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PubMed1979

Pili of Neisseria meningitidis: effect of media on maintenance of piliation, characteristics of Pili, and colonial morphology.

In contrast to information in the literature which indicates that meningococci rapidly lose pili upon cultivation in vitro, we found that piliation of meningococci could be maintained in vitro for 15 or more passages. Pili were present on all eight isolates tested, whether from asymptomatic carriers or from subjects with meningococcal disease. Complete loss of piliation occurred in the same two strains on two of the three media tested. On one medium (Thayer-Martin medium with supplement B), there was partial or complete loss of pili by all strains. The optimal medium for maintaining pili was chocolate agar with 1% IsoVitaleX; 95% or more of the microorganisms of six of the eight strains tested were piliated after 15 passages in vitro, and more than 60% of the microorganisms of the other two strains were piliated. Meningococci passed on this medium generally maintained their initial density of piliation (3 to 34 pili per diplococcus). The ability to predictably cultivate piliated meningococci in vitro and to select piliated and nonpiliated clones of the same strain should allow investigation of the biochemical and immunological properties of meningococcal pili as well as their possible role in the pathogenicity of Neisseria meningitidis.

Culture Media↗
PubMed1971

Protection against pneumococcal infection by a ribosomal preparation.

A subcellular extract prepared from nonencapsulated Diplococcus pneumoniae type 3 protected mice against a subsequent challenge with virulent D. pneumoniae types 1, 2, 3, and 7. Potency ratios ranged from 25 (type 3) to >1,175 (type 1). The immunity induced in mice by this preparation was best obtained by intraperitoneal inoculation followed by intravenous challenge. Mice immunized in this manner were protected up to 12 weeks and could withstand a challenge of several hundred LD(50). The protection was destroyed by treatment of the preparation with ribonuclease and was decreased by treatment with protease. The preparation consisted of 60.5% ribonucleic acid, 29.1% protein, 6.3% deoxyribonucleic acid, and 4.0% hexose. Ultracentrifugation studies indicated that this extract had five constituents which are compatible with ribosomal material.

Journal Article↗
PubMed1971

Respiratory pathogens in monkeys.

Respiratory disease in a dynamic colony of nonhuman primates during a 4-year period was due primarily to infections caused by Klebsiella pneumoniae, Diplococcus pneumoniae, Bordetella bronchiseptica, Pasteurella multocida, and Haemophilus influenzae. The principal secondary invaders were Escherichia coli, Staphylococcus aureus, and streptococci. A high fatality rate was associated with infections caused by each of the primary pathogens, and females appeared to be more susceptible than males. Incidence of respiratory disease was greatest in the fall and early winter; however, at all times newly colonized monkeys had a higher infection rate than conditioned monkeys. Infections were occasionally confined only to the lungs and were sometimes present without grossly observable lung lesions. The information given on susceptibility of 10 species of nonhuman primates to respiratory infections provides a basis for developing disease models.

Journal Article↗
PubMed1971

Enhancement of bacterial infections in mice by newcastle disease virus.

This report describes an attempt to define the factors which incite secondary bacterial pneumonias. Groups of mice were given bacteria intraperitoneally and, at various intervals, Newcastle disease virus intravenously. There was an increase in the number of deaths and in the rates of death in these groups, when compared with a control group which was given only bacteria. These results were obtained with Streptococcus pneumoniae (Diplococcus pneumoniae), Pseudomonas aeruginosa, and Salmonella enteritidis ser. typhimurium. Newcastle disease virus increased the mortality rate of mice with bacterial infections when the two agents were given within 24 hr.

Journal Article↗
PubMed1971

Serum Bacteriostasis of Staphylococcus aureus.

Serum bacteriostasis of Staphylococcus aureus was characterized quantitatively and quantitatively. Bacteriostasis was proportional to the concentration of serum. Reproducibility was good; freezing and thawing did not materially affect the end point. Four of six different strains, including the propagating S. aureus strain for phage 73 which does not produce coagulase, were susceptible to serum bacteriostasis in similar titers; two were not susceptible at all. All six strains were effective inhibitors of bacteriostasis. Active and inactive coagulase were also inhibitors. In contrast to sensitive S. aureus, S. epidermidis and Streptococcus salivarius were not uniformly susceptible to bacteriostasis by different serums. Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Salmonella montevideo, S. zymogenes, and Diplococcus pneumoniae were not susceptible. Among gram-positive bacteria, only D. pneumoniae inhibited S. aureus bacteriostasis. Agglutinins of S. aureus and nonspecific substances such as lysozyme, beta-lysin, C-reactive protein, and transferrin were not responsible for S. aureus serum bacteriostasis. After diethylaminoethyl column fractionation of serum, the bacteriostatic principle was eluted in proximity to blood group antibody; immunoglobulins A, G, and M appeared to be present in bacteriostatic fractions. It is suggested that S. aureus bacteriostasis by serum is due to natural antibody and that inhibitory reactions with pneumococci and coagulase are due to common antigens.

Journal Article↗
PubMed1991

Evidence for functionally distinct pili expressed by Neisseria meningitidis.

In order to investigate possible functional consequences of phase and antigenic variation of meningococci, the attachment of 15 strains of Neisseria meningitidis to human erythrocytes was studied by a nitrocellulose hemadsorption assay. This assay allows the study of individual meningococcal colonies with respect to erythrocyte attachment. Of the 15 strains studied, 7 demonstrated binding of human erythrocytes (HA+). Among these seven strains, the percentage of colonies that were HA+ ranged from 0.2 to 97%. Meningococcal colonies that did not produce pilin (the major structural subunit of pili) did not demonstrate erythrocyte binding (HA-). The HA+ colony phenotype was correlated with assembly of pilin into pili and expression of pili on the meningococcal surface. However, only some piliated colonies bound human erythrocytes. This could not be explained by differences between piliated HA+ and HA- colonies in the amount of pilin produced or by differences in number of pili expressed per diplococcus. Pili of five of the meningococcal strains with HA+ colonies were antigenically related to gonococcal pili (class I meningococcal pili), but HA+ colonies were also seen in two meningococcal strains expressing class II meningococcal pili. Changes from HA+ to HA- and from HA- to HA+, in the presence of continuing pilin production and pilus assembly, occurred at frequencies of up to 10(-2)/CFU per generation. Such frequencies resemble those of phase and antigenic variation described previously for Neisseria species pilin. These studies indicate that phase variation influences the ability of meningococci to attach to human cells and suggest that meningococci may express functionally different pili.

Antigens, Bacterial↗
PubMed1972

Nitrogen metabolism and protein synthesis during pneumococcal sepsis in rats.

Markedly increased synthesis of alpha(2) and beta globulins and alpha(1), alpha(2), and beta glycoglobulins occurs during pneumococcal sepsis in the rat simultaneously with decreased albumin formation, diminished tritiated leucine incorporation into muscle protein, and enhanced excretion of nitrogen. This augmented synthesis of specific serum proteins does not become evident until fever and bacteremia develop, and it appears to be a fundamental aspect of host response to a proliferating bacterial infection in that it occurs even in rats fed a protein-deficient (6% protein) diet after weaning and before exposure to Diplococcus pneumoniae. Although amino acid catabolism, in general, appears to be increased during infection, tryptophan degradation via the kynurenine pathway, as assessed by measuring diazotizable urinary metabolites, changes little or is, at times, significantly less than in control animals. Coincidentally, functional tryptophan oxygenase activity decreases at 16 hr after exposure. Total tryptophan oxygenase activity, however, is unchanged.

Animals↗
PubMed1998

Phage antibodies obtained by competitive selection on complement-resistant Moraxella (Branhamella) catarrhalis recognize the high-molecular-weight outer membrane protein.

We used competitive panning to select a panel of 10 different human antibodies from a large semisynthetic phage display library that distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcus Moraxella (Branhamella) catarrhalis. Western blotting analyses and inhibition enzyme-linked immunosorbent assays showed that all phage antibodies were directed against the same or closely spaced epitopes on the target protein, which is the high-molecular-weight outer membrane protein (HMW-OMP) of M. catarrhalis. HMW-OMP was found in multiple isolates of complement-resistant but not complement-sensitive M. catarrhalis strains. Nucleotide sequence analysis demonstrated that the immunoglobulin heavy- and light-chain variable-region genes encoding the 10 phage antibodies were remarkably similar, with a strong preference for basic amino acid residues in the heavy-chain CDR3 regions. This is the first report showing that competitive panning is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells.

Antibodies, Viral↗
PubMed1973

Effect of pneumococcal neuraminidase on canine cerebral cortex in experimental meningitis.

We examined the effect of experimental meningeal infection with a neuraminidase-producing strain of Diplococcus pneumoniae on the N-acetylneuraminic acid (NANA) content of canine cerebral cortical gray matter. The Warren thiobarbituric acid assay for NANA was used. There was no significant difference between the free and bound NANA levels of the tissue from control animals and from those with meningitis. This finding suggests that the neurological impairment so frequent in clinical pneumococcal meningitis is not a result of the activity of this bacterial enzyme upon cortical glycolipids.

Animals↗
PubMed1973

Filamentous capsulated streptococci from the human respiratory tract. I. Antigenic attributes of provisional capsular type 83 and its relationship to streptococci of so-called group M.

Two immunologically reactive polysaccharides have been isolated from the cell walls and from culture filtrates of a filamentous alpha-hemolytic streptococcus provisionally designated capsular type 83. Both polysaccharides were purified by diethylaminoethyl-cellulose chromatography. Analysis indicates that the capsular polysaccharide consists of galactose and phosphorus, whereas the cell wall polysaccharide contains galactosamine, glucosamine, glucose, and phosphorus. On the basis of immunochemical experiments, it is suggested that the capsular polysaccharide is composed of galactose-phosphate units with terminal galactose residues at the nonreducing end. It has also been found that the capsular antigen of streptococcus type 83 is shared by a number of streptococcal strains classified in Lancefield's group M. The cell wall polysaccharide of streptococcus type 83 cross-reacts with antibody to the C(s), or cell wall-like capsular, polysaccharide of Diplococcus pneumoniae, and this cross-reactivity may be a reflection that the streptococcal antigen possesses certain structural features which are similar to those of pneumococcal C and C(s) polysaccharides.

Antigens, Bacterial↗
PubMed1974

Nonspecific resistance to intraocular infection. I. Elicitation of resistance by Neisseria meningitidis.

Both eyes of rabbits whose right eyes had previously been injected with Neisseria meningitidis were injected with Diplococcus pneumoniae. The pneumococci failed to grow in the right eye of most rabbits challenged 2 and 4 days after the injection of the meningococci, but grew well in all left eyes. The "protective" effect was less pronounced in the rabbits challenged on days 6 and 9 and virtually absent when the interval between the two injections was 14 or 22 days. Pneumococci grew equally well in both eyes of rabbits that had received nitrogen mustard treatment prior to the intravitreal injection of meningococci, indicating a possible role for the polymorphonuclear leukocyte in the protective effect.

Agglutination Tests↗
PubMed1972

Total synthesis of acetate from CO 2 . V. Determination by mass analysis of the different types of acetate formed from 13 CO 2 by heterotrophic bacteria.

Mass analysis was used to determine the amount of acetate which is totally synthesized from (13)CO(2) during fermentations by Clostridium formicoaceticum, C. acidiurici, C. cylindrosporum, Butyribacterium rettgeri, and Diplococcus glycinophilus. In the fermentation of fructose by C. formicoaceticum, 27% of the acetate was found to be totally synthesized from CO(2), and the remaining acetate was unlabeled, having been formed from fructose. Evidence is presented that the purine-fermenting organisms, C. acidiurici and C. cylindrosporum, totally synthesized about 9% of the acetate from CO(2), and that the methyl group of an additional 9% was formed from CO(2). The remaining acetate was formed from the carbons of the purine and not via CO(2). It has been postulated that the fermentation of the purines and synthesis of acetate from CO(2) both occur via derivatives of tetrahydrofolate. Evidence is presented that a compartmentalization of these folate intermediates is required if both the purine degradation and the CO(2) utilization involve identical intermediates. Neither B. rettgeri nor D. glycinophilus incorporated sufficient (13)CO(2) into acetate to allow determination of the types of acetate by mass analysis, although they did incorporate labeled (14)CO(2) in both positions of acetate.

Acetates↗
PubMed1972

Activity of deoxyribonucleic acid fragments of defined size in Bacillus subtilis transformation.

The transforming activity of Bacillus subtilis deoxyribonucleic acid (DNA) that had been sheared and purified with respect to size by sucrose gradient sedimentation is given as a function of the DNA molecular weight. It is shown (i) that fragments of median molecular weight 1.2 x 10(6) have finite activity (10(-4)), (ii) that the shape of the activity-versus-molecular weight function is qualitatively similar to that observed previously for Diplococcus pneumoniae, and (iii) that this shape precludes interpretation in terms of critical size models.

Bacillus subtilis↗
PubMed1972

Transformation and deoxyribonucleic acid size: extent of degradation on entry varies with size of donor.

The fate of label introduced as donor deoxyribonucleic acid (DNA) into competent cells of Diplococcus pneumoniae was determined immediately after entry at 25 C, as a function of the size of the donor DNA. Part of the label is found to be acid soluble, part has been incorporated into chromosomal DNA, apparently through reincorporation of degraded donor DNA, and part is found in single strands of length smaller than that of the input donor DNA strands. The last fraction apparently constitutes the precursor for integration of intact donor genetic markers and is referred to as the intact fraction. For large donor DNA the intact fraction contains over 80% of the total intracellular label, but the median strand length has been reduced to 2.2 x 10(6) daltons. For small donor molecules (1 x 10(5) to 6 x 10(5) daltons per strand) the fraction intact increases with donor size from 10 to 50% of the total intracellular label, and the median strand length of this fraction is half that of the donor strands. By combining these results with earlier data on the size dependence of the yield of transformants per unit of total intracellular donor label, we have calculated the probability that a marker in the intact fraction will be integrated, as a function of the length of the donor strand after entry. This probability has a linear dependence on strand length for activities below 40% of maximum, and extrapolates to zero activity at 77,000 daltons per strand.

Bacillus subtilis↗
PubMed1973

5-Fluoropyrimidine-resistant mutants of pneumococcus.

Three classes of 5-fluorpyrimidine-resistant mutants of Diplococcus pneumoniae have been characterized. The mutant strain upp is resistant to high concentrations of the fluoropyrimidine bases fluorouracil (FU) and fluorocytosine (FC); strain upp has a defective uridine monophosphate pyrophosphorylase. The mutant strain udk is resistant to inhibition by fluorouridine (FUR) and exhibits defective uridine kinase activity. The mutant strain fun is resistant to inhibition by the nucleosides fluorodeoxyuridine, fluorodeoxycytidine, and FUR, but shows normal activity for all pyrimidine pathway enzymes tested. This strain may be defective in the activity of a transport system that governs the cellular uptake of pyrimidine ribo- and deoxyribonucleosides. Biochemical studies on wild-type and fluoropyrimidine-resistant pneumococci are discussed with respect to the transport and early metabolism of preformed pyrimidine precursors by this organism.

Carbon Isotopes↗
PubMed1973

Selective utilization of pyrimidine deoxyribonucleosides for deoxyribonucleic acid synthesis in pneumococcus.

When pyrimidine deoxyribonucleosides are supplied to growing cultures of Diplococcus pneumoniae, they are selectively used for incorporation into deoxyribonucleic acid (DNA). Differently labeled molecules of deoxyuridine, thymidine, and deoxycytidine were used to study the precursor pathways of this organism. Each of these preformed pyrimidine deoxynucleosides is incorporated intact (i.e., without cleavage of the glycosidic bond) and is predominantly recoverable as DNA thymidine. During the utilization of deoxycytidine and deoxyuridine by pneumococci, large proportions of the available precursor are converted to free thymidine, which is secreted back into the growth medium. The biochemical pathways for selective incorporation into DNA and the regulation of concentrations of intracellular thymidine compounds by excretion of free thymidine are discussed.

Autoradiography↗
PubMed1973

Structure of deoxyribonucleic acid on the cell surface during uptake by pneumococcus.

We exposed competent cells of Diplococcus pneumoniae to high-molecular-weight donor deoxyribonucleate (DNA) and examined the state of the DNA bound to them in forms sensitive to deoxyribonuclease I. The portion elutable with 5 M guanidine hydrochloride was shown to be native, of much lower molecular weight (4 x 10(6) to 5 x 10(6)) than the donor, and as active in further transformation as sheared DNA of the same size. The portion resistant to release by guanidine hydrochloride was also shown to be native and active in transformation. These results, along with previous ones, imply that the breaks produced outside the cell are not at genetically specific sites. Furthermore, it was found that entry past the cell barrier to deoxyribonuclease could occur at 0 C by a process sensitive to ethylenediaminetetraacetate.

Carbon Isotopes↗
PubMed1974

In situ activity of enzymes on polyacrylamide gels of a deoxyribonucleic acid-membrane fraction extracted from pneumococci.

A deoxyribonucleic acid (DNA)-membrane fraction extracted from Diplococcus pneumoniae was subjected to polyacrylamide gel electrophoresis after treatment with 0.16% sodium dodecyl sulfate. At least two DNA polymerase activities were detected by in situ assays with appropriate substrates, templates, and inhibitors, including a co-polymer of deoxyadenylic and thymidylic acid and N-ethylmaleimide. This activity coincided with a fraction in the gel containing 7.5, 9.4, and 24%, respectively of the DNA, phospholipid, and protein present in the DNA-membrane fraction before electrophoresis and sodium dodecyl sulfate treatment. Assays with minced gels showed that several nuclease activities, deoxyribonucleotide kinase activity, and DNA ligase activity also coincided with this fraction. However, ribonucleoside diphosphate reductase activity did not. These results demonstrate that a complex of enzymes involved in DNA replication is firmly bound to the DNA-membrane fraction in pneumococci.

Bacterial Proteins↗