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A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis.

ABSTRACT: In Lactococcus lactis, cell-wall polysaccharides (CWPSs) act as receptors for many bacteriophages, and their structural diversity among strains explains, at least partially, the narrow host range of these viral predators. Previous studies have reported that lactococcal CWPS consists of two distinct components, a variable chain exposed at the bacterial surface, named polysaccharide pellicle (PSP), and a more conserved rhamnan chain anchored to, and embedded inside, peptidoglycan. These two chains appear to be covalently linked to form a large heteropolysaccharide. The molecular machinery for biosynthesis of both components is encoded by a large gene cluster, named cwps In this study, using a CRISPR/Cas-based method, we performed a mutational analysis of the cwps genes. MALDI-TOF MS-based structural analysis of the mutant CWPS combined with sequence homology, transmission EM, and phage sensitivity analyses enabled us to infer a role for each protein encoded by the cwps cluster. We propose a comprehensive CWPS biosynthesis scheme in which the rhamnan and PSP chains are independently synthesized from two distinct lipid-sugar precursors and are joined at the extracellular side of the cytoplasmic membrane by a mechanism involving a membrane-embedded glycosyltransferase with a GT-C fold. The proposed scheme encompasses a system that allows extracytoplasmic modification of rhamnan by complex substituting oligo-/polysaccharides. It accounts for the extensive diversity of CWPS structures observed among lactococci and may also have relevance to the biosynthesis of complex rhamnose-containing CWPSs in other Gram-positive bacteria.

SUBMITTER: Theodorou I

PROVIDER: S-EPMC6873199 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in <i>Lactococcus lactis</i>.

Theodorou Ilias I Courtin Pascal P Palussière Simon S Kulakauskas Saulius S Bidnenko Elena E Péchoux Christine C Fenaille François F Penno Christophe C Mahony Jennifer J van Sinderen Douwe D Chapot-Chartier Marie-Pierre MP

The Journal of biological chemistry 20191003 46

In <i>Lactococcus lactis</i>, cell-wall polysaccharides (CWPSs) act as receptors for many bacteriophages, and their structural diversity among strains explains, at least partially, the narrow host range of these viral predators. Previous studies have reported that lactococcal CWPS consists of two distinct components, a variable chain exposed at the bacterial surface, named polysaccharide pellicle (PSP), and a more conserved rhamnan chain anchored to, and embedded inside, peptidoglycan. These two ...[more]

PMID: 31582566

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Project description:Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein - CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type C. jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analyzed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ Lactic Acid Bacteria (LAB) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to deliver heterologous antigens to the bird immune system. Additionally, the analysis of the structure and immunogenicity of the generated rCjaAD hybrid protein showed that the CjaA antigen can be considered as a starting point to construct multiepitope anti-Campylobacter vaccines.

Project description:Lactococcus lactis is a Gram-positive bacterium that is widely used as a cell factory for the expression of heterologous proteins that are relevant in the pharmaceutical and nutraceutical fields. The signal peptide of the major secreted protein of L. lactis, Usp45, has been employed extensively in engineering strategies to secrete proteins of interest. However, the biological function of Usp45 has remained obscure despite more than 25?years of research. Studies on Usp45 homologs in other Gram-positive bacteria suggest that Usp45 may play a role in cell wall turnover processes. Here, we show the effect of inactivation and overexpression of the usp45 gene on L. lactis growth, phenotype, and cell division. Our results are in agreement with those obtained in streptococci and demonstrate that the L. lactis Usp45 protein is essential for proper cell division. We also show that the usp45 promoter is highly activated by galactose. Overall, our results indicate that Usp45 mediates cell separation, probably by acting as a peptidoglycan hydrolase.IMPORTANCE The cell wall, composed mainly of peptidoglycan, is key to maintaining the cell shape and protecting the cell from bursting. Peptidoglycan degradation by peptidoglycan hydrolysis and autolysins occurs during growth and cell division. Since peptidoglycan hydrolases are important for virulence, envelope integrity, and regulation of cell division, it is valuable to investigate their function and regulation. Notably, PcsB-like proteins such as Usp45 have been proposed as new targets for antimicrobial drugs and could also be target for the development of food-grade suicide systems. In addition, although various other expression and secretion systems have been developed for use in Lactococcus lactis, the most-used signal peptide for protein secretion in this bacterium is that of the Usp45 protein. Thus, elucidating the biological function of Usp45 and determining the factors affecting its expression would contribute to optimize several applications.

Dataset Information

A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis.

Publications

A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in <i>Lactococcus lactis</i>.

Similar Datasets

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