Kareen J. I. Thorne

The metabolism of acetate and mevalonic acid by lactobacilli IV. Analysis of the fatty acids by gas-liquid chromatography

Abstract The fatty acids of Lactobacillus casei, Lactobacillus arabinosus and Lactobacillus acidophilus were analysed by gas-liquid chromatography using columns with polyethylene glycol adipate or Apiezon L grease as stationary phase. Two groups of acids were analysed, those prepared by saponification and those prepared by acid hydrolysis. Acid hydrolysis was necessary to obtain the total fatty acids but it resulted in the destruction of lactobacillic acid. In addition to the whole cells, the fatty acids from the bacterial membranes of L. casei , prepared by differential centrifugation, were examined and compared with those from the membranes of Micrococcus lysodeikticus and Bacillus megaterium .

Phagocytosis and killing of Trypanosoma dionisii by human neutrophils, eosinophils and monocytes.

The cell-mediated resistance of human leucocytes to Trypanosoma dionisii, a bat parasite related to T. cruzi, was investigated. Human peripheral blood neutrophils and monocytes were cytotoxic to T. dionisii as assessed by electron microscopy and by induction of 99mTc release from trypanosomes pre-labelled with [99mTc] pertechnetate. The enhancement of cytotoxicity by specific antiserum varied considerably from one individual to another. Neither blood lymphocytes nor blood eosinophils induced 99mTc release from T. dionisii. The trypanosomes were readily phagocytosed by neutrophils and monocytes even in the absence of added antiserum but the rate was enchanced when antiserum was present. Eosinophils also phagocytosed T. dionisii but only in the presence of antiserum. Investigation by electron microscopy revealed that T. dionisii is rapidly destroyed in the phagocytic vacuole of enutrophils and monocytes and by eosinophils. Phagocytosis, ultrastructural damage and induction of 99mTc release occurred more rapidly in neutrophils than in monocytes.

The metabolism of acetate and mevalonic acid by lactobacilli I. The effect of acetate and mevalonic acid on growth

Abstract The requirement of three species of lactobacillus for acetate and its replacement by mevalonic acid was investigated. It was found that, in the presence of 1% glucose, Lactobacillus acidophilus had a requirement for sodium acetate which could be replaced by 1/8000 times its concentration of mevalonic acid, but when the glucose concentration was 2%, acetate was no longer required. Lactobacillus arabinosus also required acetate, but mevalonic acid replaced only partially the acetate requirement. Acetate was not required, under the present experimental conditions for Lactobacillus casei . The acetate requirement of L. acidophilus could also be replaced by β-hydroxy-β-methylglutaric acid and by 2,2′-dimethylacrylic acid, but not by farnesol or geraniol.

The nature of the attachment of a regularly arranged surface protein to the outer membrane of an Acinetobacter sp

Acinetobacter 199A carries on the outer surface of its outer membrane a layer of regularly arranged protein subunits. The isolated surface protein assembles into the same regular array even in the absence of the underlying outer membrane. Cl- minus is required for this self-assembly. Evidence is presented that the interaction of the surface protein with the outer membrane involves the linking of a carboxyl group in the surface protein to a negatively charged group in the outer membrane protein, via a divalent cation. The surface protein could be detached from the outer membrane by the protein perturbant urea, by the chelating agent EDTA and by replacing Mg-2+ with Na+. It could not be detached by treatment with phospholipases A anc D or the detergents Tween 80 and sodium deoxycholate. The conditions favourable for reattachment of surface protein to the cell wall were the presence of divalent cations and a pH of 3-5. Conversion of carboxyl groups in the surface protein to amine with carbodiimide and ethylene diamine interfered with reattachment. The surface protein did not attach to isolated cell wall lipid or lipopolysaccharide.

Evasion of the oxidative microbicidal activity of human monocytes by trypomastigotes of Trypanosoma dionisii

Trypomastigotes of Trypanosoma dionisii, a stercorarian trypanosome from bats, are effectively killed by neutrophils from human peripheral blood but are less sensitive to the cytotoxic action of human monocytes. The mechanism of killing appears to involve peroxidase and hydrogen peroxide. Trypomastigotes are as effective as epimastigotes in inducing the formation of hydrogen peroxide by effector cells. They are, however, less sensitive than epimastigotes to the cytotoxic effect of peroxidase and hydrogen peroxide. They are therefore susceptible to the high concentrations of peroxidase found in the phagosome of the neutrophil, but resist the lower levels encountered in monocytes.

Phospholipase A2 activity of the regularly arranged surface protein of Acinetobacter sp. 199A

The regularly arranged surface protein, the a-protein, of Acinetobacter 199A has been shown to have phospholipase A2 activity. Since half of the a-protein synthesised by Acinetobacter 199A is secreted into the growth medium, the bacteria are producing extracellular phospholipase A2.

The metabolism of acetate and mevalonic acid by lactobacilli II. The incorporation of [14C]acetate and [14C]mevalonic acid into the bacterial lipids

Abstract Using [ 14 C]acetate and [ 14 C]mevalonic acid, it was shown that Lactobacillus casei, Lactibacillus arabinosus and Lactobacillus acidophilus incorporate from 20–35 times as much sodium acetate as mevalonic acid, the cellular concentration of mevalonic acid being from 0.5–0.9 μg/mg dry weight. Most of the radioactive material was found in the lipid fraction, probably bound to protein. Acetate was shown to be a precursor of both fatty acids and unsaponifiable lipids, mevalonic acid gave only unsaponifiable material. Part of the fatty acids could only be liberated from the whole lipids by acid hydrolysis and was not hydrolysed under alkaline conditions. Analysis of the unsaponifiable material by reversed-phase paper chromatography showed the presence of at least six components. The chemical nature of these substances was studied. Since mevalonic acid did not yield fatty acids, [ 4 C]glucose in absence of acetate was shown to be a precursor of the bacterial fatty acids. Biotin was found to be necessary for fatty acid synthesis from acetate but not for the synthesis of the unsaponifiable lipids from mevalonic acid.

The metabolism of acetate and mevalonic acid by lactobacilli III. Studies on the unsaponifiable lipids derived from mevalonic acid

Abstract In an investigation of the synthesis of the unsaponifiable lipids of Lactobacillus casei from mevalonic acid, it was shown that although no preferential order of synthesis of the components could be detected there was a turnover of the principal one with a simultaneous increase in the others. The presence of glucose and of all the amino acids was found to be necessary for the incorporation of mevalonic acid by L. casei and L. arabinosus . Since, in addition, the incorporation of mevalonic acid was inhibited by chloramphenicol it appears that protein synthesis is necessary for the uptake of mevalonic acid. This requirement for protein synthesis could not be explained by the necessity for inducing an enzyme before mevalonic acid could be utilised by the bacteria. Of other inhibitors tested, only p -chloromercuribenzoate completely inhibited the incorporation of mevalonic acid. Analysis of the cell fractions, prepared by differential centrifugation, suggested that the lipid from mevalonic acid is located in the soluble fraction of the cell. Only a trace of this lipid was found in the protoplast membrane of Micrococcus lysodeikticus . The unsaponifiable lipids could not replace mevalonic acid or acetate in the growth of L. acidophilus and they were not incorporated into the cell lipids by L. casei, L. arabinosus or L. acidophilus . The effect of mevalonic acid deficiency on the incorporation of various radioactive biochemicals was investigated and glucose uptake was found to be significantly reduced. A smaller reduction was found in the rate of acid production.

Bacitracin-induced changes in bacterial plasma membrane structure.

Abstract Bacitracin interacts with the plasma membranes of gram-positive and gram-negative bacteria to produce morphological changes which appear as rods, 25–35 nm in diameter, and of variable length, in freeze fractured preparations.

The interaction of human eosinophils and neutrophils with non-phagocytosable surfaces: a model for studying cell-mediated immunity in schistosomiasis

A model has been developed to simulate the surface of an antibody-coated schistosomulum. It consists of a layer of agar, containing antigen (tetanus toxoid) and a chemotactic factor (ECF). Some layers were coated with human anti-tetanus immunoglobulin. The mode of adherence of human eosinophils and neutrophils to these agar layers and the subsequent degranulation of the cells exactly paralleled the interaction of these cell types with antibody-coated schistosomula of Schistosoma mansoni. In particular, eosinophils made much more intimate contact than did neutrophils, and lysosomal enzymes were secreted extracellularly by direct fusion of granules with the plasma membrane of the cell. Biochemical evidence was also obtained for the secretion of enzymes during degranulation and the rate of enzyme release was found to be enhanced in the presence of specific antibody. This model, non-phagocytosable surface has the potential to provide basic information of the mode of action of effector cells in cell-mediated cytotoxic reactions against a wide range of parasites by incorporation of different factors into the agar layers.