Mycobacterial surface moieties are released from infected macrophages by a constitutive exocytic event

Bacterial cell wall constituents are released from mycobacterial phagosomes and actively traffic within infected macrophages. Colocalization of fluorescently tagged bacterial moieties with endocytic tracers revealed the dynamic movement of released mycobacterial constituents into the endocytic network with accumulation in tubular lysosomal-like compartments. The released bacterial constituents not only penetrated the infected host cell but were also present in an extracellular microvesicular fraction. To identify the intracellular source of these exocytic compartments, released vesicular material was isolated from culture supernatants by differential ultracentrifugation and characterized by Western blot and electron microscopy analyses. The presence of lysosomal membrane proteins and lysosomal proteases suggested that labeled mycobacterial cell wall constituents access a constitutive lysosomal exocytic pathway. An abundance of multilamellar extracellular compartments morphologically reminiscent of MHC class II-enriched compartments (MIIC) implicated a MHC class II transport pathway in the extracellular release of bacterial constituents. Increases in intracellular free calcium have previously been shown to trigger lysosomal exocytosis by inducing fusion of lysosomes with the plasma membrane. To test if an increase in calcium would stimulate exocytosis with release of mycobacterial constituents, infected macrophages were exposed to the calcium ionophore A23187. The ionophore triggered the release of a microvesicular fraction containing labeled bacterial moieties, implicating calcium-regulated lysosomal exocytosis as a trafficking pathway by which mycobacterial products are released from infected macrophages.     

Influence of temperature on complement-dependent immune damage to liposomes

Maximal release of trapped liposomal glucose, in the presence of saturating amounts of liposomal antigen (galactocerebroside), antiserum (anti-galactocerebroside), and complement, was dependent on temperature. At lower temperatures (20--25 degrees C), maximal glucose release was inversely related to liposomal phospholipid fatty acyl chain length (dimyristoyl phosphatidylcholine > dipalmitoyl phosphatidylcholine > distearoyl phosphatidylcholine > sphingomyelin). At higher temperatures (32--35 degrees C) a limiting plateau of glucose release, at approx. 60%, was reached, or approached, by all preparations. Sphingomyelin liposomes still released less glucose than those prepared from other phospholipids, even at 35 degrees C. The titers of antiserum and complement (ABL50/ml and CL50/ml) were dependent on temperature, and differences based on liposomal phospholipid fatty acyl chain length were observed. Analysis of antiserum and complement-dependence on temperature, and on phospholipid type, revealed that although antibody binding to galactocerebroside undoubtedly was subject to steric hindrance due to interference by surrounding phospholipids at 20--25 degrees C, steric hindrance did not play a major role in blocking antibody binding above 32 degrees C.     

Normal serum cytotoxicity for P32-labeled smooth Enterobacteriaceae. 3. Isolation of a gammag normal antibody and characterization of other serum factors causing P32 loss

Spitznagel, John K. (University of North Carolina School of Medicine, Chapel Hill). Normal serum cytotoxicity for P(32)-labeled smooth Enterobacteriaceae. III. Isolation of a gammaG normal antibody and characterization of other serum factors causing P(32) loss. J. Bacteriol. 91:401-408. 1966.-Gram-negative bacteria lost metabolically incorporated P(32) when suspended in serum only if the serum contained heat-labile in addition to heat-stable factors. Gram-positive bacteria labeled with P(32) and included for comparison lost P(32) in heat-inactivated as well as in fresh normal serum. Further investigation of gram-negative bacteria showed that a smooth Escherichia coli (O117:H27) lost P(32) only if suspended in serum containing complement fractions C'1, C'2, C'3, and C'4 "normal" antibody and lysozyme. The normal antibody was recovered by absorption on and subsequent elution from E. coli O117:H27 cell walls. Immunoelectrophoresis showed that it was a gammaG-globulin. Its P(32)-releasing activity was destroyed by 2-mercaptoethanol. Lysozyme was found to potentiate the P(32)-releasing action of normal antibody plus complement. Evidence was obtained suggesting that beta(1C) globulin was the component absorbed to zymosan during serum absorption at 15 C. Reduction of the beta(1C) level evidently upsets an important balance that exists in normal serum among complement, antibody, and lysozyme. This balance is essential for maximal P(32) release from labeled bacteria, or possibly for a maximal antibacterial effect from normal serum. The possible relationships of bactericidal, bacteriolytic, and opsonic action of normal serum are discussed.     

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

Biochemical changes in Bifidobacterium bifidum var. pennsylvanicus after cell wall inhibition. IX. Metabolism and release of cellular lipids in the presence of antibiotics.

Inhibiton of cell wall synthesis caused simultaneously an increase in cellular phospho-and glycolipids and a marked release of these compounds to the medium. The composition of the cellular and the released glyco-and phospholipids was almost the same. Antibiotics, which inhibit cell wall synthesis, did not influence glycolipid composition, but increased the relative and absolute amounts of disphosphatidylglycerol and its lysoderivatives. Incorporation and chase experiments demonstrated a considerable stimulation of phospholipid metabolism, and of diphosphatidylglycerol synthesis especially. Release of lipids was not accompanied by loss of cellular protein. Omission of Tween 80 from the medium decreased the release by about 50% and increased the relative amounts of the phosphogalactolipids in the cells and in the culture fluid. Inhibitors of protein synthesis and valinomycin caused a decrease in cellular lipidphosphorus content, and a relative increase of the phosphogalactolipids. No release of lipids was observed under these conditions.     

Chemical studies on damages of Escherichia coli by the immune bactericidal reaction. I. Release and degradation of phospholipids from damaged bacteria

When antibody-sensitized Escherichia coli B is treated with complement in the absence of lysozyme, bacterial phospholipids or fragments containing phospholipid appear in the surrounding medium. Almost at the start of the reaction, a little phosphatidylethanolamine (PE) appears in the lipid fraction extracted from the supernatant of the reaction mixture. Later it does not increase greatly in amount but free fatty acids (FFA) and lysophosphatidylethanolamine (LPE) appear and increase gradually. Addition of lysozyme to the reaction mixture changes the amounts of FFA and LPE released, but does not increase the numbers of the spots on a thin layer chromatogram of the lipid fraction of the supernatant. The FFA fraction contains no beta-hydroxymyristic acid from lipid A of the lipopolysaccharide complex.     

Normal serum cytotoxicity for P32-labeled smooth Enterobacteriaceae. I. Loss of label, death, and ultrastructural damage

Spitznagel, John K. (University of North Carolina, Chapel Hill), and Lawrence A. Wilson. Normal serum cytotoxicity for P(32)-labeled smooth Enterobacteriaceae. I. Loss of label, death, and ultrastructural damage. J. Bacteriol. 91:393-400. 1966.-Metabolically labeled smooth Escherichia coli lost between 10 and 90% of P(32), compared with control suspensions, when suspended for 60 min in normal serum at 37 C. Similar results were obtained with several other genera of Enterobacteriaceae. The structural nature of the cell injury in E. coli was shown by electron microscopic examination of ultrathin sections. Complex injury which included all peripheral cell structures occurred and various degrees of cytoplasmic loss resulted. Injured bacteria retained their essential shape and did not collapse, evidently because sufficient rigid cell wall remained to prevent this. The cell damage was more like that reported with ethylenediaminetetraacetic acid lysozyme than that reported with growth in penicillin. Damage sufficient to cause rupture of peripheral structures was unique in that it involved only localized areas of cell wall and left large sections relatively intact. The loss of P(32) in the fresh normal serum was closely related to bacterial viability loss.     

Cell wall-associated peroxidase in cultured cells of liverwort,Marchantia polymorpha L. Changes of peroxidase level and its localization in the cell wall

Most of the peroxidase activities from cultured cells of Marchantia polymorpha L. were found in the cell wall. The activities increased markedly after the beginning of stationary growth. Cytochemical examination using an electron microscope indicated that the peroxidase was localized in the layers of the cell wall. The increase of peroxidase released from the cells into the culture medium was closely correlated with the increase of the peroxidase level in the cell wall. The release of peroxidase seemed to be caused by fragmentation of the cell wall stripped from cell.     

Electron Microscopy of Immune Disruption of Leptospires: Action of Complement and Lysozyme

Sequential disruption of the sheath of avirulent leptospires of the serotype canicola with antibody and complement was monitored by electron microscopy. Loosening and separation of the sheath from the protoplasmic cylinder was observed as early as 2 min after exposure to complement. Virulent leptospires of this serotype were morphologically intact after 1 hr of exposure to antibody and complement. Similarly, treatment of leptospires of the serotype patoc with normal serum and complement severely damaged the sheath structure. Removal of the sheath of both serotypes permitted lysozyme to act on the wall of the protoplasmic cylinder. Thus, morphological evidence for the location of the mucopeptide-containing structure of these leptospires was obtained. Viable leptospires with intact sheaths were resistant to lysozyme alone. Sections and negatively stained preparations of sheaths of serotypes canicola and patoc revealed three dense layers with two intermediate light zones and an overall thickness of about 110 A. A periodicity of 40 A was observed in sheath fragments produced by complement. The 70 A wallmembrane complex of leptospires of both serotypes consisted of two dense layers with an intermediate light zone. Structures apparent after removal of the outer sheath included membranous bodies or mesosomes, axial filaments attached to terminal knobs at opposite ends of the cell, and electron-dense intracellular bodies.     

Interactions of alkaline phosphatase and the cell wall of Pseudomonas aeruginosa

Spheroplasts prepared by lysozyme treatment of cells of Pseudomonas aeruginosa, suspended in 20% sucrose or 0.2 m MgCl(2), were examined in detail. Preparation of spheroplasts in the presence of 0.2 m Mg(2+) released periplasmic alkaline phosphatase, whereas preparation in the presence of 20% sucrose did not, even though untreated cells released phosphatase when suspended in sucrose in the absence of lysozyme. Biochemical characterizations of the sucrose-lysozyme preparations indicated that lysozyme mediated a reassociation of the released phosphatase with the spheroplasts. In addition, the enzyme released from whole cells suspended in 20% sucrose (which represents 20 to 40% of the cell-bound phosphatase) reassociates with the cells in the presence of lysozyme. Electron microscopic examinations of various preparations revealed that phosphatase released in sucrose reassociated with the external cell wall layers in the presence of lysozyme, that sucrose-lysozyme prepared spheroplasts did not dissociate phosphatase which remained in the periplasm of sucrose-washed cells, and that phosphatase was never observed to be associated with the cytoplasmic membrane. A model to account for the binding of P. aeruginosa alkaline phosphatase to the internal portion of the tripartite layer of the cell wall rather than to the cytoplasmic membrane or peptidoglycan layer is presented.     

Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts.

Streptococcus mutans BHT was grown in a synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. Kinetic experiments demonstrated that although lysozyme alone could not liberate deoxyribonucleic acid, cellular deoxyribonucleic acid was liberated from lysozyme-treated cells by addition of low concentrations of inorganic sodium salts. When the salts were tested for their ability to dislodge cell-bound tritiated lysozyme, the extent of the initial release of enzyme by individual anions correlated with the anion potency for deoxyribonucleic acid liberation (SCN- greater than ClO4- greater than I- greater than Br- greater than NO3- greater than Cl- greater than F-), although the total amount of lysozyme dislodged did not correspond directly with cell lysis. Differences in the effectiveness of anions (SCN-, HCO3-, Cl- and F-) in potentiating cell lysis could be enhanced or minimized by varying the lysozyme, anion, and bacterial cell concentrations. As the anion concentration was increased for each enzyme concentration and cell concentration, the lysis increased, in some cases markedly, until maximum levels of released deoxyribonucleic acid were attained. The maximum levels of lysis of SCN- and HCO3- were similar and were greater than those for Cl- and F-. In addition, the maximum levels were observed to increase for each of the anions as the concentration of lysozyme increased.     

Vectorial and nonvectorial transphosphorylation catalyzed by enzymes II of the bacterial phosphotransferase system

The plasmolytic response of Bacillus licheniformis 749/C cells to the increasing osmolarity of the surrounding medium was quantitated with stereological techniques. Plasmolysis was defined as the area (in square micrometers) of the inside surface of the bacterial wall not in association with bacterial membrane per unit volume (in cubic micrometers) of bacteria. This plasmolyzed surface area was zero when the cells were suspended in a concentration of sucrose solution lower than 0.5 M, but increased linearly when the sucrose molarity rose above 0.5 M, reaching a plateau value of 3.61 micrometers2/micrometers3 in 2 M sucrose. In contrast, when the bacterial cells were treated with lysozyme plasmolysis increased abruptly from 0.06 micrometers2/micrometers3 in 0.75 M sucrose to 4.09 micrometers2/micrometers3 in 1 M sucrose. When the time of exposure was prolonged, the degree of plasmolysis increased gradually for the duration of the experiment (30 min) after exposure to 1 M sucrose without lysozyme, whereas with lysozyme plasmolysis reached a maximum (4.09 micrograms2/micrometers3) in 2 to 5 min. The examination of ultrastructure showed that the protoplast bodies of lysozyme-treated cells in 1 M sucrose and untreated cells in 2 M sucrose are maximally retracted from the intact wall of the bacteria; hardly any retraction of protoplasts could be seen for untreated cells in 1 M sucrose. The data suggest that the B. licheniformis cells are isoosmotic to 800 to 1,100 mosM solutions, but are able to withstand much greater osmotic pressure with no signs of plasmolysis because the cell wall and the plasma membrane are held in close association, perhaps by a covalent bond. It is likely that lysozyme weakens this bond by degradation of the peptidoglycan layer. Cellular autolysis also weakens this wall-membrane association.     

Metabolism of Phosphatidylglycerol, Phosphatidylethanolamine, and Cardiolipin of Bacillus stearothermophilus

The total phospholipid content of Bacillus stearothermophilus was constant during exponential growth, increased during the transition from the exponential to stationary phase of growth, and then slowly increased during the stationary phase. The first increase was a result of an increase in phosphatidylethanolamine; the second was a result of an increase in cardiolipin. Cessation of aeration of an exponentially growing culture or suspension in a nongrowth medium resulted in an immediate reduction in the rate of total phospholipid and phosphatidylethanolamine synthesis and a quantitative conversion of phosphatidylglycerol to cardiolipin. Cardiolipin appeared to be synthesized by the direct conversion of two molecules of phosphatidylglycerol to cardiolipin. After a 20-min pulse of (32)P, phosphatidylglycerol showed the most rapid loss of (32)P followed by cardiolipin, whereas phosphatidylethanolamine did not lose (32)P. The loss of (32)P from the total lipid pool, phosphatidylglycerol, and cardiolipin was biphasic, with rapid loss during the first two bacterial doublings followed by a greatly reduced rate of loss. The major loss of (32)P from the total phospholipid pool appeared to be by breakdown of cardiolipin. The loss of (32)P from the lipid pool was energy dependent (i.e., did not occur under anaerobic conditions or in the absence of an energy source) and was dependent on some factor other than the concentration of cardiolipin in the cells. The apparent conversion of phosphatidylglycerol to cardiolipin was independent of energy metabolism. Chloramphenicol reduced the rate of turnover of both phosphatidylglycerol and cardiolipin. The rate of lipid synthesis (all phospholipid components) was constant for about 10 min after the addition of chloramphenicol but diminished markedly after 20 min. Turnover of (32)P incorporated into phospholipid during a 30-min period prior to the addition of chloramphenicol was more rapid after the removal of chloramphenicol than that of (32)P incorporated during a 30-min period in the presence of chloramphenicol.     

Lysis of Staphylococcus aureus Cell Walls by a Soluble Staphylococcal Enzyme

Enzyme preparations of Staphylococcus aureus were examined for their ability to solubilize (32)P-labeled cell walls of the parent organism. Enzymatic activity was observed in the growth medium, in soluble fractions, and associated with native cell walls. Enzyme associated with isolated cell walls could be inactivated with formaldehyde without reducing the susceptibility of the walls to the action of added enzyme. When cells are frozen and thawed, 50 to 75% of the intracellular enzyme is released along with 2% of the intracellular protein. This freeze-thaw extracted enzyme has little, if any, activity on intact S. aureus cells. It appears that the enzyme resides near the cell wall and acts on the cell-wall inner surface.     

Synthesis of the inner cell wall layer of the chlamydomonad flagellate,Gloeomonas kupfferi

Summary An antibody to the inner wall layer ofGloeomonas kupfferi was isolated and used in a developmental analysis of cell wall processing, secretion and extracellular assembly. The focus of the processing of this matrix layer is the endomembrane system, in particular the Golgi apparatus (GA) and contractile vacuole (CV). During interphase, inner wall materials are processed in the GA, packaged in trans face vesicles and transported to the CV, the final internal depository of wall precursors until release to the cell surface. During cell division, significant changes occur in the inner wall layer processing. Early on in cytokinesis, the GA does not label with our antibody, suggesting that other wall layers are being processed. In later stages of cytokinesis, the GA changes in morphology and begins to produce inner wall layer materials. These wall precursors are shuttled to the CV where they are released around the daughter cell protoplasts. The first wall layer that is formed around daughter cells is the crystalline median wall layer. Once assembled, the inner wall layer condenses upon the crystalline layer and grows in size.     

Influence of pH extremes on sporulation and ultrastructure of Sarcina ventriculi.

Distinct morphological changes in the ultrastructure of Sarcina ventriculi were observed when cells were grown in medium of constant composition at pH extremes of 3.0 and 8.0. Transmission electron microscopy revealed that at low pH (less than or equal to 3.0) the cells formed regular packets and cell division was uniform. When the pH was increased (to greater than or equal to 7.0), the cells became larger and cell division resulted in irregular cells that varied in shape and size. Sporulation occurred at high pH (i.e., greater than or equal to 8.0). The sporulation cycle followed the conventional sequence of development for refractile endospores, with the appearance of a cortex and multiple wall layers. The spores were resistant to oxygen, lysozyme, or heating at 90 degrees C for 15 min. Spores germinated within the pH range of 4.6 to 7.0.     

Electron microscopic studies on protoplast release from I.C.A.-1.65 line (I.F.C.-1.65 line) of Bacillus subtilis cells.

Under lysozyme action a minicell-forming line (I.C.A.-1.65) of B. subtilis releases protoplasts. The main cytologic events which proceed protoplast releasing are described. Different areas of the cell wall prove a remarkable difference in their sensitivity to enzymatic lysozyme action. Central areas of the cell wall are most sensitive and the polar areas are most resistant. Mesosomal vesicles and tubules are extruded and released together with other cytoplasmic extrusion during the protoplasting process. The cell wall of minicells does not prove resistance particularities to lysozyme action. The minicells release protoplasts.     

Localization of alkaline phosphatase in three gram-negative rumen bacteria

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.     

Vasopressin-stimulated phosphorylation of rat liver phospholipid methyltransferase in isolated hepatocytes

Addition of vasopressin (1 microM) to isolated rat hepatocytes prelabeled with [32P]phosphate was accompanied by a 250% increase in the phosphorylation of phospholipid methyltransferase. Vasopressin-stimulated phospholipid methyltransferase phosphorylation was time- and dose-dependent. 32P-labeled phospholipid methyltransferase was recovered by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. After electrophoresis, phospholipid methyltransferase was electroeluted from the polyacrylamide gel and subjected to tryptic digestion or HCl hydrolysis. Analysis of 32P-labeled peptides reveals only one site of phosphorylation and the analysis of [32P]phosphoamino acids indicates that phosphoserine is the only labeled amino acid.