Dataset Information

Analysis of the Topology and Active-Site Residues of WbbF, a Putative O-Polysaccharide Synthase from Salmonella enterica Serovar Borreze.

ABSTRACT: Bacterial lipopolysaccharides are major components and contributors to the integrity of Gram-negative outer membranes. The more conserved lipid A-core part of this complex glycolipid is synthesized separately from the hypervariable O-antigenic polysaccharide (OPS) part, and they are joined in the periplasm prior to translocation to the outer membrane. Three different biosynthesis strategies are recognized for OPS biosynthesis, and one, the synthase-dependent pathway, is currently confined to a single example: the O:54 antigen from Salmonella enterica serovar Borreze. Synthases are complex enzymes that have the capacity to both polymerize and export bacterial polysaccharides. Although synthases like cellulose synthase are widespread, they typically polymerize a glycan without employing a lipid-linked intermediate, unlike the O:54 synthase (WbbF), which produces an undecaprenol diphosphate-linked product. This raises questions about the overall similarity between WbbF and conventional synthases. In this study, we examine the topology of WbbF, revealing four membrane-spanning helices, compared to the eight in cellulose synthase. Molecular modeling of the glycosyltransferase domain of WbbF indicates a similar architecture, and site-directed mutagenesis confirmed that residues important for catalysis and processivity in cellulose synthase are conserved in WbbF and required for its activity. These findings indicate that the glycosyltransferase mechanism of WbbF and classic synthases are likely conserved despite the use of a lipid acceptor for chain extension by WbbF.IMPORTANCE Glycosyltransferases play a critical role in the synthesis of a wide variety of bacterial polysaccharides. These include O-antigenic polysaccharides, which form the distal component of lipopolysaccharides and provide a protective barrier important for survival and host-pathogen interactions. Synthases are a subset of glycosyltransferases capable of coupled synthesis and export of glycans. Currently, the O:54 antigen of Salmonella enterica serovar Borreze involves the only example of an O-polysaccharide synthase, and its generation of a lipid-linked product differentiates it from classical synthases. Here, we explore features conserved in the O:54 enzyme and classical synthases to shed light on the structure and function of the unusual O:54 enzyme.

SUBMITTER: Wear SS

PROVIDER: S-EPMC7015711 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Analysis of the Topology and Active-Site Residues of WbbF, a Putative O-Polysaccharide Synthase from Salmonella enterica Serovar Borreze.

Wear Samantha S SS Hunt Brittany A BA Clarke Bradley R BR Whitfield Chris C

Journal of bacteriology 20200211 5

Bacterial lipopolysaccharides are major components and contributors to the integrity of Gram-negative outer membranes. The more conserved lipid A-core part of this complex glycolipid is synthesized separately from the hypervariable O-antigenic polysaccharide (OPS) part, and they are joined in the periplasm prior to translocation to the outer membrane. Three different biosynthesis strategies are recognized for OPS biosynthesis, and one, the synthase-dependent pathway, is currently confined to a s ...[more]

PMID: 31792013

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Project description:BackgroundTyphoid fever is an acute systemic infection of humans caused by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi). In chronic carriers, the bacteria survive the harsh environment of the gallbladder by producing biofilm. The phenotype of S. Typhi biofilm cells is significantly different from the free-swimming planktonic cells, and studies have shown that they are associated with antibiotic resistance, immune system evasion, and bacterial persistence. However, the mechanism of this transition and the events leading to biofilm formation are unknown. High throughput sequencing was performed to identify the genes involved in biofilm formation and to postulate the mechanism of action.ResultsPlanktonic S. Typhi cells were cultured using standard nutrient broth whereas biofilm cells were cultured in a stressful environment using high shearing-force and bile to mimic the gallbladder. Sequencing libraries were prepared from S. Typhi planktonic cells and mature biofilm cells using the Illumina HiSeq 2500 platform, and the transcriptome data obtained were processed using Cufflinks bioinformatics suite of programs to investigate differential gene expression between the two phenotypes. A total of 35 up-regulated and 29 down-regulated genes were identified. The identities of the differentially expressed genes were confirmed using NCBI BLAST and their functions were analyzed. The results showed that the genes associated with metabolic processes and biofilm regulations were down-regulated while those associated with the membrane matrix and antibiotic resistance were highly up-regulated.ConclusionsIt is proposed that the biofilm phenotype of S. Typhi allows the bacteria to increase production of the membrane matrix in order to serve as a physical shield and to adhere to surfaces, and enter an energy conservation state in response to the stressful environment. Conversely, the planktonic phenotype allows the bacteria to produce flagella and increase metabolic activity to enable the bacteria to migrate and form new colonies of infection. This data provide a basis for further studies to uncover the mechanism of biofilm formation in S. Typhi and to discover novel genes or pathways associated with the development of the typhoid carrier state.

Dataset Information

Analysis of the Topology and Active-Site Residues of WbbF, a Putative O-Polysaccharide Synthase from Salmonella enterica Serovar Borreze.

Publications

Analysis of the Topology and Active-Site Residues of WbbF, a Putative O-Polysaccharide Synthase from Salmonella enterica Serovar Borreze.

Similar Datasets

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