bluuebunny/crossref_metadata_2025 huggingface dataset:
A chemically synthesized peptide which elicits humoral and cellular immune responses to mycobacterial antigens
Authors: Minden P , Houghten R A , Spear J R , Shinnick T M
Published: January 2020

Monoclonal antibodies directed to Mycobacterium bovis BCG (BCG) and to M. tuberculosis H37Rv (H37Rv) were used in conjunction with affinity chromatography to prepare a mycobacterial component which was designated BCG-a. A synthetic peptide antigen was prepared based on the amino acid sequence of BCG-a and was designated BCG-a-P. Significant immunological similarities were found between BCG-a-P and antigens in extracts of BCG and H37Rv but not between BCG-a-P and antigens of nontuberculous mycobacteria. An enzyme-linked immunosorbent assay detected antibodies to BCG-a-P in sera from rabbits that had been immunized with BCG and H37Rv sonicates. In Western blot analysis, antibodies to BCG-a-P reacted to 10,000-molecular-weight components of extracts of BCG and H37Rv. Delayed cutaneous hypersensitivity reactions to BCG-a-P were elicited in guinea pigs immunized with sonicates of BCG and H37Rv but were weak or nonexistent in unimmunized animals or in animals immunized with sonicates of nontuberculous mycobacteria. This study points out the feasibility of using monoclonal antibodies to prepare and characterize synthetic mycobacterial peptides with a potential for immunodiagnostic purposes.
Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis
Authors: Aoki H , Shiroza T , Hayakawa M , Sato S , Kuramitsu H K
Published: January 2020

The gtfB gene coding for a glucosyltransferase (GTF) activity of Streptococcus mutans GS-5 was isolated on a 15.4-kilobase DNA fragment by using a lambda L47.1 gene library. The activity was catalyzed by gene products of 150 and 145 kilodaltons which reacted with antibodies directed against both soluble and insoluble glucan-synthesizing GTFs. The enzyme present in crude Escherichia coli extracts synthesized both soluble and insoluble glucans. The enzyme was partially purified from lysates of the lambda DS-76 clone and synthesized both types of glucans in a primer-independent fashion. In addition, the purified enzyme exhibited a pI of approximately 5.0. Southern blot analysis indicated that the cloned GTF gene represented a contiguous nucleotide sequence on the strain GS-5 chromosome. Furthermore, evidence for the existence of a distinct gene sharing partial homology with gtfB was also obtained. The gtfB gene was subcloned into plasmid pACYC184 into E. coli and exhibited GTF activity when carried on GS-5 inserts as small as 5 kilobases. The approximate location of the GTF promoter and the direction of gene transcription were also determined. The cloned enzyme was not secreted through the cytoplasmic membrane of E. coli, since most of the activity was found in the cytoplasm and, in lesser amounts, associated with the cytoplasmic membrane. The gtfB gene was insertionally inactivated by introducing a gene fragment coding for erythromycin resistance into the GTF coding region. After transformation of strain GS-5 with the altered gene, transformants defective in insoluble glucan synthesis were identified. These results indicate that the gtfB gene codes for a GTF involved in insoluble glucan synthesis in strain GS-5.
Nutritional relationships between oral bacteria
Authors: Grenier D , Mayrand D
Published: January 2020

Nutritional relationships were revealed during the coculturing of Bacteroides gingivalis with Wolinella recta and Bacteroides melaninogenicus with W. recta. W. recta produced a substance that stimulated the growth of B. gingivalis and B. melaninogenicus. Characterization by thin-layer chromatography and absorption spectrometry identified the compound as protoheme. Production of large amounts of formate by B. melaninogenicus stimulated the growth of W. recta. These nutritional relationships could represent examples of mechanisms favoring bacterial succession in periodontal sites.
Regulation of staphylococcal toxic shock syndrome toxin-1 and total exoprotein production by magnesium ion
Authors: Mills J T , Dodel A W , Kass E H
Published: January 2020

The effect of Mg2+ on in vitro production of extracellular proteins and, specifically, of toxic shock syndrome toxin-1 (TSST-1), by Staphylococcus aureus in a chemically defined medium was examined. As previously observed, the organisms did not proliferate in the absence of divalent cations. Low levels of Mg2+ (0.02 to 0.04 mM) permitted submaximal proliferation and elevated production of exoproteins. When the Mg2+ concentration was raised to 0.4 mM, multiplication was optimal and exoprotein levels were depressed. Ca2+ and Mn2+ diminished the effect of limiting Mg2+. The increased levels of exoproteins were not due to cell lysis or leakage since intracellular TSST-1 levels were not high enough to account for the increase in extracellular TSST-1 and since the intracellular enzyme, lactate dehydrogenase, was not found in culture supernatants. Cells cultured in low levels of Mg2+ remained in logarithmic growth longer than did those cultured in high concentrations of Mg2+ and, unlike the latter, produced exoproteins throughout the logarithmic growth phase. Low Mg2+ had no effect on cultures in the stationary phase, and organisms cultured in low Mg2+ recovered fully when transferred to high Mg2+. We conclude that, when cultured in medium deficient in Mg2+, S. aureus responds early in the growth cycle by increasing production of many extracellular proteins, including TSST-1.
Characterization of a protein from normal human polymorphonuclear leukocytes with bactericidal activity against Pseudomonas aeruginosa
Authors: Hovde C J , Gray B H
Published: January 2020

Purification of a bactericidal protein (BP) from the cytoplasmic granules of normal human polymorphonuclear leukocytes (PMN) with activity against Pseudomonas aeruginosa is described. Bactericidal activity from acid extracts of a mixed granule population was purified 175-fold by a two-step chromatographic procedure. The first step, dye-ligand affinity chromatography with Matrex-Gel Orange A, was followed by cation-exchange chromatography with Bio-Rex 70 resin, and this combination routinely gave a yield near 80%. Only one peak of bactericidal activity against P. aeruginosa type I was found after each chromatographic step. Characterization of BP showed a single band with an apparent molecular weight of 55,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified BP was most active against the six strains of P. aeruginosa tested and against Escherichia coli B (a deep rough mutant). Purified BP killed 5 X 10(6) CFU of P. aeruginosa type I per ml at a concentration of 60 to 80 ng/ml. Proteus mirabilis and Staphylococcus aureus were both resistant to the bactericidal activity of BP. BP was shown to be glycosylated by periodic acid staining after gel electrophoresis and to have an isoelectric point near 7.5 by chromatofocusing. The amino acid composition of BP is presented.
Antigonococcal activity of human neutrophil cathepsin G
Authors: Shafer W M , Onunka V C , Martin L E
Published: January 2020

We have shown that lysosomal cathepsin G, prepared from acid extracts of granules derived from human polymorphonuclear granulocytes, exhibits potent in vitro antimicrobial activity against Neisseria gonorrhoeae. An isolated isozyme of cathepsin G was found to exhibit antigonococcal activity by a nonenzymatic mechanism in a time-dependent manner. Moreover, we observed that the antigonococcal activity of cathepsin G was relatively independent of pH and evident over a pH range resembling that invoked for maturing phagolysosomes. Using a number of isogenic strains, we determined that certain mutations known to alter cell envelope structure rendered gonococci more susceptible to cathepsin G. This suggests that the susceptibility of gonococci to cathepsin G, and possibly other antimicrobial proteins derived from PMN granules, is genetically determined and possibly related to the structure of the gonococcal cell envelope.
Organization of genes encoding two outer membrane proteins of the Lyme disease agent Borrelia burgdorferi within a single transcriptional unit
Authors: Howe T R , LaQuier F W , Barbour A G
Published: January 2020

OspA and OspB are major outer membrane proteins and antigens of the Lyme disease spirochete, Borrelia burgdorferi. We examined the organization of ospA and ospB, the genes encoding these proteins. The location and direction of transcription of each osp gene was determined by subcloning, deletion analysis, and transposon Tn5 mutagenesis. Transposon Tn5 insertions within the ospA gene abrogated expression of ospB, suggesting that these genes are transcribed from a common promoter. Northern blot analysis of mRNA from B. burgdorferi with two DNA probes individually specific for ospA or ospB identified a 2.2-kilobase transcript that hybridized with each probe. These studies indicate that the two osp genes of B. burgdorferi constitute a single transcriptional unit.
Role of NADH oxidase in the oxidative inactivation of Streptococcus salivarius fructosyltransferase
Authors: Abbe K , Takahashi-Abbe S , Schoen R A , Wittenberger C L
Published: January 2020

A cell-associated fructosyltransferase produced by Streptococcus salivarius was irreversibly inactivated in a time-dependent manner when resting or permeabilized cell suspensions were incubated with low concentrations (less than 1.0 microM) of copper. In addition to copper, the inactivation was dependent on oxygen and on a fermentable carbon source (endogenous intracellular polysaccharide or glucose). In starved, permeabilized cell suspensions, the fermentable carbon source could be replaced by NADH but not by NADPH or ATP. Of several other S. salivarius enzymes tested, only fructosyltransferase was inactivated under these conditions. The available evidence indicated that NADH oxidase is the enzyme responsible for fructosyltransferase inactivation. Results from oxygen radical scavenger studies implicated one or more species of oxygen radicals and hydrogen peroxide in the inactivation reaction.
Analyses of rat Pneumocystis carinii antigens recognized by human and rat antibodies by using western immunoblotting
Authors: Graves D C , McNabb S J , Worley M A , Downs T D , Ivey M H
Published: January 2020

The major Pneumocystis carinii antigens inducing antibody responses in infected hosts were identified by Western immunoblotting techniques. The biochemical nature of these antigens was also elucidated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by protein staining revealed a major component with a molecular weight (MW) of greater than 205,000. This major component disappeared and a new major protein staining component of approximately 110,000 to 116,000 MW appeared when electrophoresis was done in the presence of beta-mercaptoethanol. Periodic acid-Schiff staining revealed that this major component contains carbohydrate moieties. A major component in the 55,000- to 60,000-MW region was visible with periodic acid-Schiff stain, but not with a protein stain, after electrophoresis in the presence of beta-mercaptoethanol. The majority of sera tested from humans with diagnosed pneumocystosis and from rats allowed to recover from steroid-induced pneumocystosis reacted strongly with 110,000- to 116,000-, and 55,000- to 60,000-MW components. These sera often, but not always, detected antigens with MWs of approximately 170,000, 125,000, and 30,000 to 32,000. The data suggest that the antigenic composition of P. carinii is relatively complex and that rat and human P. carinii probably share antigenic determinants. Competitive studies between infection-derived human and rat antisera for the major rat P. carinii components revealed competition; rat antisera appeared to recognize a greater range of antigenic epitopes than did human antisera. Protease treatment of the antigenic components that had been immobilized on nitrocellulose paper destroyed their antigenic reactivity with rat antibody. Treatment with sodium periodate decreased reactivity of this 110,000- to 116,000-MW component and completely destroyed the reactivity of the 55,000- to 60,000-MW component with rat antibody.
Analysis of macrophage bactericidal function in genetically resistant and susceptible mice by using the temperature-sensitive mutant of Listeria monocytogenes
Authors: Gervais F , Morris-Hooke A , Tran T A , Skamene E
Published: January 2020

Innate resistance to infection by Listeria monocytogenes is genetically controlled and is critically dependent on prompt macrophage recruitment to the sites of infection. Experiments reported here were designed to examine whether there was an additional, qualitative difference between the intrinsic bactericidal activity of the inflammatory macrophages of genetically resistant (C57BL/6J) and susceptible (A/J) hosts. To critically evaluate the bactericidal (rather than bacteriostatic) function of the macrophage, a temperature-sensitive (ts) mutant of L. monocytogenes was developed. Mutagenesis was induced with nitrosoguanidine, and the ts mutants were isolated following enrichment with penicillin-gentamicin combinations. The ts mutants were found to carry the cell surface and biochemical characteristics of the original wild-type strain of L. monocytogenes. Inflammatory peritoneal macrophages from resistant C57BL/6J mice were found to have enhanced listericidal activity when compared with inflammatory macrophages from susceptible A/J mice. However, further analysis of the macrophage populations revealed that this seemingly qualitative advantage was due to the relatively greater proportion of inflammatory macrophages present in the inflammatory exudates of resistant C57BL/6J mice. When homogeneous populations of pure inflammatory macrophages were compared, no interstrain differences in their listericidal activity in vitro were seen. These results suggest that the susceptibility of A/J strain mice to L. monocytogenes is not due to an intrinsic deficiency of the listericidal activity of the inflammatory macrophage. The slight increase in bactericidal activity of macrophages from resistant mice that was reported by others (C. J. Czuprynski, B. P. Canono, P. M. Henson, and P. A. Campbell, Immunology 55:511-518, 1985) is caused by the difference in the relative percentage of resident cells present in the peritoneal exudates from resistant and susceptible mice.
Characterization of monoclonal antibodies specific for adhesion: isolation of an adhesin of Streptococcus sanguis FW213
Authors: Elder B L , Fives-Taylor P
Published: January 2020

Monoclonal antibodies reactive to an adhesive strain of Streptococcus sanguis (FW213) and nonreactive to a nonadhesive mutant (JL7) were derived from the fusion of myeloma line X63Ag8.653 and spleen cells from BALB/c mice immunized with live S. sanguis cells. Five cell lines, belonging to subclasses of immunoglobulin G, produced monoclonal antibodies specifically directed against the adhesive strain. All five antibodies also failed to react with five additional, independently isolated, nonadhesive mutants. A spontaneous mutant of FW213 (VT508) that no longer reacted with monoclonal antibody F51 (MAbF51) was isolated by serial agglutination with the antibody. Langmuir adsorption isotherms of VT508 indicated that this mutant also had altered ability to adhere to saliva-coated hydroxyapatite further confirming the specificity of MAbF51 for adhesion. Electron microscopy revealed that VT508 had lost the peritrichous fimbriae associated with the adhesion of FW213. MAbF51 was used to purify the adhesin from lysozyme cell extracts by using an affinity column of MAbF51 linked to Sephacryl S1000. Purity was suggested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis (CIEP). The adhesin had a molecular weight greater than 150,000 and was not denatured in sodium dodecyl sulfate reducing gels. Two peaks of near electrophoretic mobility were detected in CIEP when the purified material was run against polyclonal antibody to the whole cell. Tandem CIEP analysis and immunoprecipitation provided evidence that the two peaks represented the same antigen in two different forms.
Modulation of primary antibody responses to sheep erythrocytes in Plasmodium chabaudi-infected resistant and susceptible mouse strains
Authors: Stevenson M M , Skamene E
Published: January 2020

The in vivo primary antibody response to sheep erythrocytes (SRBC) was determined in genetically resistant C57BL/6 and susceptible A/J mice during the course of infection with Plasmodium chabaudi. Spleen cells from both strains of mice, immunized with SRBC and infected on the same day, showed significant increases in the number of direct plaque-forming cells. The response of malaria-infected C57BL/6 mice was significantly enhanced in comparison with the responses of both normal C57BL/6 and malaria-infected A/J mice. When mice were immunized at later times in the infection, the level of the response declined in both strains until it was less than 50% of the response of normal mice. Thus, suppression of the primary antibody response to SRBC does not correlate with the outcome of P. chaubaudi infection in genetically resistant and susceptible hosts.
Contribution of cloned virulence factors from uropathogenic Escherichia coli strains to nephropathogenicity in an experimental rat pyelonephritis model
Authors: Marre R , Hacker J , Henkel W , Goebel W
Published: January 2020

Escherichia coli 536 (O6:K15:H31), which was isolated from a case of urinary tract infection, determines high nephropathogenicity in a rat pyelonephritis system as measured by renal bacterial counts 7 days after infection. The loss of S fimbrial adhesin formation (Sfa-) (mannose-resistant hemagglutination [Mrh-] and fimbria production [Fim-]), serum resistance (Sre-), and hemolysin production (Hly-) in the mutant 536-21 led to a dramatic reduction of bacterial counts from almost 10(5) to only 40 cells per g of kidney. The reintroduction of the cloned S fimbrial adhesin determinant (sfa) increases the virulence of the avirulent mutant strain by a factor of 20; almost the same effect was observed after restoration of serum resistance by integration of an sfa+ recombinant cosmid into the chromosome. Additional reintroduction of the Hly+ phenotype by transformation of two hly determinants increased the virulence of the strains. Hemolysin production determined increased renal elimination of leukocytes and erythrocytes. Thus all three determinants investigated, S fimbriae, serum resistance, and hemolysin, contribute to the multifactorial phenomenon of E. coli nephropathogenicity.
Antigenic characterization of the P6 protein of nontypable Haemophilus influenzae
Authors: Murphy T F , Bartos L C , Campagnari A A , Nelson M B , Apicella M A
Published: January 2020

The purpose of this study was to characterize the degree of antigenic heterogeneity or conservation of a 16,600-dalton outer membrane protein (P6) among strains of nontypable Haemophilus influenzae. Immunization of rabbits with P6 isolated from individual strains resulted in antibody to P6 of all 25 strains tested. The titers of antibody in the sera were similar among the strains. Whole organisms of two strains were used to immunize rabbits, and antibodies were produced to P6 of all strains tested. Monoclonal antibodies developed to P6 from mice immunized with whole cells of three different strains recognized determinants on P6 of all 25 strains tested. Finally, pooled normal human serum contained antibodies to P6 of all 25 strains assayed. These studies indicate that P6 is a highly conserved antigen on the outer membrane of nontypable H. influenzae.
Potential role of lysozyme in bactericidal activity of in vitro-acquired salivary pellicle against Streptococcus faecium 9790
Authors: Germaine G R , Tellefson L M
Published: January 2020

The adherence of Streptococcus faecium 9790 to hydroxyapatite (HA) coated with whole saliva supernatant proteins (S-HA) or parotid fluid proteins was studied. The organism was labeled with [3H]thymidine, and adherence was estimated as the radioactivity remaining associated with the variously coated HA preparations after incubation and removal of unbound microbes by washing the adherence substratum. Adherence was time dependent and saturable, characteristics typical of oral streptococci in this in vitro adherence model system. However, adherence to S-HA, but not bare HA, was decreased 20-fold at 4 degrees C compared with room temperature. Furthermore, adherence at 4 degrees C to S-HA was decreased 20-fold relative to bare HA at 4 degrees C. Adherence to HA coated with parotid fluid proteins also was reduced at 4 degrees C. The magnitude of the temperature dependence and the inhibitory effect at 4 degrees C of whole saliva or parotid fluid pellicles on HA was unexpected. Of several sugars and amino sugars tested, the chitin saccharides, chitotriose, chitobiose, and N-acetylglucosamine caused greater than 90% inhibition of adherence to S-HA. These same saccharides were previously shown to inhibit lysozyme, polylysine, or autolytic lysis of the organism (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985). Examination of unbound and adherent microbes revealed that lysis of the organism occurred during the adherence assays. A strong association (r = 0.83) between the extent of lysis and the extent of adherence was found under a variety of conditions. Depletion of lysozyme from saliva specimens used to coat HA resulted in a greater than 90% decrease in both cell lysis and adherence. Lysis of the microbe appeared dependent upon the presence of the saliva pellicle (coating) on HA, since solutions containing proteins desorbed from HA during mock-adherence incubations possessed lytic activity that was 2- to 10-fold too low to account for the extents of lysis observed with greater than or equal to 10(8) input cells. These results demonstrate the potential antibacterial activity of acquired salivary pellicle on enamel in vivo and the likely role of lysozyme in this activity. The data also serve to caution that this widely used in vitro adherence model will not distinguish whole-cell adherence from the adsorption of radiolabeled DNA released from lysing cells. Several additional controls are suggested that will indicate whether test microbes remain intact or lyse during adherence trials.
Role of Escherichia coli alpha-hemolysin and bacterial adherence in infection: requirement for release of inflammatory mediators from granulocytes and mast cells
Authors: König B , König W , Scheffer J , Hacker J , Goebel W
Published: January 2020

We investigated the role of bacterial mannose-resistant fimbriation of S fimbriae (Fim), mannose-resistant hemagglutination (S-Mrh), and hemolysin (Hly) production by an Escherichia coli parent and genetically cloned strains as regards their effect on histamine release from rat mast cells and generation of the chemiluminescence response, leukotriene, and enzyme release from human polymorphonuclear granulocytes. These mediators are involved in the induction of inflammatory disease processes and lead, e.g., to the enhancement of vascular permeability, chemotaxis, aggregation of granulocytes (leukotriene B4), lysosomal enzyme release, and smooth-muscle contraction (leukotrienes C4, D4, and E4). The content of azurophilic and specific granules in polymorphonuclear granulocytes consists of highly reactive enzymes which amplify inflammatory reactions. Washed bacteria (E. coli 764 Hly+/-, E. coli 21085 Hly+/- Fim+/- Mrh+/-), as well as their culture supernatants, were analyzed at various times during their growth cycle. No differences exist between parent and cloned or mutant strains with respect to their outer membrane proteins and lipopolysaccharide pattern. Washed bacteria [E. coli 764 and 21085(pANN202-312)] which produced hemolysin, unlike Hly- strains, induced high levels of histamine release from rat mast cells and led to a significant chemiluminescence response and enzyme and leukotriene release from human polymorphonuclear granulocytes. Bacterial culture supernatants from Hly+ and secreting strains showed similar results with the exception of E. coli 21085(pANN202-312), which is a hemolysin-producing but not a secretory strain. Our data suggest a potent role for hemolysin as a stimulus for noncytotoxic mediator release from various cells. Furthermore, we showed that the presence of Fim and S Mrh potentiates mediator release. The simultaneous presence of Mrh and Fim [E. coli 535/21(pANN801-4)] increased mediator release compared with Mrh+ Fim- strains [E. coli 536/21(pANN801-1)]. E. coli 536/21 (Msh- Mrh- Fim- Hly-) did not induce mediator release.
Molecular characterization of proteins from porcine spirochetes
Authors: Joens L A , Marquez R B
Published: January 2020

Sonicated preparations of Treponema hyodysenteriae and Treponema innocens were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Treponemal proteins were electrophoresed on a 10% polyacrylamide slab gel in a discontinuous Tris-glycine system and either stained with Coomassie blue dye or transferred electrophoretically at 20 mA for 16 h and 30 mA for 3 h to nitrocellulose paper. Staining of the gels revealed at least 42 distinct T. hyodysenteriae and T. innocens proteins, with molecular sizes ranging from greater than 100 to 14 kilodaltons (kDa). Each species contained 12 to 16 major protein bands; five of the proteins were common to both species. Fourteen major antigens were identified in T. hyodysenteriae isolate B204 by using serum specimens from pigs in the acute stage of swine dysentery. Twelve additional antigens were detected in isolate B204 when convalescent-phase serum specimens were reacted to the blot. A wide band at 16 kDa was identified with convalescent-phase serum specimens in T. hyodysenteriae but not in T. innocens. This 16-kDa antigen was also identified in T. hyodysenteriae with colonic secretions from convalescent pigs.
Functional characterization of extracellular vesicles produced by Bacteroides gingivalis
Authors: Grenier D , Mayrand D
Published: January 2020

Extracellular vesicles of Bacteroides gingivalis (type strain 33277) were isolated, and some of their biological activities were characterized. The vesicles were obtained from a 2-day culture after ammonium sulfate precipitation, differential centrifugation, and dialysis. When viewed by electron microscopy, vesicles of approximately 50 nm predominated. The results indicated that the enriched vesicle fraction had a high proteolytic activity against collagen, Azocoll, and N-alpha-benzoyl-DL-arginine p-nitroanilide. The polypeptide pattern of the vesicles was similar but not identical to that of the outer membrane. The membrane vesicles could also promote bacterial adherence between homologous cells as well as mediate attachment between two noncoaggregating bacterial species. These vesicles could thus play an important role in periodontal diseases by serving as a vehicle for toxins and various proteolytic enzymes, as well as being involved in adherence.
Expression of Streptococcus sanguis antigens in Escherichia coli: cloning of a structural gene for adhesion fimbriae
Authors: Fives-Taylor P M , Macrina F L , Pritchard T J , Peene S S
Published: January 2020

Chromosomal DNA from Streptococcus sanguis FW213 was partially digested with EcoRI and ligated into the positive-selection cloning vector pOP203(A2+). The ligation mixture was used to transform Escherichia coli K-12, and 4,500 transformants were examined. The tetracycline-resistant colonies had inserts averaging 3.2 kilobases. The entire colony bank was screened by colony immunoassay with polyclonal rabbit serum raised against S. sanguis FW213 whole cells. Thirty recombinant colonies produced stable positive reactions of various intensities, indicating that S. sanguis antigens could be expressed in E. coli. Restriction endonuclease digestion of these clones suggested that 26 of the clones were unique. Only two clones, VT616 and VT618, gave positive reactions with fimbria-specific antisera. That the gene coding for the antigen was located on the plasmid was confirmed by demonstrating that the presence of the plasmid was linked to antigen production. Western immunoblot analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that both clones produced a fimbrial peptide of Mr 30,000. The two recombinant plasmids were shown by Southern analysis and restriction mapping to contain the same 6-kilobase EcoRI fragment inserted in opposite orientations. Southern hybridization confirmed that this fragment is present in S. sanguis genomic DNA. The Mr 30,000 protein gene was expressed in both orientations, suggesting that the fimbrial promoter is located on the 6-kilobase fragment. These results show that at least one streptococcal fimbrial gene can be cloned and expressed in E. coli.
Protein sources of heme for Haemophilus influenzae
Authors: Stull T L
Published: January 2020

Although Haemophilus influenzae requires heme for growth, the source of heme during invasive infections is not known. We compared heme, lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin as sources of heme for growth in defined media. The minimum concentration of heme permitting unrestricted growth of strain E1a, an H. influenzae type b isolate from cerebrospinal fluid, was 0.02 micrograms/ml. Using molar equivalents of heme as lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin, we determined that myoglobin and hemoglobin permitted unrestricted growth at this concentration. To determine the ability of host defenses to sequester heme from H. influenzae, we used affinity chromatography to purify human haptoglobin and hemopexin, serum proteins which bind hemoglobin and heme. Plate assays revealed that 12 strains of H. influenzae acquired heme from hemoglobin, hemoglobin-haptoglobin, heme-hemopexin, and heme-albumin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of strain E1a grown in heme-replete and heme-restricted conditions revealed a heme-repressible outer membrane protein with an apparent molecular mass of 38 kilodaltons. These results demonstrated that, unlike Escherichia coli, H. influenzae may acquire heme from hemoglobin-haptoglobin. H. influenzae also may acquire heme from hemopexin and albumin, which have not been previously investigated. The role of outer membrane proteins in the acquisition of heme is not yet clear.
Formation of transmural complement pores in serum-sensitive Escherichia coli
Authors: Bhakdi S , Kuller G , Muhly M , Fromm S , Seibert G , Parrisius J
Published: January 2020

The binding of C9 at 0 and 37 degrees C to viable Escherichia coli K-12 cells carrying C5b-8 complexes was quantified. At low temperature, limited average binding of only 1 to 1.4 molecules of C9 per C8 molecule occurred, whereas 6 to 8 C9 molecules were bound per C8 molecule at 37 degrees C. Despite incorporation of C9 into C5b-9 complexes at 0 degrees C, these terminal complexes caused no loss of bacterial viability even when present in very large numbers (1,000 to 1,500 per CFU) on the bacterial cells. In contrast, generation of 50 to 100 C5b-9 complexes carrying multiple C9 molecules per CFU caused loss of viability. The failure of C5b-81C91 complexes to generate transmural pores was confirmed by measurements of o-nitrophenyl-beta-D-galactoside influx into the cells. Whereas treatment of C5b-8-laden cells with C9 at 32 degrees C caused virtually instantaneous influx of the marker, almost no influx was registered in cells receiving C9 at 0 degrees C. When cells carrying C5b-7 were brought into the stationary phase and given C8 and C9 at 32 degrees C, a C9-dependent disruption of the outer membrane permeability barrier immediately occurred as demonstrated by cleavage of a chromogenic substrate by periplasmic beta-lactamase. In sharp contrast, o-nitrophenyl-beta-D-galactoside influx was markedly retarded over a prolonged period, with abrupt permeability increases of the inner membrane toward this molecule being noted just before bacterial cell division occurred. We conclude that killing of E. coli requires binding of C5b-9 complexes containing C9 oligomers to the outer membrane and suggest that formation of pores in the inner membrane occurs when these complexes are "hit" by transiently forming zones of bioadhesion. Formation of the latter may be a dynamic process that is accentuated during cell division and quiescent during the stationary phase.
Role of sialic acid in saliva-mediated aggregation of Pseudomonas aeruginosa isolated from cystic fibrosis patients
Authors: Komiyama K , Habbick B F , Tumber S K
Published: January 2020

The mechanism of saliva-mediated aggregation of Pseudomonas aeruginosa in subjects with and without cystic fibrosis (CF) was investigated. Virtually all saliva from CF patients that we tested strongly agglutinated the Pseudomonas cells and was heat stable to 56 degrees C, whereas saliva from subjects without CF had a decreased aggregating ability and was heat sensitive. When saliva was treated with neuraminidase and proteases, and also when P. aeruginosa cells were treated with mixed gangliosides, there was a decrease in aggregating activities. However, neither the addition of the acid-hydrolyzed ganglioside nor the treatment of the P. aeruginosa cells by sugars had any effect on subsequent aggregating activities. Therefore, the release of sialic acid by enzymatic treatments of saliva, as well as the blockage of the sialic acid-binding sites on the cell wall by mixed gangliosides, resulted in the parallel loss of saliva-mediated aggregating activity of P. aeruginosa. The level of free sialic acid released by endogenous neuraminidase was higher in the saliva from CF patients than in that from the non-CF subjects examined. The increased aggregation of P. aeruginosa mediated by saliva from patients with CF seems to be directly related to the sialic acid content present, suggesting that this acid molecule acts as the salivary receptor for P. aeruginosa.
Comparative analysis of the localization of lipoteichoic acid in Streptococcus agalactiae and Streptococcus pyogenes
Authors: Mattingly S J , Johnston B P
Published: January 2020

The cellular locations of deacylated lipoteichoic acid (dLTA) and lipoteichoic acid (LTA) were examined in late-exponential-phase cells of a serotype III strain of Streptococcus agalactiae (group B streptococci [GBS]) isolated from an infant with late-onset meningitis and compared with a fresh clinical isolate of Streptococcus pyogenes (group A streptococci [GAS]). LTA and dLTA were found to be associated with the protoplast membranes of both organisms, with only dLTA found in mutanolysin cell wall digests. Both organisms released dLTA during growth, but only the GAS released substantial levels of LTA into the culture medium. However, penicillin treatment (5 micrograms/ml for 60 min) of GBS resulted in the recovery of LTA in cell wall digests as well as in the culture medium. These results suggest that under normal growth conditions, the hydrophobic region (glycolipid) of LTA remains associated with the cytoplasmic membrane of GBS and unavailable for hydrophobic interactions at the cell surface with epithelial cells. In contrast, release of LTA into the environment by the GAS allows the fatty acid moieties to interact with hydrophobic domains on the surface of epithelial cells. These results may help explain the marked differences in the specificity of binding between these two major streptococcal pathogens for human fetal and adult epithelial cells.
Interaction of soluble fibronectin with group B streptococci
Authors: Butler K M , Baker C J , Edwards M S
Published: January 2020

Fibronectin binds to a variety of bacterial species, and we hypothesized that differential fibronectin binding might influence the invasive potential of group B streptococci (GBS). Human plasma fibronectin purified by a standard two-step chromatographic procedure was radiolabeled with 3H. Fifty GBS strains (invasive, colonizing, or bovine) representing serotypes Ia (10 strains), Ib (6 strains), Ia/c (6 strains), II (10 strains), III (11 strains), IV (1 strain), and nontypable (6 strains) were tested. No source or serotype variability was detected among GBS strains, and binding was uniformly less than 1.5% of available fibronectin. Lack of detectable binding occurred at both the log and stationary growth phases and persisted despite treatment with trypsin or neuraminidase or opsonization with immunoglobulin G containing high levels (greater than 40 micrograms/ml) of antibody specific for the Ia, II, or III GBS capsular polysaccharides. Incubation with GBS did not inhibit fibronectin binding to the Cowan 1 strain of Staphylococcus aureus. Strain COH 31-15, an isogenic, type III, capsule-deficient mutant of COH 31r/s, also failed to bind fibronectin. In contrast to other streptococci, GBS do not have readily detectable receptors for soluble fibronectin as part of their surface structures. If present, binding sites for soluble fibronectin are deep to surface structures, obscured from host defense systems, or require the presence of other factors to facilitate their recognition of fibronectin. The uniform ability of GBS to resist binding to soluble fibronectin could be a significant virulence factor in the pathogenesis of invasive infections of infants.
Lipid X ameliorates pulmonary hypertension and protects sheep from death due to endotoxin
Authors: Golenbock D T , Will J A , Raetz C R , Proctor R A
Published: January 2020

Lipid X (2,3-diacylglucosamine-1-phosphate) is a novel monosaccharide precursor of lipid A that has some of the physiologic activities of endotoxin but little toxicity. To determine whether lipid X would interfere with the toxic effects of endotoxin, we pretreated sheep with either 100 or 200 micrograms of lipid X per kg of body weight and then challenged them with a potentially fatal dose of Escherichia coli endotoxin (20 micrograms/kg). Twenty-one sheep underwent pulmonary artery catheterization and were monitored for changes in pulmonary artery pressure, temperature, pH, partial O2 pressure, partial CO2 pressure, blood pressure, and cell counts over 7 h. Overall mortality for control animals was 37% versus 5.3% for pretreated animals. None of the 13 animals pretreated with 100 micrograms of lipid X per kg died. These differences in survival were significant (P less than 0.05). Animals pretreated with 100 micrograms of lipid X per kg had significantly lower pulmonary artery pressure during both phases 1 and 2 of endotoxin-induced pulmonary artery hypertension. A higher dose of lipid X, 200 micrograms/kg, produced pulmonary hypertension. Perhaps because lipid X is a subunit of lipid A, lipid X shows a partial pyrogenic effect while also decreasing the pyrogenic activity of complete lipopolysaccharide (LPS). Lipid X did not prevent endotoxin-induced neutropenia or moderate hypotension in response to LPS. Lipid X is a potential prototype compound for a new type of chemotherapy directed at blocking the harmful effects of LPS during bacterial septicemia.
Pulmonary clearance of Mycoplasma pulmonis in C57BL/6N and C3H/HeN mice
Authors: Parker R F , Davis J K , Blalock D K , Thorp R B , Simecka J W , Cassell G H
Published: January 2020

In C57BL/6N and C3H/HeN mice known to be free of all murine pathogens and matched for age, sex, and environmental factors, pulmonary clearance was measured over a 72-h time period after exposure to infectious aerosols of 35S-labeled Mycoplasma pulmonis. Reduced clearance of M. pulmonis in C3H/HeN mice relative to C57BL/6N mice was primarily due to impaired mycoplasmacidal activity in the lungs of the C3H/HeN mice. The C3H/HeN mice also had a slightly slower rate of mechanical transport of radiolabel from the lungs in the first 4 h after infection relative to the C57BL/6N mice but not at any later times. By 72 h after infection (relative to 0 h, C3H/HeN mice had an over 4,000% (1.75 X 10(7) versus 4.30 X 10(5] increase in neutrophils and an over 18,000% (more than 2 orders of magnitude) increase in numbers of M. pulmonis recovered from mechanically disaggregated lungs. In contrast, C57BL/6N mice reduced the number of M. pulmonis present by over 83% (nearly 2 orders of magnitude) before any increase in inflammatory cells, which was only a slight increase in lymphocytes and macrophages at 24 h after infection. These results directly link decreased mycoplasmal pulmonary clearance in C3H/HeN mice with the increased susceptibility to, and severity of, murine respiratory mycoplasmosis observed in this strain. The resistance of C57BL/6N mice appears to be related to nonspecific host defense mechanisms responsible for limiting the extent of infection.
Ability of intestinal Escherichia coli to survive within mesenteric lymph nodes
Authors: Wells C L , Maddaus M A , Jechorek R P , Simmons R L
Published: January 2020

Identification of mesenteric lymph node (MLN) bacteria showed that indigenous streptomycin-sensitive Escherichia coli could be recovered from MLN at least 48 h after this organism had been essentially eliminated from the cecal flora by antibiotics and replaced with exogenous streptomycin-sensitive E. coli JK. Additional experiments with antibiotic-treated rats also showed that indigenous streptomycin-sensitive E. coli could be recovered from the MLN 4 days after elimination of this organism from the cecal flora. These findings suggest that the time of bacterial translocation to MLN may be kinetically different from the time of recovery of bacteria from MLN and that the MLN may be a focus of infection with intestinal bacteria.
Colonial dissociation and susceptibility to phagocytosis of Pseudomonas aeruginosa grown in a chamber implant model in mice
Authors: Kelly N M , Battershill J L , Kuo S , Arbuthnott J P , Hancock R E
Published: January 2020

Pseudomonas aeruginosa strains were grown in 1-cm plastic chambers sealed at both ends with porous Millipore filters and implanted in the peritonea of mice. Mucoid and nonmucoid strains of P. aeruginosa isolated from a patient with cystic fibrosis largely retained their phenotypes when grown for up to 1 year in this in vivo system, although colonial dissociation occurred, as observed in chronic lung infections of patients with cystic fibrosis. In the absence of added opsonins, P. aeruginosa M2 cells taken directly from the in vivo system were significantly more susceptible to phagocytosis than were the same P. aeruginosa cells after being washed in buffer. Phagocytosis of in vivo-grown P. aeruginosa cells could be further enhanced by using a porin protein F-specific monoclonal antibody.
Inhibition by 1,25(OH)2-vitamin D3 of the multiplication of virulent tubercle bacilli in cultured human macrophages
Authors: Crowle A J , Ross E J , May M H
Published: January 2020

Historically, sunlight has seemed to fortify antituberculosis resistance. Evidence is presented here suggesting a role for vitamin D in this effect. The active metabolite of this photosynthesized vitamin, 1,25-dihydroxy-vitamin D3 (1,25D), promotes maturation and activation of human monocytes and macrophages (MPs). Therefore, it was tested for ability to protect MPs against virulent tubercle bacilli. MPs were derived by 7-day culture from blood monocytes, infected with the bacilli, and exposed to 1,25D in several regimens. Their inhibition of bacilli was measured by lysing samples of the cultures at 0, 4, and 7 days after infection and making bacillary CFU counts from serial dilutions of the lysates. 1,25D enabled MPs to slow or stop bacillary replication. Autologous serum supported the 1,25D-induced protection because the vitamin was not effective in medium supplemented with a serum substitute and was less effective in a heterologous AB serum than in autologous serum. The protection developed rapidly and could be induced even when 1,25D was added 3 days after infection. A concentration on the order of 4 micrograms/ml was needed for protection by the regimens used in these experiments. That is considerably higher than normal circulating concentrations of 1,25D but could be reached in infectious granulomas, because MPs can make 1,25D from precursor 25-hydroxyvitamin D3. The precursor circulates at levels 10(3) higher than those of 1,25D and is directly influenced by dietary intake or photosynthetic production of vitamin D. These results identify 1,25D as an immunomodulator which can reproducibly activate human MPs to express tuberculoimmunity. They connect vitamin D, sunlight, and tuberculoimmunity and suggest that vitamin D should be considered a vital factor in the practical control of tuberculosis.
Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic
Authors: Curtiss R , Kelly S M
Published: January 2020

Salmonella typhimurium SR-11 mutants with cya::Tn10 or crp::Tn10 mutations were found to be avirulent and immunogenic for BALB/c mice. Fusaric acid-resistant derivatives with deletions of the Tn10 and adjacent DNA sequences were constructed in S. typhimurium SR-11 strains with or without the virulence plasmid pStSR100. These delta cya delta crp strains grew more slowly than wild-type strains. They possessed wild-type ability to attach to, invade, and persist in gut-associated lymphoid tissue for up to a week but exhibited a diminished ability to reach mesenteric lymph nodes and the spleen. Mice 4 to 8 weeks old were resistant to oral infection with 10(9) cells of several different delta cya and delta cya delta crp strains (the equivalent to 10(4) 50% lethal doses of wild-type S. typhimurium SR-11) and 30 days after immunization became resistant to oral challenge with 10(3) to 10(4) 50% lethal doses of wild-type S. typhimurium SR-11.
Elaboration by mammalian mesenchymal cells infected with Trypanosoma cruzi of a fibroblast-stimulating factor that may contribute to chagasic cardiomyopathy
Authors: Wyler D J , Libby P , Prakash S , Prioli R P , Pereira M E
Published: January 2020

Myocardial fibrosis can occur as a complication of chronic infection of the heart with Trypanosoma cruzi (Chagas' disease) and can lead to serious disability. To assess whether there might be a direct relationship between intracellular parasitization and subsequent tissue fibrosis in this disease, we tested serum-free conditioned media from cultures of fibroblasts, vascular smooth-muscle cells, and myocardial cells for fibroblast-stimulating activity. Conditioned media from all infected cultures, but not from uninfected cultures, stimulated fibroblast [3H]thymidine incorporation, DNA and protein synthesis, and cell proliferation. Fibroblast-stimulating activity was also detected in extracts of amastigotes but not of trypomastigotes or epimastigotes. We conclude that parasitization of mesenchymal cells, including myocardial cells, results in elaboration of a fibroblast-stimulating factor(s), perhaps of parasite origin. We postulate that this factor may play a role in initiation of myocardial fibrosis in Chagas' disease.
Selective pressures and lipopolysaccharide subunits as determinants of resistance of clinical isolates of gram-negative bacilli to human serum
Authors: Porat R , Johns M A , McCabe W R
Published: January 2020

Differences in molecular composition of lipopolysaccharides (LPS) between serum-sensitive (S) clinical isolates of Escherichia coli and serum-resistant (R) clones derived by serial passage in serum were demonstrated to determine sensitivity or resistance to killing by normal human serum (NHS). LPS from R clones had a greater proportion of higher-molecular-weight, more highly O-antigen-substituted subunits than LPS from their serum S parents. Utilization of a liposomal model with inserted LPS simulating bacterial cell walls established LPS as the site of serum bactericidal action. Liposomes containing S LPS were lysed, while liposomes containing R LPS were unaffected by NHS. R and S LPS were fractionated into higher (F1)- and lower (F2)-molecular-weight fractions. Liposomes containing R LPS or the F1 fraction of S and R LPS were not lysed by serum. Liposomes containing the F2 fraction of S or R LPS were lysed by serum analogous to that observed with liposomes containing intact S LPS. These findings establish LPS to be one site of serum bactericidal activity and demonstrate that the higher-molecular-weight, highly O-antigen-substituted LPS subunits mediate resistance to killing by NHS.