such as lipopolysaccharides, capsular polysaccharides, and exopolysaccharides, play crucial roles in conferring resistance against the host immune response, as well as in colonization, surface adhesion, and for protection against antibiotics and bacteriophages. These extracellular polysaccharides are major virulent determinants and are highly divergent with respect to their antigenic properties. Wzx/Wzy-, ABC-, and synthase-dependent proteinaceous nano-machineries are involved in the biosynthesis, transport, and cell surface expression of these sugar molecules. Although the proteins involved in the biosynthesis and surface expression of these sugar molecules represent potential drug targets, variation in the amino acid sequences of some of these proteins, in combination with diversity in their sugar composition, poses a major challenge to the design of a universal drug for Klebsiella infections. This review discusses the challenges in universal Klebsiella vaccine and drug development from the perspective of antigen sugar compositions and the proteins involved in extracellular antigen transport.

Keywords: Klebsiella species; antibiotics; capsular polysaccharide; complement system; exopolysaccharide; lipopolysaccharide; multidrug resistance; vaccine.

Figure 1

Schematic representation of Klebsiella spp. CPS biosynthesis and surface export machinery. The sugar precursors biosynthesized in the cytoplasm are subsequently assembled in the cytoplasmic face of the inner membrane to form the repeating unit with the help of sugar-specific glycosyl transferases WbaP (or WcaJ), followed by WbaZ, WcaN, WcaJ, and WcaO. The recognition of the CPS repeating unit by the first sugar linked to undecaprenol-pyrophosphate (Und-PP) by Wzx (a flippase) facilitates the flipping of the repeating unit to the periplasmic side. Subsequent to this event, Wzy (a copolymerase) polymerizes the repeating units. Finally, Wza (an outer-membrane translocon), Wzc (a tyrosin autokinase), and Wzb (a phosphatase) synergistically transport CPS onto the bacterial surface and anchor the CPS onto the outer-membrane protein Wzi (a lecto-aqua-porin). As structural information on the representative proteins from Kp is unknown, the structures of Wzi, Wza, Wzb, Wzc (cytoplasmic domain), and Wzx have been modeled from available reference structures through the SWISS-MODEL server (Schwede et al., 2003). Klebsiella pneumoniae (K20) accession numbers corresponding to Wzi, Wza, Wzb, Wzc, and Wzx are BAF47011.1, BAF47012.1, BAF4703.1, BAF47029.1, and BAT24471.1, respectively. The corresponding PDB IDs used as templates in the modeling are 2YNK (99.78%), 2J58 (99.44%), 2WMY (99.32%), 3LA6 (57.93%), and 3MKU (14.11%), respectively. The sequence identity between the query and template is indicated in the bracket.

Figure 2

Schematic representation of Klebsiella spp. ABC transporter-dependent LPS assembly and transport. The O-antigen repeating unit is synthesized in the cytosol with the help of the corresponding glycosyltransferases and is subsequently polymerized by Wzy and transferred to the periplasm by an ABC transporter (Wzm/Wzt complex). In a similar fashion, the Kdo₂-lipid A–core oligosaccharide biosynthesized in the cytoplasmic region is flipped to the periplasmic region through the ATP-driven MsbA. Following this, the matured O-polysaccharide and lipid A-core-oligosaccharide are ligated by WaaL ligase in the periplasmic region. The completely grown LPS is transported to the bacterial surface through LptA-G assembly as indicated. For the purpose of illustration, the LptB2FG (PDB ID: 5L75) and LptDE (PDB ID: 5IV9) structures are taken directly from Kp, while the Wzm/Wzt complex and MsbA proteins are homology-modeled using structures available in other organisms as templates. The reference PDB IDs for Wzm, Wzt-NBD, and Wzt-CBD are 6AN7 (34.5%), 6AN5 (46.32%), and 2R5O (100%), respectively. The sequence identity between the template and the Kp are given in brackets. The NCBI accession numbers corresponding to the Kp protein sequences are CZQ25306.1 (Wzm) and CZQ25307.1 (Wzt-NBD and Wzt-CBD). LptA and LptC are indicated by schematic representation. The helical and beta-jelly conformation of LptC is shown in red. The beta-jelly conformation of LptA is colored dark gold. As WaaL structural information for any Gram-negative organism is unavailable, the two domains of WaaL are represented in yellow and peach-colored ovals. Note that for the purpose of illustration, O3 has been considered as a case in point. Individual parts of the LPS and O-antigen are annotated separately at the bottom of the figure.

Figure 3

An amino acid sequence logo constructed using the multiple sequence alignment of 139 Wza protein sequences (A) and 138 Wzi protein sequences (B). Note the variation observed in the C-terminal region (transmembrane region) of Wza that faces the extracellular region of the bacterial cell (dash-box) (A). In contrast, Wzi sequences are highly conserved between different serotypes of Klebsiella spp. Note that non-redundant sequences that have a defined K-type are used to generate sequence logo.