Determinants of murein hydrolase targeting to cross-wall of Staphylococcus aureus peptidoglycan

Cells of eukaryotic or prokaryotic origin express proteins with LysM domains that associate with the cell wall envelope of bacteria. The molecular properties that enable LysM domains to interact with microbial cell walls are not yet established. Staphylococcus aureus, a spherical microbe, secretes two murein hydrolases with LysM domains, Sle1 and LytN. We show here that the LysM domains of Sle1 and LytN direct murein hydrolases to the staphylococcal envelope in the vicinity of the cross-wall, the mid-cell compartment for peptidoglycan synthesis. LysM domains associate with the repeating disaccharide β-N-acetylmuramic acid, (1→4)-β-N-acetylglucosamine of staphylococcal peptidoglycan. Modification of N-acetylmuramic acid with wall teichoic acid, a ribitol-phosphate polymer tethered to murein linkage units, prevents the LysM domain from binding to peptidoglycan. The localization of LytN and Sle1 to the cross-wall is abolished in staphylococcal tagO mutants, which are defective for wall teichoic acid synthesis. We propose a model whereby the LysM domain ensures septal localization of LytN and Sle1 followed by processive cleavage of peptidoglycan, thereby exposing new LysM binding sites in the cross-wall and separating bacterial cells.     

pH driven conformational dynamics and dimer-to-monomer transition in DLC8

Dynein light chain protein, a part of the cytoplasmic motor assembly, is a homodimer at physiological pH and dissociates below pH 4.5 to a monomer. The dimer binds to a variety of cargo, whereas the monomer does not bind any of the target proteins. We report here the pH induced stepwise structural and motional changes in the protein, as derived from line broadening and 15N transverse relaxation measurements. At pH 7 and below until 5, partial protonation and consequent interconversion between molecules carrying protonated and neutral histidines, causes conformational dynamics in the dimeric protein and this increases with decreasing pH. Enhanced dynamics in turn leads to partial loosening of the structure. This would have implications for different efficacies of binding by target proteins due to small variations in pH in different parts of the cell, and hence for cargo trafficking from one part to another. Below pH 5, enhanced charge repulsions, partial loss of hydrophobic interactions, and destabilization of H-bonds across the dimer interface cause further loosening of the dimeric structure, leading eventually to the dissociation of the dimer.     

The crystal structure of a family GH25 lysozyme from Bacillus anthracis implies a neighboring-group catalytic mechanism with retention of anomeric configuration

Lysozymes are found in many of the sequence-based families of glycoside hydrolases (www.cazy.org) where they show considerable structural and mechanistic diversity. Lysozymes from glycoside hydrolase family GH25 adopt a (α/β)₅(β)₃-barrel-like fold with a proposal in the literature that these enzymes act with inversion of anomeric configuration; the lack of a suitable substrate, however, means that no group has successfully demonstrated the configuration of the product. Here we report the 3-D structure of the GH25 enzyme from Bacillus anthracis at 1.4 Å resolution. We show that the active center is extremely similar to those from glycoside hydrolase families GH18, GH20, GH56, GH84, and GH85 implying that, in the absence of evidence to the contrary, GH25 enzymes also act with net retention of anomeric configuration using the neighboring-group catalytic mechanism that is common to this ‘super-family’ of enzymes.     

Porphyromonas gingivalis Peptidoglycans Induce Excessive Activation of the Innate Immune System in Silkworm Larvae

Porphyromonas gingivalis, a pathogen that causes inflammation in human periodontal tissue, killed silkworm (Bombyx mori, Lepidoptera) larvae when injected into the blood (hemolymph). Silkworm lethality was not rescued by antibiotic treatment, and heat-killed bacteria were also lethal. Heat-killed bacteria of mutant P. gingivalis strains lacking virulence factors also killed silkworms. Silkworms died after injection of peptidoglycans purified from P. gingivalis (pPG), and pPG toxicity was blocked by treatment with mutanolysin, a peptidoglycan-degrading enzyme. pPG induced silkworm hemolymph melanization at the same dose as that required to kill the animal. pPG injection increased caspase activity in silkworm tissues. pPG-induced silkworm death was delayed by injecting melanization-inhibiting reagents (a serine protease inhibitor and 1-phenyl-2-thiourea), antioxidants (N-acetyl-l-cysteine, glutathione, and catalase), and a caspase inhibitor (Ac-DEVD-CHO). Thus, pPG induces excessive activation of the innate immune response, which leads to the generation of reactive oxygen species and apoptotic cell death in the host tissue.     

Extragenic suppression of the requirement for diaminopimelate in diaminopimelate auxotrophs of Mycobacterium smegmatis

Mycobacteria, like many prokaryotes, have a peptidoglycan with peptides composed of L-alanine (or glycine), D-iso-glutamine, meso-diaminopimelate, and D-alanine. We sought to study mycobacterial peptidoglycan biosynthesis by constructing diaminopimelate (DAP) auxotrophs of Mycobacterium smegmatis and then isolating spontaneous mutants of these auxotrophs that can grow in the absence of DAP. Here we report the isolation and characterization of seven classes of spontaneous M. smegmatis mutants with extragenic mutations that can suppress the DAP requirement of DAP auxotrophs.     

The Evolution of Parasite Recognition Genes in the Innate Immune System: Purifying Selection on <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Peptidoglycan Recognition Proteins

Genes involved in the recognition of parasites by the acquired immune system are often subject to intense selection pressures. In some cases, selection to recognize a diverse range of parasites has resulted in high levels of polymorphism, while elsewhere the protein sequence has changed rapidly under directional selection. We tested whether parasite recognition genes in the innate immune system show similar patterns of evolution. We sequenced seven peptidoglycan recognition protein genes (PGRPs) from 12 lines of Drosophila melanogaster and one line of D. simulans and used a variety of tests to determine whether the observed mutations were selectively neutral. We were unable to detect either balancing or directional selection. This suggests that the molecular cues used by insects to detect parasites are highly conserved and probably under strong functional constraints which prevent their evolving to evade the host immune response. Therefore, interactions between these genes are unlikely to be the focus of host–parasite coevolution, at least in Drosophila. We also found evidence of gene conversion occurring between two genes, PGRP-SC1A and PGRP-SC1B.     

Regiospecific alkaline protease-catalyzed divinyl acyl transesterifications of primary hydroxyl groups of mono- and di-saccharides in pyridine

This paper describes highly selective transesterification reactions, catalyzed by an alkaline protease from Bacillus subtilis in pyridine, of several mono- and di-saccharides with divinyl dicarboxylates ranging from 4 to 10 carbon atoms. A series of polymerizable vinyl fatty acid sugar esters were obtained with good selectivity and high yields. Most products had high proportions of the alpha anomer. The influences of the enzymes, solvents, temperature, and acyl donor chain length on the reaction were studied. Vinyl sugar esters offer a new family of functional water-soluble monomers for preparation of sugar-containing polymers.     

Stable monomeric intermediate with exposed Cys-119 is formed during heat denaturation of beta-lactoglobulin

The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented.