Dataset Information

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.

SUBMITTER: Kelly SD

PROVIDER: S-EPMC6635448 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The lipopolysaccharide O antigens of Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16 both contain a repeating unit disaccharide of [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]; the resulting polymer is known as D-galactan I. In K. pneumoniae serotype O1, the genes responsible for the synthesis of D-galactan I are found in the rfb gene cluster (rfbKpO1). We report here the cloning and analysis of the rfb cluster from S. marcescens serotype O16 (rfbSmO16). This is the first rfb gene cluster examined for the genus Serratia. Synthesis of D-galactan I is an rfe-dependent process for both K. pneumoniae serotype O1 and S. marcescens serotype O16. Hybridization experiments with probes derived from each of the six rfbKpO1 genes indicate that the cloned rfbSmO16 cluster contains homologous genes arranged in the same order. However, the degree of homology at the nucleotide sequence level was sufficiently low that hybridization was detected only under low-stringency conditions. rfbABSmO16 genes were subcloned and shown to encode an ABC-2 (ATP-binding cassette) transporter which is functionally identical to the one encoded by the corresponding rfb genes from K. pneumoniae serotype O1. The amino acid sequences of the predicted RfbA and RfbB homologs showed identities of 75.7% (87.9% total similarity) and 78.0% (86.5% total similarity), respectively. The last gene of the rfbKpO1 cluster, rfbFKpO1, encodes a bifunctional galactosyltransferase which initiates the formation of D-galactan I. RfbFKpO1 and RfbFSmO16 are 57.6% identical (with 71.1% total similarity), and both show similarity with RfpB, the galactosyltransferase involved in the synthesis of Shigella dysenteriae type I O-polysaccharide. The G+C contents of the rfbAB genes from each organism are quite similar, and values are lower than those typical for the species. However, the G+C content of rfbFSmO16 (47.6%) was much higher than that of rfbFKpO1 (37.3%), despite the fact that the average for each species (52 to 60%) falls within the same range.

Project description:Klebsiella pneumoniae can be broadly classified into classical strains that cause drug-resistant, hospital-associated infections and hypervirulent strains that cause invasive, community-acquired, drug-susceptible infections. Hypermucoviscosity in Klebsiella pneumoniae has been associated with immune evasion and hypervirulence. A string-test-positive, hypermucoviscous strain of Klebsiella pneumoniae, P34, was isolated from the cystic lesion of a patient who reported to a tertiary care hospital in Jodhpur, Rajasthan, India. Given the antibiotic-susceptible and hypermucoviscous nature of the isolate, it was suspected to belong to the hypervirulent lineage of Klebsiella pneumoniae. However, P34 did not overproduce capsular polysaccharides and also remained susceptible to the antimicrobial effects of human serum when tested alongside strains that were non-hypermucoviscous. Sequencing of the genome of P34 revealed the absence of any large virulence plasmids or integrative conjugative elements that usually carry hypermucoviscosity- and hypervirulence-associated genes. P34 also lacked key virulence determinants such as aerobactin, yersiniabactin, and salmochelin biosynthesis clusters. In addition, P34 lacked homologs for genes associated with enhanced capsule synthesis and hypermucoviscosity, such as rmpA, rmpA2, rmpC, and rmpD (regulator of mucoid phenotype). These observations suggest that P34 may harbor novel genetic determinants of hypermucoviscosity independent of the indirectly acting rmpA and the recently described rmpD. IMPORTANCE Hypermucoviscosity is a characteristic of hypervirulent Klebsiella pneumoniae strains, which are capable of causing invasive disease in community settings. This study reports phenotyping and genomic analysis of an unusual clinical isolate of Klebsiella pneumoniae, P34, which exhibits hypermucoviscosity and yet does not harbor rmp (regulator of mucoid phenotype) genes, which are known determinants of hypermucoviscosity (rmpA and rmpD). Similar clinical isolates belonging to the K. pneumoniae complex that are hypermucoviscous but do not harbor the rmp loci have been reported from India and abroad, indicating the prevalence of unknown determinants contributing to hypermucoviscosity. Therefore, strains like P34 will serve as model systems to mechanistically study potentially novel determinants of hypermucoviscosity in the K. pneumoniae complex.

Project description:Klebsiella pneumoniae is a Gram-negative, ubiquitous bacterium capable of causing severe nosocomial infections in individuals with impaired immune system. Emerging multi-drug resistant strains of this species and particularly carbapenem-resistant strains pose an urgent threat to public health. The lipopolysaccharide (LPS) O-antigen is the main surface antigen. It contributes to the virulence of this species and determines the O-serotype of K. pneumoniae isolates. Among the nine main O-serotypes of K. pneumoniae, O1-and O2-type pathogens are causative agents of over 50% of all infections. Serotype O1, the most common O-serotype, expresses complex LPS consisting of d-galactan-I (a polymer built of → 3)-β-d-Galf-(1 → 3)-α-d-Galp-(1 → repeating units) capped by d-galactan-II (built of [ → 3)-α-d-Galp-(1 → 3)-β-d-Galp-(1 →] repeating units). Galactan-I is present as the sole polymer in O2 serotype. Recently, in case of serotype O2, conversion of galactan-I to galactan-III (→ 3)-β-d-Galf-(1 → 3)-[α-d-Galp-(1 → 4)]-α-d-Galp-(1 →) was reported. Substitution of → 3)-α-d-Galp by a branching terminal α-d-Galp was dependent on the presence of the gmlABC operon and had a major impact on the antigenicity of the galactan polymer. Genetic analysis indicated that 40% of the O1 clinical isolates also carry the gmlABC locus; therefore we aimed to characterize the corresponding phenotype of LPS O-antigens. The presence of galactan-III among O1 strains was proven using galactan-III-specific monoclonal antibodies and confirmed by structural analyses performed using sugar and methylation analysis as well as classical and high-resolution magic angle spinning NMR spectroscopy. By using an isogenic mutant pair, we demonstrated that galactan-III expression was dependent on the presence of glycosyltransferases encoded by gmlABC, as was shown previously for the O2 serotype. Furthermore, the galactan-II structures in O1gml+ strains remained unaffected corroborating no functional interactions between the biosynthesis of galactan-III and galactan-II polymers.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets