Dataset Information

Study of the cwaRS-ldcA Operon Coding a Two-Component System and a Putative L,D-Carboxypeptidase in Lactobacillus paracasei.

ABSTRACT: The cell surface is the primary recognition site between the bacterium and the host. An operon of three genes, LSEI_0219 (cwaR), LSEI_0220 (cwaS), and LSEI_0221 (ldcA), has been previously identified as required for the establishment of Lactobacillus paracasei in the gut. The genes cwaR and cwaS encode a predicted two-component system (TCS) and ldcA a predicted D-alanyl-D-alanine carboxypeptidase which is a peptidoglycan (PG) biosynthesis enzyme. We explored the functionality and the physiological role of these three genes, particularly their impact on the bacterial cell wall architecture and on the bacterial adaptation to environmental perturbations in the gut. The functionality of CwaS/R proteins as a TCS has been demonstrated by biochemical analysis. It is involved in the transcriptional regulation of several genes of the PG biosynthesis. Analysis of the muropeptides of PG in mutants allowed us to re-annotate LSEI_0221 as a putative L,D-carboxypeptidase (LdcA). The absence of this protein coincided with a decrease of two surface antigens: LSEI_0020, corresponding to p40 or msp2 whose implication in the host epithelial homeostasis has been recently studied, and LSEI_2029 which has never been functionally characterized. The inactivation of each of these three genes induces susceptibility to antimicrobial peptides (hBD1, hBD2, and CCL20), which could be the main cause of the gut establishment deficiency. Thus, this operon is necessary for the presence of two surface antigens and for a suitable cell wall architecture.

SUBMITTER: Scornec H

PROVIDER: S-EPMC7062640 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Study of the cwaRS-ldcA Operon Coding a Two-Component System and a Putative L,D-Carboxypeptidase in Lactobacillus paracasei.

Scornec Hélène H Palud Aurore A Pédron Thierry T Wheeler Richard R Petitgonnet Clément C Boneca Ivo Gomperts IG Cavin Jean-François JF Sansonetti Philippe J PJ Licandro Hélène H

Frontiers in microbiology 20200303

The cell surface is the primary recognition site between the bacterium and the host. An operon of three genes, LSEI_0219 (cwaR), LSEI_0220 (cwaS), and LSEI_0221 (ldcA), has been previously identified as required for the establishment of Lactobacillus paracasei in the gut. The genes cwaR and cwaS encode a predicted two-component system (TCS) and ldcA a predicted D-alanyl-D-alanine carboxypeptidase which is a peptidoglycan (PG) biosynthesis enzyme. ...[more]

PMID: 32194510

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Project description:Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplement industries, and specific strains, such as Lactobacillus acidophilus NCFM, have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR (clustered regularly interspaced palindromic repeats)-SpyCas9D10A nickase (Cas9N)-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9N vector harboring cas9N under the regulation of a Lactobacillus promoter and a cloning region for a customized single guide RNA (sgRNA) and editing template. The functionality of the pLbCas9N system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS, and ltaS), yielding 35 to 100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9N system. To showcase the versatility of this system, we also inserted an mCherry fluorescent-protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9N system was further deployed in other species to generate a concurrent single-base substitution and gene deletion in Lactobacillus gasseri ATCC 33323 and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered with by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9N system for genome manipulations in diverse lactobacilli and open new avenues for the engineering of health-promoting lactic acid bacteria.IMPORTANCE This work describes the development of a lactobacillus CRISPR-based editing system for genome manipulations in three Lactobacillus species belonging to the lactic acid bacteria (LAB), which are commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiotas and for their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species. This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems. Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.

Dataset Information

Study of the cwaRS-ldcA Operon Coding a Two-Component System and a Putative L,D-Carboxypeptidase in Lactobacillus paracasei.

Publications

Study of the <i>cwaRS-ldcA</i> Operon Coding a Two-Component System and a Putative L,D-Carboxypeptidase in <i>Lactobacillus paracasei</i>.

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