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As part of an ongoing study concerned with improving human vaccines against Streptococcus pneumoniae, the genes for two defined pneumolysin (PL) toxoids (pneumolysoids), Pd-A (PL with a Cys----Gly substitution at amino acid 428) and Pd-B (PL with a Trp----Phe substitution at position 433), were inserted into the high-expression vector pKK233-2 in Escherichia coli and the pneumolysoids were purified. Groups of mice which had been immunized with either Pd-A, Pd-B, or native PL purified from S. pneumoniae were then challenged either intranasally or intraperitoneally with virulent pneumococci. Mice in all immunized groups survived significantly longer than sham-immunized controls. Both pneumolysoids were more effective than PL as protective immunogens. Pneumolysoid Pd-B was conjugated covalently with pneumococcal type 19F capsular polysaccharide (19F PS), and the immunogenicities of both the protein and the PS moieties of the conjugate in mice were determined. Significant anti-PL titers were obtained, and the immunogenicity of the 19F PS moiety was markedly enhanced compared with that of unconjugated PS. Conjugation also appears to have converted the 19F PS into an antigen capable of inducing a booster effect. These results support the notion that the efficacy of human, PS-based antipneumococcal vaccines might be improved by supplementation with pneumolysoid in the form of a covalent pneumolysoid-PS conjugate.

The immunoregulatory function of the complement system has been the focus of many investigations. In particular, fragments of complement factor C3 have been shown to play a role in B-lymphocyte activation and proliferation, lymphokine production, and the generation of in vitro antibody production. Purified pneumococcal polysaccharides (PS) can induce direct activation of C3 via the alternative pathway. Using sera of C1q-deficient patients and healthy subjects, we demonstrated that C3d, a split product of C3 that is generated after degradation of iC3b, can be bound to PS antigens. The binding of C3d to PS can occur in the absence of specific antibodies. Subsequently, we showed that PS complexed with C3d can be recognized by complement receptor type 2 that is expressed on B cells. Treatment of B cells with a monoclonal antibody recognizing the C3d-binding site of complement receptor type 2 reduces the binding of PS-C3d to the cells. In addition, we showed that PS4 complexed with C3d exerted an increased immunogenicity compared with free PS4. Our results show that the complement system plays a role in the activation of PS-specific B cells, carrying membrane receptors for C3d. Consequently, the complement system plays a regulatory role in the antibody response to T-cell-independent type 2 antigens such as PS.

PspA is an antigenically variable surface protein of Streptococcus pneumoniae that appears to be essential for full pneumococcal virulence. In addition, monoclonal antibodies to PspA protect mice against infection with specific strains of pneumococci virulent for mice. In this study, we have isolated the 43-kDa N-terminal half of the native 84-kDa PspA and determined the sequence of the first 45 amino acids. This sequence, the first obtained for a pneumococcal surface protein, is consistent with that of an amphiphatic coiled-coil alpha helix with a 7-residue periodicity common to fibrous proteins such as tropomyosin and streptococcal M protein. The 7-residue periodicity begins with residue 8 and extends throughout the remaining sequence for nearly 11 turns of the helix. Mice immunized with this purified PspA segment were protected from fatal pneumococcal challenge, thus demonstrating that those PspA epitopes eliciting protection were present in the N-terminal half of the molecule.

Analysis of polysaccharide components of meningococcal- and pneumococcal vaccines was carried out by proton n.m.r. spectroscopy and gas chromatography. The meningococcal polysaccharides were of high purity but showed differences in degree and position of O-acetylation between manufacturers. The level of contamination of the pneumococcal polysaccharides by C-substance was quantified. These methods provide an alternative to immunological methods for determining serotype and purity.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.

The composition of the extracts of the cultures of individual streptococcal strains, studied by immunoblotting techniques, has been shown to contain proteins with a molecular weight of 70-80 KD. These proteins have pronounced affinity to human macroglobulins: alpha-macroglobulin, alpha-glycoprotein associated with pregnancy and protein A. The significance of this phenomenon on the cellular and somatic levels is discussed.

The attachment of capsular polysaccharide to Streptococcus pneumoniae was examined using monoclonal and polyclonal antibodies. Among the strains examined, the capsular polysaccharide of types 2, 4, 6A, 6B, 7F, 8, 14, 19F and 23F was bound to the pneumococci whereas that of a type 3 strain was not. Sequential treatment with 2% SDS at 100 degrees C, pronase, and EDTA did not dissociate the capsular polysaccharide from the pneumococci. Treatment of the cells with mutanolysin, a muramidase that degrades the cell wall peptidoglycan of pneumococci and other streptococci, released both the capsular and the cell wall C-polysaccharide (C-Ps). Type 6A capsular polysaccharide released from cell walls by mutanolysin treatment, was fractionated by high performance liquid chromatography and examined by immunoelectrophoresis. It was found to be bound to both the C-Ps and the peptidoglycan. The bond between the capsular polysaccharide and the peptidoglycan has not yet been identified but is probably covalent, as the two components could not be dissociated after boiling in SDS. Based on our studies with type 6A, we propose that capsular polysaccharide and C-Ps of the pneumococcus are linked to the peptidoglycan at different sites and, thereby, indirectly to each other. Studies in mice showed that the peptidoglycan enhanced the serum antibody response to C-Ps but not to type 6A polysaccharide.

Application of methylation analysis, specific degradations, and n.m.r. spectroscopy to the capsular polysaccharide elaborated by Streptococcus pneumoniae type 7A indicates a hexasaccharide repeating-unit with the structure (Formula; see text).

Bacterial capsular polysaccharides are major virulence factors and some are used as vaccinal antigens. Their molecular size is an important physicochemical criterion which correlates with immunogenicity. This article describes a new application of high performance liquid chromatography (HPLC), based on molecular sieving, for such an evaluation. This HPLC method is rapid, accurate, reproducible, requires only very low amounts of product and presents good correlation with conventional gel permeation chromatography.

Pneumococcal surface protein A (PspA) has been shown to be a virulence factor of pneumococci and to elicit protective anti-pneumococcal antibodies in mice. PspAs from different pneumococcal isolates have been shown to exhibit antigenic variability. In previous studies with three strains, two different apparent molecular weights of PspA were observed. In this report we have studied the variation in molecular weight of PspA from 43 pneumococcal strains reactive with anti-PspA monoclonal antibodies, Xi64 and/or Xi126. The relative molecular mass (Mr) of the major PspA band ranged from 67 k to 99 k in the different strains. Variations in Mr of PspA were observed even within strains of the same capsular type. The molecular size of PspA from strain Rx1 was not affected by treatment with a variety of chemical, enzymatic, and physical procedures, suggesting that the differences in Mr of PspA among different strains, was not due to uncontrolled variations in PspA preparation. The Mr of PspA of a given strain was found to be stable both in vivo and in vitro. As a result variations in the Mr of PspA from clinical isolates, should allow discrimination between strains within a given capsular type in epidemiologic studies.

Measurement of killing kinetics of azithromycin against strains of Streptococcus pneumoniae and Klebsiella pneumoniae in vitro showed that it had a limited bactericidal activity (greater than 90% kill) for the first eight hours of incubation, but developed complete bactericidal activity (greater than 99.9% kill) by 24 h incubation. Since high and sustained tissue levels of azithromycin occur in animals and humans, it was proposed that it might produce a bactericidal effect in vivo. This was demonstrated in a lung infection model in mice, designed to mimic the in-vitro killing studies. A 25 mg/kg dose of azithromycin given 24 h before intranasal challenge reduced the recoverable Str. pneumoniae population by greater than 99.9%, in comparison with untreated controls. Erythromycin did not produce a bactericidal effect at 100 mg/kg, and roxithromycin only reduced the viable count by 96%, at a dose of 50 mg/kg. Against a K. pneumoniae lung infection, a 50 mg/kg dose of azithromycin reduced the bacterial count by 99%. The bactericidal effect was correlated with lung tissue concentrations of azithromycin. In a proliferating Escherichia coli paper disc infection model, extravascular fluid concentrations of azithromycin were correlated with a 99.9% reduction in bacterial count, while corresponding serum concentrations were always less than the MIC. Dosing with azithromycin eradicated Haemophilus influenzae from the bulla (middle ear) of gerbils, as was not the case with erythromycin and roxithromycin. This effect was correlated with the antibiotic concentration in bulla lavage.

A 33-year-old woman with quiescent systemic lupus erythematosus developed meningitis due to a penicillin-resistant strain of Streptococcus pneumoniae. After an initial failure of penicillin therapy, the patient responded to cefotaxime. The meningitis was complicated by total neurosensory hearing loss. Howell-Jolly bodies noted on peripheral blood smear led to a radionuclide spleen scan, which documented functional asplenia. This woman is the second patient in the United States with meningitis caused by a strain of S. pneumoniae moderately resistant to penicillin G (minimal inhibitory concentration, 1.0 microgram/mL). The resistant isolate displayed resistance to penicillin that was not of enzymatic origin but rather was due to an alteration of penicillin-binding proteins. This experience illustrates the importance of testing the in vitro susceptibility of the pneumococcus to penicillin G.

The well-known immologically active component of pneumococci, C-polysaccharide, is a teichoic acid that can be isolated from the cell walls and purified by Sephadex and ion-exchange chromatography. Further details of the structure of C-teichoic acid were established by chemical degradation, including hydrolysis in acid and alkali, treatment with HF, periodate oxidation and methylation. In addition, the use of 13C n.m.r. has confirmed some of these structural features and resulted in a proposal for the order of substituents, the location of positions of substitution and the configuration of anomeric centres in the repeating unit of the polymer.

The structure of the cell wall of Streptococcus pneumoniae R36a was investigated by means of chemical analysis of the solubilized products formed during autoplast formation. By autoplast formation, almost all of the cell wall components were solubilized as the end products within several hours. Analysis of the solubilized products revealed that the pneumococcal cell wall consists of three macromolecular components, teichoic acid-glycopeptide I (TA-GP I), teichoic acid-glycopeptide II (TA-GP II), and glycopeptide (GP-III). The molecular size of TA-GP I was larger than that of TA-GP II. TA-GP I and TA-GP II were constituted of similar components, galactosamine, 2-acetamido-4-amino-2, 4, 6-trideoxyhexose, glucose, ribitol, choline, phosphate, and peptidoglycan components, but the ratio of teichoic acid to glycopeptide in TA-GP II was higher than that in TA-GP I. TA-GP II was solubilized more slowly than TA-GP I and GP III during autoplast formation. The assembly of the cell wall by TA-GP I and II, and GP III is discussed in connection with the action of autolysin.

Methods are described that resulted in the selective release of deoxyribonucleic acid (DNA)-binding factor from the surface of competent pneumococci. The same methods caused a parallel inactivation of the DNA-binding capacity of the extracted bacteria. Genetically or physiologically incompetent pneumococci did not yield binding factor upon exposure to the same methods. The solubilized binding factor appeared to be a protein; it could be assayed by a membrane filter binding procedure. The binding factor had properties reminiscent of those of the DNA receptors of transformable pneumococci (Seto et al., 1975).

The in vitro kinetics of muramic acid-alanine bond hydrolysis and pneumococcal purpura-producing principle generation by incubation of Streptococcus pneumoniae cell wall preparations with the bacterial autolysin N-acetylmuramyl-L-alanine amidase were similar. The generated purpura-producing principle preparation had a weight-average molecular weight of ca. 2.6 X 10(7) and possessed the glycan and teichoic acid constituents of the pneumococcal cell wall. The results support the idea that the pneumococcal purpura-producing principle is a high-molecular-weight, glycan-teichoic acid fragment released by hydrolysis of the muramic acid-alanine bonds in the bacterial cell wall.

1. The type-specific substance, S. 33B, from Pneumococcus type 33B contains P, 2.89; hexose, 51; total sugar, 69; galactosamine, 18; and d-glucose, 20%. 2. After degradation with alkali, followed by enzymic dephosphorylation, S. 33B yielded a hexasaccharide. 3. The hexasaccharide was assigned the structure O-beta-d-glucopyranosyl- (1-->5)-O-beta-d-galactofuranosyl- (1-->3)-O-2-acetamido-2-deoxy-beta-d- galactopyranosyl-(1-->4)-O-[alpha-d- galactopyranosyl-(1-->2)]-alpha-d-galactopyranosyl- (1-->2)-ribitol. 4. Phosphate residues in S. 33B are located on the hydroxyl groups at position 5 of ribitol units and on the hydroxyl groups at position 6 of hexopyranose residues.

Penicillin-binding proteins of Streptococcus pneumoniae were labeled with [3H] propionyl-ampicillin and treated with trypsin. The fragments were separated on sodium dodecyl sulfate/polyacrylamide gels, and peptides containing the beta-lactam-binding site visualized by fluorography. From native penicillin-binding proteins (PBP), either membrane-bound or solubilized with Triton X-100, relatively stable end products of proteolysis were obtained. The smallest radioactive peptides from PBP 1a (92 kDa), PBP 2b (77 kDa), and PBP 3 (43 kDa ) had sizes of 36.5 kDa, 26 kDa, and 29 kDa, respectively. When the PBP were trypsin treated prior to labeling with the radioactive beta-lactam, these small peptides were still able to bind the antibiotic. Under conditions of limited proteolysis, membrane-bound PBP 2b and PBP 3 were converted into soluble, hydrophilic derivatives after loss of a peptide of only 2 kDa and 1.5 kDa, respectively. These two PBP are therefore anchored in the membrane by a small terminal peptide. In contrast, PBP 1a could be digested to a Mr of 48000 without becoming water-soluble; the only hydrophilic tryptic peptide that could be found was the 36.5 kDa fragment. Therefore, large domains of this PBP seem to be embedded in the membrane.

A simple and sensitive automated method has been developed to separate monosaccharides by reverse-phase chromatography using the amino acid analyzer column in the Li+ form with 90% alcohol as eluent. The column can be regenerated with LiBr and 90% ethanol to achieve highly reproducible resolution of sugar components. Glycoproteins and polysaccharides are hydrolyzed by a combination of methanesulfonic acid/Dowex 50 X 8 resin in water. Glucose and mannose are distinguished by treatment of the hydrolysate with glucose oxidase prior to analysis.

The structure of the Pneumococcus type 19A (57) capsular polysaccharide has been reinvestigated by using methylation analysis and n.m.r. spectroscopy. It is composed of residues of 2-acetamido-2-deoxy-D-mannose, D-glucose, L-rhamnose, and phosphate in the molar ratios of 1:1:1:1. The polysaccharide is linear, and is composed of these components in a repeating unit of the following structure. ---- 4)-beta-D-ManpNAc-(1 ---- 4)-alpha-D-Glcp-(1 ---- 3)-alpha-L- Rhap-(1-PO4-) ---- The type 19A polysaccharide (Na+ salt) was depolymerized by heating it in water at 100 degrees, conditions that also hydrolyzed the newly formed phosphoric monoesters.

The capsular polysaccharide from Streptococcus pneumoniae type 1 is composed of D-galactopyranosyluronic acid residues and 2-acetamido-4-amino-2,4,6-trideoxy-D-galactopyranosyl residues. The latter sugar, previously unknown in Nature, was not isolated but was identified from the products obtained on deamination of the polymer. Using n.m.r. spectroscopy, methylation analysis, and Smith degradation as the principal methods of structural investigation, it is concluded that the polysaccharide is composed of trisaccharide repeating-units having the structure: leads to 3)-alpha-Sugp-(1 leads to 4)-alpha-D-GalpA-(1 leads to 3)-alpha-D-GalpA-(1 leads to, in which Sug denotes the new sugar.

The specific capsular polysaccharide of type 45 Streptococcus pneumoniae was isolated in pure form by chemical and chromatographic methods and found to be a high-molecular-weight, glycosidically linked polymer of a hexasaccharide repeating unit composed of D-galactose (2 mol), L-rhamnose (1 mol), N-acetyl-D-glucosamine (1 mol), N-acetyl-D-galactosamine (1 mol), N-acetyl-L-fucosamine (1 mol), and phosphate (1 mol).

The immunological relation, physicochemical characteristics, and chemical composition of type 19F(19) and 19A(57) within the cross-reactive group 19 pneumococcal capsular polysaccharides were studied. By using rabbit hyperimmune diagnostic antisera in agglutination, immunodiffusion, quantitative precipitation, and absorption assays, extensive cross-antigenicity and cross-immunogenicity were demonstrable between the disease-associated types 19F(19) and 19A(57). Types 19B(58) and 19C(59), rarely associated with human disease, were extensively cross-reactive with each other but reacted weakly with types 19F(19) and 19A(57). Both types 19F(19) and 19A(57) polysaccharides contained trace amounts of protein and nucleic acid and had comparable molecular sizes as determined by gel filtration. Compositional analysis showed type 19F(19) to contain rhamnose, glucose, N-acetylmannosamine, and a phosphate ester. Type 19A(57) contained these four moieties, and in addition, contained fucose, galactose, and N-acetylglucosamine. Plans for using this information to evaluate current and proposed formulation of multivalent pneumococcal polysaccharide vaccines are discussed.

Donor deoxyribonucleic acid (DNA) single strands exist in a complex during the eclipse phase in pneumococcal transformation. This eclipse complex exhibited specific physical properties distinct from those of both pure DNA single strands and native DNA. These included a lower affinity for diethylaminoethyl-cellulose and hydroxylapatite than that of single-strand DNA, faster sedimentation than the DNA chains that it contains, and a buoyant density in Cs2SO4 lower than that of native DNA. The complex was dissociated by treatments with sodium dodecyl sulfate, NaOH, guanidine-hydrochloride, chloroform, and proteinase K but was insensitive to ribonuclease.

Purpura was grossly observable in albino mice 6 to 8 h after the intraperitoneal injection of sterile, deoxyribonuclease-treated, cell-free extracts prepared by sodium deoxycholate-induced lysis, sonic disruption, Parr bomb treatment, autolysis without sodium deoxycholate, or alternate freezing and thawing of washed suspensions of Streptococcus pneumoniae type I. Cell-free extracts obtained from sonically disrupted, heat-killed cells (100 degrees C for 20 min) did not contain purpurogenic activity. The reaction was maximal at approximately 24 h postinjection, started to fade slowly after 24 to 48 h, and usually was not grossly observable by 4 to 6 days postinjection. The purpura-producing principle (PPP) in the cell-free extract was purified by sequential ammonium sulfate precipitation, protamine sulfate precipitation, Sepharose 6B gel filtration, wheat germ lectin-Sepharose 6MB affinity chromatography, ribonuclease and trypsin treatment, and a second Sepharose 6B gel filtration step. The final preparation (i) contained glucosamine (5.6%), muramic acid (8.0%), neutral carbohydrate (12.8%), phosphate (8.0%), orcinol-reactive material (6.0%), and Lowry-reactive material (1.6%), and (ii) was free of detectable amounts of deoxyribonucleic acid, capsular polysaccharide, neuraminidase, cytolysin, and hyaluronidase. The isoelectric point and molecular size of the PPP were approximately pI 3.0 and several million daltons, respectively, and the activity remained in the supernatant fluid after centrifugation for 1 day at 105,000 x g. PPP activity was destroyed by incubation with egg white lysozyme and sodium metaperiodate but was resistant to trypsin, pronase, alpha-amylase, deoxyribonuclease, ribonuclease, alkaline phosphatase, pancreatic lipase, 7% trichloroacetic acid, 6 M urea, autoclaving (121 degrees C) for 30 min, and mild acid and alkali exposure. Our observations indicate that the PPP requires intact beta-1,4-glucosidic linkages for activity and support the working hypothesis that activity is associated with pneumococcal peptidoglycan solubilized by the bacterium's autolysin.

Pneumococcal cell walls in which the normal phosphorylcholine component of the wall teichoic acids is replaced with phosphorylethanolamine cannot absorb the homologous autolytic enzyme and are completely resistant to autolytic degradation (S. Giudicelli and A. Tomasz, J. Bacteriol. 158:1188-1190, 1984). We have now isolated and characterized soluble teichoic acid-containing muropeptides from such cell walls and tested them as substrates for the pneumococcal autolytic enzyme. Both choline- and ethanolamine-containing muropeptides were hydrolyzed to the same extent by the enzyme. Furthermore, free choline concentrations that totally inhibited the digestion of pneumococcal cell walls in vivo and in vitro were without effect when the soluble substrates were used.

Adherence of microorganisms to mucosal surfaces is a general phenomenon among microorganisms infecting the human host. Its role for persistence and colonization as well as production of local inflammation is well established. This paper describes the adhesion of Streptococcus pneumoniae to human epithelial cells. Strains from various anatomical sites or diseases are compared for attaching capacity. Isolates from the same host but at different times are also compared. The molecular mechanisms, the so-called adhesin-receptor interactions, are partially described. The pneumococcus recognizes a sugar sequence; GlcNAc beta 1-3Gal; on the surface of the host epithelial cell. Glycoconjugates containing this disaccharide act as receptors for adhering pneumococci. The adhesin in pneumococcal attachment is less well characterized. It is a heat and trypsin sensitive component, most likely a peptide, which forms a bridge between the receptor and an anchoring site in the pneumococcal cell wall. Receptor active saccharides are part of the adhesion-inhibitory activity found in human milk.

Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.

The specific capsular polysaccharide produced by Streptococcus pneumoniae type 9V (American type 68) is composed of D-glucuronic acid (1 part), D-galactose (1 part), 2-acetamido-2-deoxy-D-mannose (1 part), D-glucose (2 parts), and O-acetyl (1.6 parts). Methylation, periodate oxidation, optical rotation, and nuclear magnetic resonance studies, and partial hydrolysis showed that the polysaccharide is an unbranched high molecular weight linear polymer of a partially O-acetylated pentasaccharide repeating unit having the structure indicated below. (Formula: see text).

Two mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-L-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 degrees C or at 30 degrees C, although only the cell extracts obtained at 30 degrees C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 degrees C, mutant cultures grown at 30 degrees C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 degrees C.

The structure of the repeating oligosaccharide of the pneumococcal capsular polysaccharide type 18C has been investigated. The repeating oligosaccharide, isolated from an aqueous hydrofluoric acid hydrolyzate of the polysaccharide, was shown, by fast atom bombardment-mass spectrometry, to have a molecular weight of 928, and to contain an O-acetyl group and a glycerol residue. Information about the sequence in the per-O-methylated oligosaccharide was derived from electron-impact mass spectrometry. Supporting data were obtained from methylation analysis, periodate and chromium trioxide oxidations, and enzymic and acid hydrolyses of the oligosaccharide. These studies indicated that the polysaccharide consists of the following pentasaccharide repeating unit. (formula see text)

A neutrophil elastase-inhibitor isolated from lysed pneumococcal cells, as well as trypsin-digest peptides derived from this factor, were tested for their ability to suppress acute lung injury in mice treated with human neutrophil granule extracts. Injury was assessed by measuring pulmonary sequestration of circulating 125I-labeled albumin, lung water, and lung hemoglobin. Both the native inhibitor and the tryptic-peptides gave good protection when preincubated with granule extract for brief periods before intrapulmonary instillation. Lesser, but still significant, protection was observed in the absence of preincubation. Protection was not simply due to addition of exogenous proteins to the granule extract because substitution of goat immunoglobulin for pneumococcal fraction was ineffective. These results suggest that pneumococcal elastase-inhibitors, recently described by us, may play a role in minimizing lung injury during pneumococcal pneumonia.

Intracellular locations of 11 proteins associated with the development of competence in Streptococcus pneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of subcellular fractions prepared from protoplasts. Controls showed that the competence-induced proteins were stable during the formation of protoplasts at 25 degrees C even though some had a half-life of only 8 min at 37 degrees C. Five competence-induced proteins p38, p27, p19.5, p16, and p14.5, were found in the cytoplasm. Two, p52 and p41, were associated with the membrane, and one, p10, was extracellular. Three others, p50, p36, and p29, were recovered in both cytoplasmic and membrane fractions. No competence-induced protein was detected in the periplasmic fraction except under conditions where leakage of all components was occurring, a phenomenon that was seen in many preparations. Similar fractionation of competent cells soon after uptake of [3H]DNA showed the "eclipse complex" of single-stranded DNA and p19.5 was associated approximately one-third with membranes and two-thirds with cytoplasmic fractions, with almost none in the periplasm. This result suggests strongly that at the time the donor DNA entered the cytosol it was in single-stranded form and it had not yet paired with the recipient DNA.

The specific capsular polysaccharide of type 46 Streptococcus pneumoniae (American type 73) was isolated in pure form and shown to be a high-molecular-weight, glycosidically linked polymer composed of d-galactose (2 mol), N-acetyl-d-glucosamine (1 mol), N-acetyl-d-galactosamine (1 mol), and N-acetyl-l-fucosamine (1 mol).

The polar lipids of Streptococcus pneumoniae wild type and aminopterin-resistant strains were analysed. The membrane contained only two acid phospholipids, phosphatidylglycerol and cardiolipin, and a large amount of two glycolipids, glucosyldiglyceride and galactosylglucosyldiglyceride. The unsaturated acyl chains ranged from 58 to 87% of total fatty acids, depending on the strain and on growth conditions. No relation could be established between aminopterin resistance and polar lipid or fatty acid compositions. However, in the presence of bacteriostatic concentrations of aminopterin, the wild type and the resistant mutant did not have the same behavior. The resistant strain maintained its fatty acid composition and a normal [32P]phosphate distribution among phospholipids while the wild type shifted to a higher content in unsaturated fatty acids and to a high relative cardiolipin labelling. Such a differencein [32P] distribution was not observed when bacteriostatic concentrations of chloramphenicol were used, or when growth was stopped after amino acid deprivation induced by high concentrations of isoleucine. The biochemical basis of the aminopterin resistant character of the amiA mutants are not yet well understood but the present study establishes that the mutation confers a certain insensitivity of the lipid metabolism to aminopterin.

The cellular localization of enzymes in Diplococcus pneumoniae was examined by fractionation of spheroplasts. A deoxyribonuclease implicated in the entry of deoxyribonucleic acid (DNA) into the cell during genetic transformation was located in the cell membrane. This enzyme, the major endonuclease of the cell (endonuclease I), which is necessary for the conversion of donor DNA to single strands inside the cell and oligonucleotides outside, thus could act at the cell surface. Another enzyme, the cell wall lysin (autolysin), was also found in the membrane fraction. Other enzymes, including amylomaltase, two exonucleases, and adenosine triphosphate-dependent deoxyribonuclease, and a restriction type endonuclease, were located in the cytosol within the cell. None of the enzymes examined were predominantly periplasmic in location. Spheroplasts were obtained spontaneously on incubation of pneumococcal cells in concentrated sugar solutions. The autolytic enzyme appears to be involved in this process. Cells that were physiologically competent to take up DNA formed osmotically sensitive spheroplasts two to three times faster than cells that were not in the competent state. Although some genetically incompetent mutants also formed spheroplasts more slowly, other such mutants formed them at the faster rate.

A large number of pneumococcal isolates (over 80 strains) from a variety of geographic locales and representing a spectrum of resistance levels from a penicillin MIC of 0.003 microgram/ml up to an MIC of 16 micrograms/ml were analyzed for their penicillin-binding protein (PBP) patterns. With a few exceptions, the great majority of strains with penicillin MICs up to about 0.05 microgram/ml contained the same set of five PBPs with molecular sizes typical of those of susceptible pneumococci. In strains with penicillin MICs of about 0.1 microgram/ml and up, virtually all isolates showed two common features: (i) all isolates showed loss of PBP 1A (98 kilodaltons) with or without a parallel appearance of a "new" PBP that ranged in molecular size between 96 and 97 kilodaltons; and (ii) in strains with penicillin MICs of 0.5 microgram/ml or more, PBP 2B could not be detected on the fluorograms even with very high concentrations of radioactive penicillin. Beyond these two common features, resistant strains with similar penicillin MICs showed a surprising variety of PBP profiles (i.e., in the number and molecular sizes of PBPs), each characteristic of a given isolate. We suggest that in pneumococci remodeling of critical PBPs in more than one way may result in comparable levels of penicillin resistance.

The specific capsular polysaccharide produced by Streptococcus pneumoniae type 15F (American type 15) is composed of D-galactose (3 parts), D-glucose (1 part), 2-acetamido-2-deoxy-D-glucose (1 part), phosphate (1 part) and O-acetyl (2 parts). Methylation, periodate oxidation, nitrous acid deamination, optimal rotation and nuclear magnetic resonance studies showed that the polysaccharide is a high mol. wt linear polymer of a pentasaccharide repeating unit having the structure.