Normal serum cytotoxicity for P32-labeled smooth Enterobacteriaceae. II. Fate of macromolecular and lipid phosphorus of damaged cells

Spitznagel, John K. (University of North Carolina, Chapel Hill). Normal serum cytotoxicity for P(32)-labeled smooth Enterobacteriaceae. II. Fate of macromolecular and lipid phosphorus of damaged cells. J. Bacteriol. 91:148-152. 1966.-Fresh, normal adult guinea pig serum released micro- and macromolecular forms of metabolically incorporated P(32) from smooth Escherichia coli. A significant amount of P(32)-labeled phospholipid was also released. The overall acid solubility distribution of the P(32) label in bacterial suspensions was very little affected in fresh serum, although P(32)-labeled cytoplasmic contents and cell walls of E. coli lost some P(32) when they were suspended in fresh or heat-inactivated normal serum. These findings confirmed and extended earlier evidence that the peripheral wall and membrane structures of smooth E. coli are extensively damaged by factors in fresh normal serum.     

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.     

Molecular and Structural Damage to Escherichia coli Produced by Antibody, Complement, and Lysozyme Systems

The antibacterial action of antibody (normal and hyperimmune), complement, and lysozyme has been studied by correlating the ultrastructural and biochemical changes that they cause in smooth Escherichia coli. Both normal and hyperimmune antibody, in the absence of lysozyme, produced complement-dependent release, into the suspending medium, of 63 to 72% of the (32)P-labeled phospholipid and 74 to 85% of the small molecular bacterial constituents. Macromolecular nucleic acid labeled with (32)P was not released. By phase microscopy, these cells appeared as bacilli but their ultrastructure showed general swelling, with smoothing of the normally wrinkled outer cell wall layers. Cytoplasmic membranes were damaged and the internal cell structure was disorganized. Membranous spherules, apparently from the outermost putatively lipopolysaccharide cell layer, were released into the medium. When lysozyme was added to antibody and complement, (32)P-labeled macromolecular constituents were released from the cells. Damage to ultrastructure then included loss of cell wall rigidity, cell wall breakage, and some spheroplast formation. Characteristic fibrillar fragmentation was seen in cell wall mucopeptide layers. The relationships between antibody-complement dependent release of bacterial phospholipid, loss of selective cell permeability, and increase in sensitivity to lysozyme are discussed.     

Phospholipid metabolism of monkey smooth muscle cells grown in hyperlipemic serum.

The phospholipid content and synthesis of monkey smooth muscle cells grown in tissue culture with normal or hyperlipemic monkey serum were examined. The pattern of incorporation of radioactively labeled inorganic phosphate into the phospholipids of these cells was measured using a 4 h pulse of 32P. Phosphatidylcholine and phosphatidylinositol were the predominant phospholipids labeled. Although phosphatidylcholine constituted 45% of the cellular phospholipid, phosphatidylinositol had the highest specific activity. Exposure of the smooth muscle cells to hyperlipemic monkey serum did not alter the phospholipid content, composition or synthesis of these cells. The total phospholipid content of the smooth muscle cells was independent of the concentration of lipid in the media. The distribution of 32P into the phospholipids of monkey alveolar macrophages, L-cell mouse fibroblasts, and segments of the intima-media from monkey aortas is reported.     

Electron Microscopy and Viability of Lysostaphin-induced Staphylococcal Spheroplasts, Protoplast-like Bodies, and Protoplasts

The cell walls of a selected isolate of Staphylococcus aureus FDA 209P were observed undergoing progressive disintegration when exposed to lysostaphin (1 unit/ml) in 24% NaCl solution. Electron micrographs of ultrathin sections of test cells after exposure to lysostaphin for 2 min showed only superficial evidence of lytic damage. However, an average of 89% of these cells were osmotically fragile, and 21% were damaged beyond their capacity to regenerate cell walls and to grow as normal staphylococci. The 68% (average) of the osmotically fragile cells which retained the capacity to revert to normal staphylococci were designated spheroplasts. Neither perforations of the cell walls nor separation of the cell walls from the plasma membranes were observed in the micrographs of these 2-min spheroplasts. Thus, it appears that the osmotic fragility of these and possibly all lysostaphin-induced staphylococcal spheroplasts results from the hydrolysis of a critical number of the pentapeptide cross-linkages of the murein of the cell wall. Electron micrographs of cells exposed to lysostaphin for 5 to 10 min showed perforations and more extensive damage, including the separation of walls from the plasma membranes and the disintegration of large sections of the walls. Smaller numbers of spheroplasts (21 and 8%) were recovered from these 5- and 10-min preparations; those recovered probably represent cells which were attacked more slowly than the majority by the lytic enzyme. The nonrevertible, osmotically fragile cells that retained segments of cell wall were designated protoplast-like bodies. After 20-min exposure to lysostaphin, all of the cell wall was digested away from most of the cells, and true staphylococcal protoplasts were produced. These lysostaphin-induced, osmotically fragile forms appear to have different osmotic properties from the staphylococcal "protoplasts" reported by other investigators and should serve as the basis for a variety of fundamental investigations.     

Protective effect of Urtica dioica L. on renal ischemia/reperfusion injury in rat

Renal ischemia–reperfusion (I/R) injury may occur after renal transplantation, thoracoabdominal aortic surgery, and renal artery interventions. This study was designed to investigate the effect of Urtica dioica L. (UD), in I/R induced renal injury. A total of 32 male Sprague–Dawley rats were divided into four groups: control, UD alone, I/R and I/R?+?UD; each group contain 8 animals. A rat model of renal I/R injury was induced by 45-min occlusion of the bilateral renal pedicles and 24-h reperfusion. In the UD group, 3?days before I/R, UD (2?ml/kg/day intraperitoneal) was administered by gastric gavage. All animals were sacrificed at the end of reperfusion and kidney tissues samples were obtained for histopathological investigation in all groups. To date, no more histopathological changes on intestinal I/R injury in rats by UD treatment have been reported. Renal I/R caused severe histopathological injury including tubular damage, atrophy dilatation, loss of brush border and hydropic epithelial cell degenerations, renal corpuscle atrophy, glomerular shrinkage, markedly focal mononuclear cell infiltrations in the kidney. UD treatment significantly attenuated the severity of intestinal I/R injury and significantly lowered tubulointerstitial damage score than the I/R group. The number of PCNA and TUNEL positive cells in the control and UD alone groups was negligible. When kidney sections were PCNA and TUNEL stained, there was a clear increase in the number of positive cells in the I/R group rats in the renal cortical tissues. However, there is a significant reduction in the activity of PCNA and TUNEL in kidney tissue of renal injury induced by renal I/R with UD therapy. Our results suggest that administration of UD attenuates renal I/R injury. These results suggest that UD treatment has a protective effect against renal damage induced by renal I/R. This protective effect is possibly due to its ability to inhibit I/R induced renal damage, apoptosis and cell proliferation.     

Structure of Micrococcus denitrificans and M. halodenitrificans Revealed by Freeze-etching

S ummary . After freeze-etching, cells of Micrococcus denitrificans and M. halodenitrificans revealed structures similar to those observed in ultrathin sections. Both organisms had a similar cell wall structure. The cell wall was double layered, the smooth surface of which had a delicate granular structure. The cytoplasmic membrane was in 2 parts, both covered with spherical particles 8–12 nm diam. The cytoplasmic membrane possessed rod-shaped invaginations (100–300 × 30–50 nm). The cytoplasm of both species contained inclusions of poly-β-hydroxybutyric acid.     

Coronary Injury Score Correlates with Proliferating Cells and Alpha-Smooth Muscle Actin Expression in Stented Porcine Coronary Arteries

Neointimal formation and cell proliferation resulting into in-stent restenosis is a major pathophysiological event following the deployment of stents in the coronary arteries. In this study, we assessed the degree of injury, based on damage to internal elastic lamina, media, external elastic lamina, and adventitia following the intravascular stenting, and its relationship with the degree of smooth muscle cell proliferation. We examined the smooth muscle cell proliferation and their phenotype at different levels of stent injury in the coronary arteries of domestic swine fed a normal swine diet. Five weeks after stent implantation, swine with and without stents were euthanized and coronaries were excised. Arteries were embedded in methyl methacrylate and sections were stained with H&E, trichrome, and Movat’s pentachrome. The expression of Ki67, α-smooth muscle actin (SMA), vimentin, and HMGB1 was evaluated by immunofluorescence. There was a positive correlation between percent area stenosis and injury score. The distribution of SMA and vimentin was correlated with the degree of arterial injury such that arteries that had an injury score >2 did not have immunoreactivity to SMA in the neointimal cells near the stent struts, but these neointimal cells were positive for vimentin, suggesting a change in the smooth muscle cell phenotype. The Ki67 and HMGB1 immunoreactivity was highly correlated with the fragmentation of the IEL and injury in the tunica media. Thus, the extent of coronary arterial injury during interventional procedure will dictate the degree of neointimal hyperplasia, in-stent restenosis, and smooth muscle cell phenotype.     

Homology of the Gene Coding for Outer Membrane Lipoprotein Within Various Gram-Negative Bacteria

The mRNA for a major outer membrane lipoprotein from Escherichia coli was found to hybridize specifically with one of the EcoRI and one of the HindIII restriction endonuclease-generated fragments of total DNA from nine bacteria in the family Enterobacteriaceae: E. coli, Shigella dysenteriae, Salmonella typhimurium, Citrobacter freundii, Klebsiella aerogenes, Enterobacter aerogenes, Edwardsiella tarda, Serratia marcescens, and Erwinia amylovora. However, among the Enterobacteriaceae, DNA from two species of Proteus (P. mirabilis and P. morganii) did not contain any restriction endonuclease fragments that hybridized with the E. coli lipoprotein mRNA. Furthermore, no hybrid bands were detected in four other gram-negative bacteria outside the family Enterobacteriaceae: Pseudomonas aeruginosa, Acinetobacter sp. HO1-N, Caulobacter crescentus, and Myxococcus xanthus. Envelope fractions from all bacteria in the family Enterobacteriaceae tested above cross-reacted with antiserum against the purified E. coli free-form lipoprotein in the Ouchterlony immunodiffusion test. Both species of Proteus, however, gave considerably weaker precipitation lines, in comparison with the intense lines produced by the other members of the family. All of the above four bacteria outside the family Enterobacteriaceae did not cross-react with anti-E. coli lipoprotein serum. From these results, the rate of evolutionary changes in the lipoprotein gene seems to be closely related to that observed for various soluble enzymes of the Enterobacteriaceae.     

Locus of the Action of Serum and the Role of Lysozyme in the Serum Bactericidal Reaction

The mechanism of the lethal action of human serum on a rough strain of Escherichia coli was investigated by use of serum with and without lysozyme, in medium of low and high osmotic pressure, with cells radioactively labeled in the peptidoglycan polymer, and by electron microscopy. The results suggested that there are two separate components in the bacterial cell wall that afford structural support for the cell. Lysozyme attacked one of these, the peptidoglycan polymer. Serum damaged the other, which is probably the peripherally located lipopolysaccharide-phospholipid complex. The cell wall damage caused by lysozyme-free serum promptly resulted in cell death under usual conditions. In plasmolyzed cells, however, the wall damage was not lethal, presumably because the membrane of the plasmolyzed cell was protected from secondary lethal changes which otherwise occur.     

Salmonella typhi cell wall protein showing antigenic similarity to transferrin.

Antiserum to cell wall proteins of S. typhi showed immunoprecipitin lines of identity with homologous antigen, normal human serum and human transferrin. Also, anti-transferrin antibody showed immunoprecipitin band with S. typhi cell wall proteins and agglutinated S. typhi whole cells, but not Eschericia coli. It is speculated that transferrin or a transferrin-like protein present in the cell wall may play a role in iron-uptake by S. typhi.     

An outer membrane-disorganizing peptide PMBN sensitizes E. coli strains to serum bactericidal action

The small cationic outer membrane-disorganizing peptide PMBN sensitized four smooth, encapsulated strains of Escherichia coli (serotypes 02:K1, 04:K12, 018:K1, and 018:K5) to the lethal action of serum. The concentrations of PMBN required were low (0.3 to 1.0 microgram/ml). One E. coli strain (IH 11030; 075:K5) remained virtually resistant to serum and also to anti-075 hyperimmune serum plus complement (C) even in the presence of PMBN. This strain was nevertheless sensitive to the outer membrane permeability-increasing action of PMBN. In the bactericidal system, PMBN could be replaced by high concentrations of lysine20 or protamine but not lysine4. The PMBN-dependent bactericidal activity of GPS was abolished by heating or zymosan treatment that inactivate its C but not by lack of the action of the classical pathway of the C in C4-deficient GPS. PMBN formed a bactericidal system also with normal rabbit, rat, and human serum but not with mouse serum. The bactericidal system against E. coli 018:K1 and its derivative EH 817 (018:K1-) was found to require a factor that can be removed from normal sera by absorption with a rough E. coli strain. This factor could be replaced by specific anti-018 antibodies. The bactericidal activity of fetal calf serum plus PMBN against E. coli 018:K1 was enhanced by normal rabbit or anti-E. coli 018 hyperimmune serum. We suggest that PMBN unshields the deep structures and the hydrophobic membrane milieu of the outer membrane and facilitates the insertion of the membrane attack complex of the C into this milieu.     

Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction.

An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7). The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.     

Formation of septum-like structures at locations remote from the budding sites in cytokinesis-defective mutants of Saccharomyces cerevisiae.

Cell wall structures that partition membrane-bound portions of cytoplasm were formed at sites along the peripheral wall when a cytokinesis-defective cell division cycle mutant (cdc3) of Saccharomyces cerevisiae was grown at a restrictive temperature. The appearance of these structures, as observed in electron micrographs, was similar to that of normal septa. Aberrant septa were also detected in cytokinesis mutants harboring mutations cdc10, cdc11, and cdc12, after growth at 37 degrees C. Formation of the abnormal septa was abolished by the introduction, in a cdc3-containing strain, of additional cell cycle mutations that precluded events leading to cytokinesis and cell division. These results showed that septum formation can occur in the absence of cytokinesis. Formation of the abnormal structures was controlled by the same sequences of cell cycle events as formation of normal septa but was not subject to the spatial controls that ensure association of the septum with the budding site.     

Ultrastructure of Pseudomonas saccharophila at Early and Late Log Phase of Growth

The fine structure of Pseudomonas saccharophila, a soil bacterium, is similar to that of the marine Pseudomonas reported by Wiebe and Chapman. The unit membrane of the plasma membrane is clearly seen in some areas of thin sections. The ribonucleoprotein granules are distributed in the cytoplasm of the cell. Cells of P. saccharophila during early exponential phase are large, and most of them contain a large number of poly-beta-hydroxybutyrate granules. Some of the granules are quite large and occupy up to three-fourths of the cross section of the cell. Thin sections of the cells in the late log phase, however, show fewer and smaller poly-beta-hydroxybutyrate granules located in the central region of the cell. Negative-stained and freeze-fracture preparations show that the outer surface of the cell wall of P. saccharophila is covered with a large number of tiny granules and long, slender flagella. The outer surface of the plasma membrane appears to be smoother than the outer surface of the cell wall, and it also contains numerous granules. Since the outer surface of the cell wall is quite smooth in freeze-fracture preparations, the wrinkled appearance in thin sections is probably an artifact of fixation and dehydration. The poly-beta-hydroxybutyrate did not solidify at the freezing temperature used (approximately -150 C), and it was consequently pulled out in a spikelike structure during the fracturing process. P. saccharophila, under the conditions in our study, appears to multiply by the constrictive type of cell division.     

Suppressors of gene 32 am mutants that specifically overproduce P32 (unwinding protein) in bacteriophage T4.

A gene 32 amber (am) mutant, amNG364, fails to grow on Escherichia coli Su3+ high temperatures, suggesting that the tyrosine residue inserted at the am codon by Su3+ leads to a temperature-sensitive gene 32 protein (P32). By plating amNG364 on E. coli Su3+ 45 degrees C, several pseudorevertants were found that proved to contain a suppressor (su) mutant in addition to the original am mutation. Crosses of two of these amNG364su strains to am+ phage indicated that the suppressors themselves are in or close to gene 32. Phage strains carrying either of the two su mutations, without amNG364, grew normally. When cells were infected by these su mutants and the proteins produced were examined by sodium dodecyl sulfate-gel electrophroesis, specific overproduction of P32 was found. Maximum overproduction compared to am+ phage was 6.6-fold for one su mutant and 2.4-fold for the other. Other proteins were produced in normal amounts and in normal time sequence. When amNG364su phage were allowed to infect E. coli S/6/5(Su-), the gene 32 am fragments produced were present at the same derepressed levels as in an infection by amNG364 without a suppressor. The suppressor mutations are interpreted as causing derepression of P32 by altering sites in this autogenously regulated protein involved in template recognition. Previously, specific derepression of gene 32 had only been shown using gene 32 conditional lethal mutants grown under restrictive conditions. We have shown that P32 can also be derepressed under permissive conditions, indicating that loss of P32 function is not necessary for specific derepression.     

Locus of the Lethal Event in the Serum Bactericidal Reaction

Hypertonic sucrose inhibited the bactericidal activity of lysozyme-free serum against a rough strain of Escherichia coli. The duration of the inhibition correlated with the duration of plasmolysis caused by the sucrose. Although the lethal action of the serum was delayed, the prompt release of alkaline phosphatase by the cells suggested that nonlethal damage to the cell wall had taken place under these conditions. In contrast, the crypticity of the cells for beta-galactosidase did not deteriorate until the viability of the bacteria began to decrease. It is concluded that the primary site of action of serum is at the bacterial cell wall; however, in the absence of lysozyme, the lethal event was subsequent damage to the bacterial cell membrane.     

The ompH gene of Yersinia enterocolitica: cloning, sequencing, expression, and comparison with known enterobacterial ompH sequences

We have recently described a previously uncharacterized outer membrane protein of Salmonella typhimurium and Escherichia coli and cloned and sequenced the corresponding gene, the ompH gene, of S. typhimurium (P. Koski, M. Rhen, J. Kantele, and M. Vaara, J. Biol. Chem. 264:18973-18980, 1989). We report here the cloning, sequencing, and expression of the corresponding gene of Yersinia enterocolitica. It is significantly homologous to the ompH genes of E. coli and S. typhimurium (homology percentages, 65 and 64%, respectively), has a promoter region strongly homologous to the E. coli 17-bp class consensus promoter, and encodes a protein consisting of 165 amino acids (22 of which form the signal sequence). The plasmid-borne Y. enterocolitica ompH was found to be expressed both in the E. coli host and in minicells. The isolated outer membrane of Y. enterocolitica was shown to contain OmpH. The homology of the Y. enterocolitica OmpH protein is 66% with E. coli OmpH and 64% with S. typhimurium OmpH. All OmpH proteins have almost identical hydrophobic profiles, charge distributions, and predicted secondary structures. Because yersiniae are considered rather distant relatives of E. coli and S. typhimurium in the Enterobacteriaceae family, these results might indicate that most or all strains of the family Enterobacteriaceae have OmpH proteins remarkably homologous to those now sequenced.     

Structure of Escherichia coli After Freeze-Etching

Survival of Escherichia coli, quick-frozen under conditions similar to those employed for freeze-etching, is close to 100%. For determination of cell shrinkage, the diameters of freeze-etched E. coli cells (average, 0.99 mum) were compared with those of preparations after negative staining and after ultrathin sectioning. Negatively stained cells measured from 0.65 to 1.0 mum in diameter, and ultrathin sections showed average cell diameters of 0.70 mum. Freeze-etched replicas of logarithmically growing, as well as stationary, E. coli B cells revealed a smooth, finely pitted cell surface in contrast to cell surfaces seen with other preparative methods. The frozen cell wall may cleave in two planes, exposing (i) a smooth fracture face within the lipid layer and (ii) in rare instances an ill-defined particulate layer. Most frequently, however, cleavage of the envelope occurred between wall and protoplasmic membrane; large areas of the membrane were then exposed and showed a surface studded with predominantly spherical particles, an appearance which did not significantly change when the cells were fixed in formaldehyde and osmium tetroxide before freeze-etching. The distribution of these particles differed between logarithmically growing cells and stationary cells.     

Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabinogalactan-peptidoglycan linker, leads to irreversible loss of viability

Temperature-sensitive mutant 2-20/32 of Mycobacterium smegmatis mc(2)155 was isolated and genetically complemented with a Mycobacterium tuberculosis H37Rv DNA fragment that contained a single open reading frame. This open reading frame is designated Rv3265c in the M. tuberculosis H37Rv genome. Rv3265c shows homology to the Escherichia coli gene wbbL, which encodes a dTDP-Rha:alpha-D-GlcNAc-pyrophosphate polyprenol, alpha-3-L-rhamnosyltransferase. In E. coli this enzyme is involved in O-antigen synthesis, but in mycobacteria it is required for the rhamnosyl-containing linker unit responsible for the attachment of the cell wall polymer mycolyl-arabinogalactan to the peptidoglycan. The M. tuberculosis wbbL homologue, encoded by Rv3265c, was shown to be capable of restoring an E. coli K12 strain containing an insertionally inactivated wbbL to O-antigen positive. Likewise, the E. coli wbbL gene allowed 2-20/32 to grow at higher non-permissive temperatures. The rhamnosyltransferase activity of M. tuberculosis WbbL was demonstrated in 2-20/32 as was the loss of this transferase activity in 2-20/32 at elevated temperatures. The wbbL of the temperature-sensitive mutant contained a single-base change that converted what was a proline in mc(2)155 to a serine residue. Exposure of 2-20/32 to higher non-permissive temperatures resulted in bacteria that could not be recovered at the lower permissive temperatures.