Structure of the cell wall of Lactobacilli. Role of muramic acid phosphate in Lactobacillus fermenti.
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ABSTRACT:
SUBMITTER: Knox KW
PROVIDER: S-EPMC1198820 | biostudies-other | 1968 Jul
REPOSITORIES: biostudies-other
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Project description:The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.
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Project description:BACKGROUND: Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background. RESULTS: Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-?B reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-? after stimulation with the WTA mutants as compared to the wild-type. CONCLUSIONS: The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality.
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Project description:The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72?Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical ? and ? D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.
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Project description:To be able to function as a probiotic, bacteria have to survive the passage through the gastrointestinal tract. We have examined survival and gene expression of Lactobacillus reuteri ATCC 55730 after a sudden shift in environmental acidity to a pH close to the conditions in the human stomach. More than 80% of the L. reuteri cells survived at pH 2.7 for 1 h. A genomewide expression analysis experiment using microarrays displayed 72 differentially expressed genes at this pH. The early response to severe acid shock in L. reuteri differed from long-term acid adaptation to milder acid stress studied in other lactic acid bacteria. The genes induced included the following: clpL, genes putatively involved in alterations of the cell membrane and the cell wall; genes encoding transcriptional regulators; phage genes; and genes of unknown function. Two genes, clpL, encoding an ATPase with chaperone activity, and lr1516, encoding a putative esterase, were selected for mutation analyses. The mutants were significantly more sensitive to acid than the wild type was. Thus, these genes could contribute to the survival of L. reuteri in the gastrointestinal tract.
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Project description:Lactobacillus helveticus strains possess an efficient proteolytic system that releases peptides which are essential for lactobacillus growth in various fermented dairy products and also affect textural properties or biological activities. Cell envelope proteinases (CEPs) are bacterial enzymes that hydrolyze milk proteins. In the case of L. helveticus, two CEPs with low percentages of amino acid identity have been described, i.e., PrtH and PrtH2. However, the distribution of the genes that encode CEPs still remains unclear, rendering it difficult to further control the formation of particular peptides. This study evaluated the diversity of genes that encode CEPs in a collection of strains of L. helveticus isolated from various biotopes, both in terms of the presence or absence of these genes and in terms of nucleotide sequence, and studied their transcription in dairy matrices. After defining three sets of primers for both the prtH and prtH2 genes, we studied the distribution of the genes by using PCR and Southern blotting experiments. The prtH2 gene was ubiquitous in the 29 strains of L. helveticus studied, whereas only 18 of them also exhibited the prtH gene. Sequencing of a 350-bp internal fragment of these genes revealed the existence of intraspecific diversity. Finally, expression of these two CEP-encoding genes was followed during the growth in dairy matrices of two strains, ITG LH77 and CNRZ32, which possess one and two CEP-encoding genes, respectively. Both genes were shown to be expressed by L. helveticus at each stage of growth in milk and at different stages of mini-Swiss-type cheese making and ripening.
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Project description:1. The polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei have been separated by mild conditions of acid hydrolysis. 2. Removal of the polysaccharide renders the mucopeptide susceptible to lysozyme. 3. The mucopeptide and polysaccharide components have been analysed and the results compared with those obtained previously. 4. The polysaccharides responsible for group specificity have a terminal reducing N-acetylgalactosamine residue substituted on C((3)) by the adjacent sugar; estimation of this component gave an indication of the molecular weight of the polysaccharides. 5. Evidence has been obtained for the presence of rhamnosyl-(1-->3)-N-acetylgalactosamine among the products of acid hydrolysis of the group B polysaccharide.
| S-EPMC1207041 | biostudies-other
Project description:The cell wall of most Gram-positive bacteria contains equal amounts of peptidoglycan and the phosphate-rich glycopolymer wall teichoic acid (WTA). During phosphate-limited growth of the Gram-positive model organism Bacillus subtilis 168, WTA is lost from the cell wall in a response mediated by the PhoPR two-component system, which regulates genes involved in phosphate conservation and acquisition. It has been thought that WTA provides a phosphate source to sustain growth during starvation conditions; however, WTA degradative pathways have not been described for this or any condition of bacterial growth. Here, we uncover roles for the Bacillus subtilis PhoP regulon genes glpQ and phoD as encoding secreted phosphodiesterases that function in WTA metabolism during phosphate starvation. Unlike the parent 168 strain, ?glpQ or ?phoD mutants retained WTA and ceased growth upon phosphate limitation. Characterization of GlpQ and PhoD enzymatic activities, in addition to X-ray crystal structures of GlpQ, revealed distinct mechanisms of WTA depolymerization for the two enzymes; GlpQ catalyzes exolytic cleavage of individual monomer units, and PhoD catalyzes endo-hydrolysis at nonspecific sites throughout the polymer. The combination of these activities appears requisite for the utilization of WTA as a phosphate reserve. Phenotypic characterization of the ?glpQ and ?phoD mutants revealed altered cell morphologies and effects on autolytic activity and antibiotic susceptibilities that, unexpectedly, also occurred in phosphate-replete conditions. Our findings offer novel insight into the B. subtilis phosphate starvation response and implicate WTA hydrolase activity as a determinant of functional properties of the Gram-positive cell envelope.
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Project description:KD is an acute febrile illness and systemic vasculitis of unknown etiology among young children, which can cause coronary artery abnormalities and aneurysms (CAA) and is the leading cause of acquired heart disease among children in the US. Lactobacillus casei cell wall extract (LCWE) induces in mice a vasculitis following intraperitoneal injection defined by the activation of macrophages, dendritic cells and CD8+ cytotoxic T cells leading to aortitis, coronary arteritis, aneurysms and myocarditis that strongly mimic the immunopathology and the cardiac lesions observed in children with Kawasaki disease (KD). To address a potential pathogenic role of LCWE-specific T cells in human vascular inflammation, we studied the activation of circulating CD4+ and CD8+ T cells ex vivo in response to LCWE in 3 cohorts: (1) KD children 2-3 weeks after fever onset, (2) age-similar healthy children controls, (3) healthy adult controls. In all subjects studied, pro-inflammatory CD4+ and CD8+T cells responded to LCWE with no significant differences. Peripherally-induced regulatory T cells (iTreg) also responded to LCWE and potentially reverted to Th17, as suggested by the detection of IL-17 in culture supernatants. Central memory T cells were also detectable and were more abundant in adults. The potential homing to the vessels of LCWE-specific T cells was suggested by the expression of CCR6 and CD31. In conclusion, a non-pathogenic, LCWE-specific T cell repertoire could lead to KD depending upon priming conditions, genetic factors and immune activation by other antigens.
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