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Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons.

ABSTRACT: Beneficial microbes that target molecules and pathways, such as oxidative stress, which can negatively affect both host and microbiota, may hold promise as an inflammatory bowel disease therapy. Prior work showed that a five-strain fermented milk product (FMP) improved colitis in T-bet(-/-) Rag2(-/-) mice. By varying the number of strains used in the FMP, we found that Lactococcus lactis I-1631 was sufficient to ameliorate colitis. Using comparative genomic analyses, we identified genes unique to L. lactis I-1631 involved in oxygen respiration. Respiration of oxygen results in reactive oxygen species (ROS) generation. Also, ROS are produced at high levels during intestinal inflammation and cause tissue damage. L. lactis I-1631 possesses genes encoding enzymes that detoxify ROS, such as superoxide dismutase (SodA). Thus, we hypothesized that lactococcal SodA played a role in attenuating colitis. Inactivation of the sodA gene abolished L. lactis I-1631's beneficial effect in the T-bet(-/-) Rag2(-/-) model. Similar effects were obtained in two additional colonic inflammation models, Il10(-/-) mice and dextran sulfate sodium-treated mice. Efforts to understand how a lipophobic superoxide anion (O2 (-)) can be detoxified by cytoplasmic lactoccocal SodA led to the finding that host antimicrobial-mediated lysis is a prerequisite for SodA release and SodA's extracytoplasmic O2 (-) scavenging. L. lactis I-1631 may represent a promising vehicle to deliver antioxidant, colitis-attenuating SodA to the inflamed intestinal mucosa, and host antimicrobials may play a critical role in mediating SodA's bioaccessibility.

SUBMITTER: Ballal SA

PROVIDER: S-EPMC4485081 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons.

Ballal Sonia A SA Veiga Patrick P Fenn Kathrin K Michaud Monia M Kim Jason H JH Gallini Carey Ann CA Glickman Jonathan N JN Quéré Gaëlle G Garault Peggy P Béal Chloé C Derrien Muriel M Courtin Pascal P Kulakauskas Saulius S Chapot-Chartier Marie-Pierre MP van Hylckama Vlieg Johan J Garrett Wendy S WS

Proceedings of the National Academy of Sciences of the United States of America 20150608 25

Beneficial microbes that target molecules and pathways, such as oxidative stress, which can negatively affect both host and microbiota, may hold promise as an inflammatory bowel disease therapy. Prior work showed that a five-strain fermented milk product (FMP) improved colitis in T-bet(-/-) Rag2(-/-) mice. By varying the number of strains used in the FMP, we found that Lactococcus lactis I-1631 was sufficient to ameliorate colitis. Using comparative genomic analyses, we identified genes unique t ...[more]

PMID: 26056274

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Project description:Background:Characterizing the structure and function of superoxide dismutase (SOD), as an antioxidant enzyme providing opportunities for its application in food supplements. Objectives:In this study, the features of the Manganese-SOD of Lactococcus Lactis (SDLL), subsp. Cremoris MG1363, as probiotic bacteria, were determined on the basis of the computational methods. Materials and Methods:The protein's physicochemical properties and the prediction of its secondary structure were determined via the ProtParam server and the GOR program respectively. Moreover, the 3D structures of the proteins were constructed via the MODELLER on the basis of the homology method and the threading algorithm MUSTER. On the other hand, the structural stability of the models was assayed under the quasi-physiological conditions by the GROMACS program via the GROMOS96 43a1 force field in Linux system. Finally, using the Molecular Docking Studies, the functionality features of the models were predicted through their affinity with the corresponding substrates. Results:The results revealed the physicochemical properties of the SDLL and a 3D model of a chain of the enzyme being similar to the SOD from the Bacillus Subtilis (SDBS). The model of the SDLL was checked for quality control purposes including the Ramachandran plot, the ERRAT and the Verifiy3D. The model was suggestive of the structural stability in quasi-physiological conditions; yet, less than that of the SDBS. Assessing the cause of the instability in the SDLL model was indicative of two unstable regions in the area far from the enzyme's active position, they were considered suitable for mutagenesis. Accordingly, the loop substitution for the corresponding region of SDBS and the deletion of the loop positioned at the C terminal of SDLL resulted in a mutant of SDLL with more stability and appropriate affinity with the corresponding substrate. Conclusion:In general, the study provides a new model of SDLL with certain thermostable features, and a new mutant with suitable stability and functionality on the basis of the direct mutagenesis being used in different applications.

Project description:Lactococcus lactis subsp. cremoris MG1363 is a model for the lactic acid bacteria (LAB) used in the dairy industry. The proteolytic system, consisting of a proteinase, several peptide and amino acid uptake systems, and a host of intracellular peptidases, plays a vital role in nitrogen metabolism and is of eminent importance for flavor formation in dairy products. The dipeptidase PepV functions in the last stages of proteolysis. A link between nitrogen metabolism and peptidoglycan (PG) biosynthesis was underlined by the finding that deletion of the dipeptidase gene pepV (creating strain MGΔpepV) resulted in a prolonged lag phase when the mutant strain was grown with a high concentration of glycine. In addition, most MGΔpepV cells lyse and have serious defects in their shape. This phenotype is due to a shortage of alanine, since adding alanine can rescue the growth and shape defects. Strain MGΔpepV is more resistant to vancomycin, an antibiotic targeting peptidoglycan d-Ala-d-Ala ends, which confirmed that MGΔpepV has an abnormal PG composition. A mutant of MGΔpepV was obtained in which growth inhibition and cell shape defects were alleviated. Genome sequencing showed that this mutant has a single point mutation in the codY gene, resulting in an arginine residue at position 218 in the DNA-binding motif of CodY being replaced by a cysteine residue. Thus, this strain was named MGΔpepVcodYR218C Transcriptome sequencing (RNA-seq) data revealed a dramatic derepression in peptide uptake and amino acid utilization in MGΔpepVcodYR218C A model of the connections among PepV activity, CodY regulation, and PG synthesis of L. lactis is proposed.IMPORTANCE Precise control of peptidoglycan synthesis is essential in Gram-positive bacteria for maintaining cell shape and integrity as well as resisting stresses. Although neither the dipeptidase PepV nor alanine is essential for L. lactis MG1363, adequate availability of either ensures proper cell wall synthesis. We broaden the knowledge about the dipeptidase PepV, which acts as a linker between nitrogen metabolism and cell wall synthesis in L. lactis.

Dataset Information

Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons.

Publications

Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons.

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