Detection of Penicillin-binding Proteins in the Endosymbiont of the Trypanosomatid Crithidia deanei

ABSTRACT. Growth by serial transfers of the trypanosomatid Crithidia deanei in culture medium containing 1 mg/ml of the β-lactam antibiotics ampicillin or cephalexin resulted in shape distortion of its endosymbiont. The endosymbiont first appeared as filamentous structures with restricted areas of membrane damage. An increase of electron lucid areas was also observed in the endosymbiont matrix. The continuous treatment with β-lactam antibiotics, resulted in endosymbiont membranes fragmentation; and later on the space previously occupied by the symbiont was identified as an electron lucid area in the host cytoplasm. The putative targets of β-lactam antibiotic were two membrane-bound penicillin-binding proteins (PBPs) detected in the Sarkosyl-soluble fraction of purified symbionts labeled with [³H]-benzylpenicillin. The apparent molecular weight of the proteins were 90 kDa (PBP1) and 45 kDa (PBP2). PBP2 represented 85% of the total PBP content in the membrane fraction of the endosymbionts. Competition experiments using the tested antibiotics and [³H]-benzylpenicillin showed that ampicillin and cephalexin have half saturating concentrations considerably higher than [³H]-benzylpenicillin and indicated that PBP1 is the probable lethal target of the antibiotics tested. These results suggest that a physiologically active PBP is present in the cell envelope of C. deanei endosymbionts and may play important roles in the control of processes such as cell division and shape determination.     

Mechanisms of nodule-specific melanization in the hemocoel of the silkworm,Bombyx mori

In the insect immune system, nodules are known to be a product of the cellular response against microorganisms and may be a preferential target for melanization. However, the mechanism of nodule-preferential melanization remains to be explored. In this study, we identified several mechanisms of nodule-preferential melanization by analyzing congregation and the activation of several factors involved in the prophenoloxidase (proPO)-activating system in the silkworm, Bombyx mori. Microorganism-binding assays revealed that B. mori larval plasma have an effective invading microorganism-surveillance network consisting of at least six pattern-recognition receptors (PRRs). We also found that a hemolymph serine proteinase, BmHP14, can bind to Saccharomyces cerevisiae. Pull-down assays showed that PRR C-type lectins form protein complexes with serine proteinase homologs, BmSPH1 and BmSPH2, which leads to the activated forms of BmSPH1 and BmSPH2 being gathered on microorganisms and trapped in nodules. Immunostaining analysis revealed that most factors in the proPO-activating system and some factors in the triggering system for antimicrobial peptide production exist in the granules of hemocytes which can gather in nodules. Western blot analysis showed that factors in the proPO-activating system are congregated in formed nodules by their concentration in plasma and aggregating hemocytes.     

Fungal invasion of epithelial cells

Interaction between host cells and invasive Candida plays a large role in the pathogenicity of Candida species. Fungal-induced endocytosis and active penetration are the two distinct, yet complementary invasion mechanisms of invasive candidiasis. Induced endocytosis is a microorganism-triggered, epithelial-driven, clathrin-mediated and actin-dependent process. During the fundamental pathological process of induced endocytosis, invasins (Als3 and Ssa1), which mediate the binding of host epithelial surface proteins, are expressed by Candida species on the hyphal surface. Sequentially, the interaction between invasins and host epithelial surface proteins stimulates the recruitment of clathrin, dynamin and cortactin to the sites where Candida enters epithelial cells, which in turn induce the actin cytoskeleton reorganization. Actin cytoskeleton provides the force required for fungal internalization. Parallely, active penetration of Candida can directly pass through epithelial cells possibly due to progressive elongation of hyphae and physical forces. Several molecules, such as secreted hydrolases and Als3, can affect the protective barrier of the epithelium and make Candida actively penetrate into epithelial cells through intercellular gaps of epithelial layers.     

The enzymic degradation of lipids resulting from physical disruption of cucumber (Cucumis sativus) fruit

Homogenization of fresh tissue from cucumber fruits results in a loss of endogenous lipid catalysed by acyl hydrolase enzymes. Deacylation of lipids is not accompanied by accumulation of free fatty acids. The levels of both saturated (mainly palmitic) and polyunsaturated (linoleic and linolenic) fatty acids in the lipids are reduced. Losses of the major acyl lipid constituents of cucumber (triacylglycerols and phospholipids) are mainly responsible for the observed hydrolysis. Triacylglycerol acyl hydrolase (lipase), phospholipase D and polar lipid acyl hydrolase enzyme activities were demonstrated. It is suggested that hydrolytic attack on endogenous lipids is the initial event on disruption of cucumber tissue, in the formation of lipid degradation products, amongst which are the volatile carbonyl compounds responsible for the characteristic flavour of cucumber.     

Degradation of soluble collagen by ozone or hydroxyl radicals

Collagen exposed to ozone or hydroxyl radicals was degraded in a time- and dose-dependent manner. This degradation was inhibited by free radical scavengers. Furthermore, lower levels of these oxidants did not degrade the molecule, but caused it to become susceptible to proteolytic degradation. We suggest an alternative mechanism by which oxygen-derived free radicals participate in the destruction of extracellular matrix observed during acute lung injury by oxidant gas, in addition to the commonly accepted proteinase-antiproteinase theory of lung injury.     

A novel type of meso-diaminopimelic acid-based peptidoglycan and novel poly (erythritol phosphate) teichoic acids in cell walls of two coryneform isolates from the surface flora of French cooked cheeses

The primary structure of the peptidoglycan and the teichoic acids of two coryneform isolates from the surface flora of French cooked cheeses, CNRZ 925 and CNRZ 926, have been determined. In the peptidoglycan, meso-diaminopimelic acid was localized in position three of the peptide subunit. It contained an d-glutamyl-d-aspartyl interpeptide bridge, connecting meso-diaminopimelic acid and d-alanine residues of adjacent peptide subunits. The -carboxyl group of d-glutamic acid in position two of peptide subunits was substituted with glycine amide. The teichoic acid pattern and composition differed between the strains: both contained an erythritol teichoic acid and strain CNRZ 925 also contained an N-acetylglucosaminylphosphate polymer. The erythritol teichoic acids differed in terms of the quality and quantity of substituents, but they both had N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid in common.Abbreviations DNP dinitrophenyl - Ery erythritol - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - GlcUANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid - Hex UANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyhexuronic - acid m-Dpm, meso-diaminopimelic acid - Mur muramic acid - MurNAc N-acetylmuramic acid     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96?hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral–alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.