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 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.

Cell-mediated killing of protozoa.

Cell-mediated immunity represents an important host defence mechanism against protozoal infections. The effector cells directly involved are neutrophils, macrophages and, ultimately, activated macrophages. Within this simple scheme there are, however, considerable variations in activity. Effector cells from different animal species, and even from different strains of the same species, may be more or less effective in controlling a certain protozoal infection. Different protozoa differ in their susceptibility to cell-mediated killing according to genus, species, strain and morphological form. The most susceptible morphological form is that which occurs in the insect vector, and which has not yet adapted to protect itself from the vertebrate host. Epimastigotes of Trypanosoma and promastigotes of Leishmania are readily killed by phagocytic cells, while the corresponding trypomastigote and amastigote forms are considerably more resistant. Protozoa which live in macrophages, such as amastigotes of Leishmania, endozoites (tachyzoites) of Toxoplasma and amastigotes of reticulotropic strains of T. cruzi, have developed a remarkable resistance to the microbicidal activity of the host cell. Conversely, amastigotes of myotropic strains of T. cruzi, which live in muscle cells, have not developed this resistance to cell-mediated killing by macrophages. Readily accessible protozoa, such as T. brucei trypomastigotes and Plasmodium merozoites in the bloodstream, while they lack the marked resistance developed by reticulotropic protozoa, have a partial protection since they are attacked by phagocytic cells only when specific antibody is present. Granulocyte-mediated killing can be largely attributed to neutrophils. Eosinophils appear to play only a minor role and compete ineffectually when neutrophils are also present. The only group of protozoal species which may be significantly controlled by eosinophils are the stercorarian species of Trypanosoma. In vitro experiments show that antibody-coated trypomastigotes of T. cruzi can be killed by eosinophils, although there is little evidence that this occurs in vivo. Interestingly, this is the only species that has been reported to be susceptible to the major basic protein of eosinophils, a toxic component of the lysosomal granules which is very active against helminths. Neutrophils are not very active against endozoites of Toxoplasma gondii, Trypanosoma, trypomastigotes of salivarian Trypanosoma, free merozoites of Plasmodium, and promastigotes and amastigotes of Leishmania.(ABSTRACT TRUNCATED AT 400 WORDS)

A new method for measuring eosinophil activating factors, based on the increased expression of CR3 alpha chain (CD11b) on the surface of activated eosinophils.

The observation that activation of eosinophils in vitro with PAF increases the surface expression of the alpha chain of the complement receptor CR3 (CD11b) has been extended to other eosinophil activating factors. CD11b may be detected on activated eosinophils by reaction with mouse monoclonal anti-human CD11b IgG, following the addition of urease-conjugated sheep anti-mouse IgG. CD11b levels were increased on eosinophils after incubation with (a) recombinant colony stimulating factors, IL-3, GM-CSF and IL-5, at concentrations of 100 U/ml, or (b) with eosinophil activating factors, recombinant TNF alpha (1000 U/ml), EAF purified from mononuclear cell supernatants and PAF (10(-6) M). CD11b levels were not affected by IL-1 alpha, IL-2 or IFN-gamma. Unstimulated neutrophils had higher levels of CD11b than unstimulated eosinophils, but neutrophil CD11b was unaffected by IL-3, GM-CSF and IL-5 and was only slightly affected by TNF, EAF and PAF. Polyclonal rabbit antibodies to IL-3 and TNF neutralised their CD11b enhancing activities. The PAF antagonists WEB 2086 and WEB 2170 neutralised the CD11b enhancing activity of PAF. We conclude that measurement of CD11b expression on eosinophils is a convenient method for the assay of eosinophil activating activity.

Inhibition of phagocytosis of Trypanosome dionisii by pregnancy alpha-2 glycoprotein.

Trypanosome phagocytosis by normal polymorphonuclear leucocytes was studied in the presence of the pregnancy-associated alpha 2-glycoprotein (PAG). A serum fraction containing PAG at a concentration of 13.5 micrograms/ml significantly inhibited the uptake of Trypanosoma dionisii when contrasted with an identically obtained male serum fraction devoid of PAG (P less than .01). This finding of diminished trypanosome uptake in physiologic concentrations of PAG could account for the suggested increase in the frequency and severity of protozoal infections during gestation.

Identification of prenol intermediates of wall biosynthesis in growing cells of Lactobacillus plantarum.

The incorporation of (14)C-mevalonic acid by Lactobacillus plantarum predominantly into C(55) prenol made it possible to determine the distribution of (14)C-prenol between all its derivatives. In logarithmic-phase cells, 25% of the prenol was free, 31% was as monophosphate, 4% as pyrophosphate, 12% as peptidoglycan precursor, and 28% as glyco-phospho-prenol. The glyco-phospho-prenol contained rhamnose, and probably glucose, galactose, and ribitol phosphate, and it may, therefore, be involved in polysaccharide and teichoic acid biosynthesis. The proportion of free prenol increased, up to 73%, as the cell culture aged. Free prenol was also formed when cells were incubated in buffer. The free prenol was readily reutilized when cells were returned to growth medium.

Incorporation of radioactive mevalonate into C50 and C55 phenols by Streptococcus mutans.

Growing cells of Streptococcus mutans Ingbritt incorporate radioactive mevalonate into unsaponifiable lipid. Of the radioactive lipid 40% was shown by chromatography and mass spectrometry to be C(50) and C(55) prenol.

Synthesis of radioactive dolichol from (4S-3H)mevalonate in the regenerating rat liver.

The dolichol of rat liver was labelled by injecting [4S-(3)H]mevalonate, the precursor of cis-isoprene residues, into partially hepatectomized animals. The optimum conditions for labelling the dolichol were to inject the animals with radioactive mevalonate 48h after hepatectomy and to kill them 12h later. The concentration of radioactive dolichol was five times as great in regenerating rat liver as in normal liver. The highest concentration of radioactive dolichol was found in the crude mitochondrial and nuclear-debris fractions of the cell. The crude microsomal fractions also contained radioactive dolichol, but at a lower concentration.

The involvement of endogenous dolichol in the formation of lipid-linked precursors of glycoprotein in rat liver.

Endogenous dolichol was shown to function as a natural acceptor of mannose residues by using regenerating rat liver containing [(3)H]dolichol. When subcellular fractions from this liver were incubated with GDP-[(14)C]mannose a double-labelled lipid, which represented 30% of the total [(14)C]mannolipid, could be isolated. This lipid was shown to be identical with the dolichol phosphate mannose formed from exogenous dolichol phosphate, by chromatography, stability to alkali and by chemical cleavage to mannose and dolichol derivatives. It was formed by the rough endoplasmic reticulum and mitochondria. If it is concerned in glycoprotein synthesis this would suggest that it functions in the formation of both secreted and mitochondrial glycoproteins. When both the dolichol and retinol of rat tissue were radioactive they made similar contributions to the synthesis of the lipid by liver microsomal fractions and intestinal epithelial cells.

The structure of bactoprenol, a lipid formed by lactobacilli from mevalonic acid.

1. The name ;bactoprenol' has been given to the most abundant lipid formed by three species of lactobacilli from mevalonic acid. 2. A method for the preparation of pure bactoprenol is described. 3. The thin-layer chromatographic properties of bactoprenol and of its acetylated and hydrogenated derivatives resembled those of dolichol. 4. Analysis by mass spectrometry and by nuclear magnetic resonance showed that the molecule is formed by condensation of 10 unsaturated isoprene units and 1 saturated isoprene unit. 5. Its molecular weight is 768 and it has 10 double bonds/molecule. 6. Infrared spectroscopy and the uptake of acetyl groups indicated that the molecule contains a hydroxyl group. 7. It is concluded that bactoprenol is a C(55) isoprenoid alcohol.

Chemical characterization of the regularly arranged surface layers of Clostridium thermosaccharolyticum and Clostridium thermohydrosulfuricum.

Clostridum thermosaccharolyticum and Clostridium thermohydrosulfuricum possess as outermost cell wall layer a tetragonal or hexagonal ordered array of macromolecules. The subunits of the surface layer can be detached from isolated cell walls with urea (8M) or guanidine-HCl (4 to 5 M). Triton X-100, dithiothreitol, ethylenediaminetetracetate, and KCl (3 M) had no visible effect on the regular arrays. Sodium dodecyl sulfate-polyacrylamide electrophroesis showed that, in both organisms, the surface layer is composed of glycoprotein of molecular weight 140,000. The glycoprotein from both microorganisms has a predominantly acidic amino acid composition and an acidic isoelectric point after isoelectric focusing on polyacrylamide gels. The glycocomponent is composed of glucose, galactose, mannose, and rhamnose.

Importance of oxidative metabolism in T cell cytotoxicity: a comparison of cloned T cells and spleen cells.

To distinguish between direct and indirect involvement of oxygen metabolites in CTL cytotoxicity a comparison was made of cloned CTLs and mixed cells from mouse spleen. Tumour cells could be protected from cloned CTLs and from spleen CTLs by the thiol protecting reducing agents DTT and DETC. Cytotoxic activity was inhibited by diversion of reducing power from NAD(P)H to artificial electron acceptors and by the inhibitor of NAD(P) linked enzymes cibacron blue. Although H2O2 formation could be detected during the lysis of P815 by spleen CTLs it did not prove to be a necessary requirement for cytolysis since it was not formed when glutaraldehyde-treated P815 cells were lysed. Of the scavengers of toxic oxygen metabolites tested only the hydroxyl radical scavenger sodium benzoate inhibited cytotoxicity.

Surface proteins of the human eosinophil. I. Isolation of eosinophil IgG binding proteins.

Proteins involved in the attachment of eosinophils to immobilized antigen-antibody complexes were isolated. Eosinophils, purified from normal human peripheral blood, were surface labelled with 125I-iodide in the presence of lactoperoxidase and hydrogen peroxide. Immobilized immune complexes were prepared by covalent coupling of tetanus toxoid antigen to cellulose and treatment of the fixed antigen with human anti-tetanus IgG. Intact cells were allowed to interact with the antigen-antibody complex and the cells were then lysed in situ with a salt solution containing the non-ionic detergent NP40. After exhaustive washing to remove unattached proteins, the bound proteins were eluted with a buffer containing mercaptoethanol with or without SDS. A major protein of mol. wt 16K and a minor protein of mol. wt 18K were isolated. These proteins were unaffected by the temperature of attachment of eosinophils to the fixed IgG antigen complexes or by the presence of protease inhibitors and did not therefore appear to be proteolytic cleavage products.

Role of hydrogen peroxide in the cytotoxic reaction of T lymphocytes.

Evidence is presented that T lymphocyte cytotoxicity is mediated by hydrogen peroxide (H2O2). At a concentration of 5 x 10(-4) M H2O2 induced 51Cr release from pre-labelled P815 mastocytoma cells. H2O2 was generated when T lymphocytes from mouse spleen were exposed to P815 cells. The concentration of H2O produced was apparently one thousand times lower than the concentration required to lyse the P815 cells. This suggests that the H2O2 is produced and acts at a highly localized site on the target cell. Sulphydryl groups on the target cell were particularly sensitive both to H2O2 and to spleen cell attack. The activity of the spleen cells was inhibited by cyanide and azide and by reducing agents which protected the target cells. Cytotoxicity was enhanced by agents which prevented H2O2 breakdown.

Detachment and chemical characterization of the regularly arranged subunits from the surface of an Acinetobacter.

Acinetobacter sp. strain MJT/F5/199A carries an array of tetragonally arranged subunits on its outer surface. The subunits can be detached from isolated cell walls by incubation with 1 M urea or by washing with water after treatment with 10 mM ethylenediaminetetraacetic acid or ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid. After removal of the urea, they reaggregate into the same ordered array at air-water interfaces in the presence of MgCl(2). The detached subunits were characterized as an acidic protein of molecular weight 65,000. They represent one-fifth of the total cell wall protein.

Chemical analysis of the outer membrane and other layers of the cell envelope of Acinetobacter sp.

Chemical analysis of fractions of the cell envelope of Acinetobacter sp. strain MJT/F5/199A, prepared by breakage in the French press and removal of plasma membranes, followed by sequential treatment with lysozyme and with papain, confirmed the existence of layers previously identified by electron microscopy. Outside the plasma membrane and periplasmic space, the envelope is composed of (i) a peptidoglycan-containing dense layer, (ii) an intermediate layer, (iii) a lipopolysaccharide-containing outer membrane, and (iv) an ordered array of protein subunits. A small amount of carbohydrate (3%) is found associated with protein in the fraction containing both the surface subunits and the intermediate layer. The papain-treated outer membranes contain 67% protein, 24% lipid, together with 11% lipopolysaccharide, and about 6% of non-lipopolysaccharide hexosamine. Lipid is located only in the papain-treated outer-membrane and is mainly phospholipid: 29% phosphatidyl glycerol, 30% phosphatidyl ethanolamine, and 40% cardiolipin. The principal fatty acid is C(18:1). Significant amounts of alcohols(16:1) and alcohols(18:1), which are found in Acinetobacter waxes, were recovered from the outer membrane.

Changes in the surface properties of rabbit polymorphonuclear leucocytes, induced by bacteria and bacterial endotoxin.

Rabbit peritoneal polymorphonuclear leucocytes were induced to aggregate by a variety of bacterial species. In the absence of serum, Gram-negative bacteria were more effective at inducing aggregation than Gram-positive. The most effective micro-organism tested, Acinetobacter sp. 199A, was readily phagocytosed and also induced extracellular secretion of the granule enzymes peroxidase and lysozyme. Isolated endotoxin from this bacterial species was highly effective in inducing aggregation and granule enzyme release. Endotoxin-induced aggregation was associated with a large increase in the amount of lactoperoxidase-catalysed iodination of surface protein. Only one iodinatable protein was detected, of molecular weight 150 000. It is postulated that phagocytosis of Gram-negative bacteria, followed by granule enzyme release, accelerates the rate of membrane recycling and that this brings new adhesive protein to the surface more rapidly.

Synthesis and turnover of the regularly arranged surface protein of Acinetobacter sp. relative to the other components of the cell envelope.

The formation of the components of the cell envelope of Acinetobacter sp. 199A was investigated by measuring the incorporation of [3H]leucine into protein, [14C]galactose into lipopolysaccharide, 32P into phospholipid, and [3H]diaminopimelic acid into peptidoglycan. Whereas the lipopolysaccharide and intrinsic protein of the outer membrane were stable, some of the regularly arranged surface protein, the alpha-protein, was lost into the growth medium. Only newly synthesized alpha-protein was lost. The peptidoglycan of the murein layer was also labile. Selective inhibition of the formation of individual components of the cell envelope with penicillin, chloramphenicol, and bacitracin showed that incorporation of protein into the outer membrane required the simultaneous formation of complete lipopolysaccharide. The converse was not true: protein synthesis was not required for lipopolysaccharide incorporation. Formation of the outer membrane and the murein layer proceeded independently.