bluuebunny/crossref_metadata_2025 huggingface dataset:
Pseudomonas aeruginosa elastase does not inactivate alpha 1-proteinase inhibitor in the presence of leukocyte elastase
Authors: Padrines M , Bieth J G
Published: January 2020

Pseudomonas aeruginosa elastase rapidly inactivates alpha 1-proteinase inhibitor by splitting its Pro-357-Met-358 peptide bond. The present study was aimed at testing whether this reaction takes place in the presence of leukocyte elastase. To this end was added alpha 1-proteinase inhibitor to a mixture of the two elastases, and we performed the following assays: (i) measurement of the residual leukocyte elastase activity, (ii) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) immunoassay of the leukocyte elastase-alpha 1-proteinase inhibitor complex. These experiments were done with various concentrations of the three proteins. All experiments gave the same result: leukocyte elastase was fully inhibited by alpha 1-proteinase inhibitor in the presence of P. aeruginosa elastase even when the bacterial enzyme was 10-fold more concentrated than the neutrophil enzyme. We also measured the initial rate of the P. aeruginosa elastase-catalyzed inactivation of alpha 1-proteinase inhibitor as a function of the inhibitor concentration. The kcat/Km value derived from this experiment was 9 x 10(4) M-1 s-1, a value much lower than the rate constant for the leukocyte elastase-inhibitor association (kass, 1.7 x 10(7) M-1 s-1). This rationalizes the above results. In conclusion, when alpha 1-proteinase inhibitor is faced with its target enzyme, leukocyte elastase, it will perform its physiologic antielastase function even if the bacterial elastase is present in excess.
Purification of a Streptococcus mutans protein that binds to heart tissue and glycosaminoglycans
Authors: Choi S H , Stinson M W
Published: January 2020

Proteins of Streptococcus mutans MT703 were isolated by differential filtration from chemically defined culture medium following growth of the bacteria. Incubation of this preparation with cryostat-cut sections of fresh rabbit cardiac muscle resulted in deposition of streptococcal components on basement membranes of sarcolemmal sheaths and capillary walls, as indicated by indirect immunofluorescence assay. Binding of radioiodinated streptococcal proteins to heart in vitro was time dependent and saturable. Unlabeled S. mutans proteins competitively inhibited 72% of heart binding by the radiolabeled proteins, indicating a high level of binding specificity. A selection of components common to tissue basement membranes was tested for their abilities to inhibit the binding of streptococcal proteins to heart tissue. Of the glycosaminoglycans, heparin was the most effective inhibitor, followed by heparan sulfate and chondroitin sulfate. Hyaluronic acid was not inhibitory. Of the glycoproteins tested, laminin and collagen type IV were weakly inhibitory, whereas fibronectin was ineffective. A single polypeptide was purified to homogeneity by affinity chromatography on a column of heparin-agarose. Gel filtration chromatography of the purified protein under nondissociating conditions showed a single component at 31 kilodaltons (kDa), whereas in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one band appeared at 8 kDa. This indicates that the tissue-binding protein may either be a linear polypeptide or be released into the environment by the bacterium as a tetramer of the 8-kDa polypeptide. The purified protein had an isoelectric point of 9.5 and showed binding activity for basement membranes in thin sections of heart. Chemical analyses of the purified binding protein showed it to have high contents of lysine and alanine and to be devoid of half-cystine, methionine, tyrosine, histidine, and both neutral and amino sugars.
Isolation and characterization of a human monoclonal antibody that recognizes epitopes shared by Pseudomonas aeruginosa immunotype 1, 3, 4, and 6 lipopolysaccharides
Authors: Lang A B , Fürer E , Larrick J W , Cryz S J
Published: January 2020

A hybridoma line secreting a human monoclonal antibody (HMAb) capable of recognizing Fisher immunotype (IT) 1, 3, 4, and 6 lipopolysaccharide (LPS) in vitro was isolated. Peripheral blood lymphocytes (PBL) were obtained from volunteers immunized with an experimental Pseudomonas aeruginosa O polysaccharide-toxin A vaccine. PBL-expressing surface antibodies able to bind to P. aeruginosa LPS were isolated by adsorption onto LPS-coated plastic wells. Such PBL were transformed with Epstein-Barr virus. Lymphoblastoid cell lines secreting anti-P. aeruginosa LPS antibodies were identified by an enzyme-linked immunosorbent assay and fused to the F3B6 heteromyeloma line. A hybridoma line producing a HMAb (2-8AH79) able to bind IT-1, IT-3, IT-4, and IT-6 LPS was identified by an enzyme-linked immunosorbent assay. This HMAb was found to bind to the O-polysaccharide regions of IT-1, IT-3, IT-4, and IT-6 LPS, as determined by immunoblotting. By using an immunofluorescence microscopy assay, the cell surfaces of IT-3 and IT-4 bacteria were strongly stained by HMAb 2-8AH79, whereas those of IT-1 and IT-6 bacteria were weakly stained. This HMAb was found to promote the uptake and killing of IT-3 and IT-4 bacteria, but not IT-1 or IT-6 organisms, by human polymorphonuclear leukocytes. Similarly, the passive transfer of HMAb 2-8AH79 to mice afforded significant protection only against a challenge with IT-3 and IT-4 bacteria.
Construction and characterization of a Vi-positive variant of the Salmonella typhi live oral vaccine strain Ty21a
Authors: Cryz S J , Fürer E , Baron L S , Noon K F , Rubin F A , Kopecko D J
Published: January 2020

The viaB locus coding for the Vi antigen of Salmonella typhi Ty2 was cloned on a 40.6-kilobase fragment into the cosmid vector pHC79. The live, oral, attenuated Vi-negative S. typhi Ty21a vaccine strain was transformed with the recombinant cosmid encoding the viaB locus. Homologous recombination of the viaB locus into the chromosome of S. typhi Ty21a was induced by UV irradiation, and Vi-positive recombinants were selected in the presence of D-cycloserine. One such isolate, termed WR4103, contained no plasmids or the attendant antibiotic resistance markers and expressed the Vi antigen stably. Vi antigen extracted from WR4103 was immunologically indistinguishable from Vi antigen purified from S. typhi Ty2. The only detectable difference between Ty21a and WR4103 was in the production of Vi antigen. The mean lethal doses of Ty21a and WR4103 for mice were nearly identical. Immunization of mice with WR4103 engendered a Vi antibody response and afforded complete protection against fatal infection with virulent S. typhi Ty2. Thus, S. typhi WR4103 may serve as an improved oral vaccine for protection against typhoid fever.
Characterization of Cryptococcus neoformans capsular glucuronoxylomannan polysaccharide with monoclonal antibodies
Authors: Todaro-Luck F , Reiss E , Cherniak R , Kaufman L
Published: January 2020

Mice were immunized with Cryptococcus neoformans serotype A capsular glucuronoxylomannan (GXM) conjugated to bovine serum albumin-adipic dihydrazide. Two splenocyte fusions yielded two monoclonal antibodies (MAbs) that were highly reactive in dot enzyme immunoassay, immunofluorescence, and sandwich enzyme immunoassay. The first MAb, BD-1 [immunoglobulin G1 (kappa) [IgG1(kappa)]], was GXM-A and GXM-D specific, whereas the second MAb, BA-4 (IgM), reacted with GXM-A and GXM-B. A third MAb, CD-6 [IgG1(kappa)], originated from mice immunized with O-deacetylated GXM-C-bovine serum albumin and reacted with GXMs of all four serotypes. Two of the MAbs (CD-6 and BD-1) were further characterized with chemically modified GXMs. Removal of glucuronosyl residues completely inhibited the binding of both MAbs, implicating (1----2)-beta-glucuronic acid as a key component of the epitope. Removal of (1----2)-beta-xylosyl residues decreased reactivity to an intermediate extent. O deacetylation led to a measurable decrease but had the least inhibitory effect of the three GXM derivatives tested. The combining site for these two MAbs appears to be a complex antigenic determinant involving more than one glycosidic residue.
O antigen and lipid A phosphoryl groups in resistance of Salmonella typhimurium LT-2 to nonoxidative killing in human polymorphonuclear neutrophils
Authors: Stinavage P , Martin L E , Spitznagel J K
Published: January 2020

We have compared the intraleukocytic survival of isogenic strains of Salmonella typhimurium, whose outer membrane lipopolysaccharide differed in O antigen and lipid A composition and whose susceptibility to nonoxidative antimicrobial granule proteins of human polymorphonuclear neutrophilis (PMN) could be established. We found that the order of resistance to the bactericidal activity of intact PMN of the three bacterial strains utilized closely resembled their ordered resistance to the purified human cationic antimicrobial 57,000-dalton protein (CAP57). LT-2, a smooth wild-type strain, was far more resistant than SH9178, its rough (Rb LPS) mutant. It was most significant that SH7426, a polymyxin B-resistant pmrA mutant of SH9178, not only was substantially more resistant to CAP57 and to intraphagocytic killing than SH9178 but also came close to being as resistant as LT-2. These experiments confirm earlier work that showed the importance of the glycosyl groups of O antigens of S. typhimurium for their resistance to O2-independent antimicrobial phagocytosis by PMN. The surprising result was that a rough strain, very susceptible to bactericide, became substantially more resistant when a mutation led to its lipid A phosphoryl groups being 100% substituted with amino pentoses. Yet unresolved is whether the protection is due to the loss of negative charges on the lipid A, the substitution of sugar molecules in vulnerable loci in the outer membrane, or both.
Structure and copy number of gene clusters related to the pap P-adhesin operon of uropathogenic Escherichia coli
Authors: Arthur M , Campanelli C , Arbeit R D , Kim C , Steinbach S , Johnson C E , Rubin R H , Goldstein R
Published: January 2020

The structurally related pap and prs operons of the uropathogenic Escherichia coli isolate J96 encode a P and an F adhesin that mediate bacterial attachment to the human P blood group antigen and the Forssman antigen, respectively. Using probes prepared from different segments of the pap operon, Southern blot hybridizations were performed to characterize pap-related sequences of 30 E. coli clinical isolates expressing different adhesin phenotypes. Gene clusters encoding P and F adhesins displayed no restriction site polymorphism in sequences homologous to the papH, papC, and papD genes that encode proteins essential to the transport and polymerization of the subunits of the P-pilus adhesin. In contrast, pap-related genetic elements associated with a null phenotype either lacked homology to the papH, papC, and papD genes or displayed a restriction site polymorphism in this region. Sequences within and surrounding the J96 papG and prsG adhesin genes that determine the binding specificities to the P and F antigens, respectively, were not conserved. However, gene clusters encoding different binding specificities could not be distinguished based on such restriction site polymorphisms. The majority of clinical isolates had more than one copy of pap-related sequences that involved gene clusters similar to the J96 pap operon, as well as genetic elements that were related only to a part of this operon. The implications of this unexpected copy number polymorphism with respect to possible recombination events involving pap-related sequences are discussed.
Hemagglutination activities of group B, C, D, and G streptococci: demonstration of novel sugar-specific cell-binding activities in Streptococcus suis
Authors: Kurl D N , Haataja S , Finne J
Published: January 2020

A total of 378 streptococcal isolates of Lancefield groups B, C, D and G were tested for their ability to hemagglutinate untreated, sialidase-treated, and endo-beta-galactosidase-treated human erythrocytes. Of the 43 strains showing positive hemagglutination, 9 were inhibitable with neutral monosaccharides. Four strains were inhibited with galactose and N-acetylgalactosamine, whereas five were inhibited with galactose only. A third, sialic acid-specific adhesion activity was suggested for two additional strains on the basis of their agglutination of native and endo-beta-galactosidase-treated but not sialidase-treated erythrocytes. All the sugar-specific agglutination activities detected were confined to Streptococcus suis strains of group D streptococci, whereas streptococci of other groups did not exhibit these types of hemagglutination activities. The adhesins were sensitive to proteases and heat treatment, which indicates that they were proteins. The hemagglutinating isolates of S. suis originated from pig brain and lung, human brain, and the tonsils of healthy pigs. No clear correlation with a particular serotype was observed. These results demonstrate the occurrence of unique sugar-specific adherence activities in S. suis, an important pig pathogen with occasional human pathogenicity.
Human immune response to Vibrio cholerae O1 whole cells and isolated outer membrane antigens
Authors: Richardson K , Kaper J B , Levine M M
Published: January 2020

The serum immunoglobulin G (IgG) and mucosal secretory IgA (SIgA) response of human volunteers challenged with Vibrio cholerae O1 was analyzed for reactivity to V. cholerae O1 antigens by the immunoblot technique. Components of both in vitro- and in vivo (rabbit ligated ileal loop)-grown V. cholerae O1 were separated by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Postchallenge serum IgG reacted uniquely with 15 antigens and with greater intensity than did prechallenge serum with at least 16 antigens. Serum IgG and SIgA reacted with antigens present in preparations from the homologous challenge strain of V. cholerae as well as antigens from strains of heterologous biotype or serotype. These heterologous antigens may represent antigens responsible for protection to rechallenge with a heterologous strain of V. cholerae. All the antigens detected by postchallenge jejunal fluid SIgA had an apparent molecular size of less than 25 kilodaltons. Serum IgG and jejunal fluid SIgA also reacted with antigens unique to in vivo-grown cells and several antigens in outer membrane preparations, suggesting that studies of protective immunity and V. cholerae O1 pathogenesis should include examination of both in vitro- and in vivo-grown V. cholerae O1 cellular antigens.
Control of Escherichia coli populations by a combination of indigenous clostridia and lactobacilli in gnotobiotic mice and continuous-flow cultures
Authors: Itoh K , Freter R
Published: January 2020

The function of indigenous lactobacilli in the control of other intestinal microbial species is not clear. Still more controversial is the effect of dietary bacterial supplements containing lactobacilli or other species. This situation is unlikely to change unless the mechanisms that control the colonization of ingested bacteria are better understood, and until more detailed information becomes available on the mechanisms by which certain populations of indigenous bacteria can affect the population sizes of other species. We used gnotobiotic mice and a continuous-flow culture system to study the interactions between Escherichia coli and (i) clostridia (in chloroform-treated cecal suspensions from conventional mice) and (ii) three strains of lactobacilli isolated from conventional mice. In gnotobiotic mice, the lactobacilli suppressed E. coli multiplication in the stomach and the small intestine, but had no demonstrable effect on E. coli multiplication in the large intestine. In contrast, clostridia were most effective in controlling E. coli multiplication in the large intestine. In the presence of both lactobacilli and clostridia, E. coli populations in the various regions of the gastrointestinal tract resembled those found in conventionalized control animals. The control of E. coli populations was not related to changes in pH or intestinal motility. In vitro stimulation of the above-described in vivo interactions required a two-stage continuous-flow culture in which the effluent from the first stage represented the influx to the second. The first stage was inoculated with lactobacilli, and the second stage was inoculated with either a pure culture of E. coli or E. coli and clostridia. In these instances, the E. coli populations in the second stage of the culture resembled in size those found in the large intestine of gnotobiotic mice harboring a similar flora. Although there are some current shortcomings of this in vitro model, we expect that a multistage continuous-flow culture can be developed to satisfactorily model the interactions among bacterial populations along the entire gastrointestinal tract.
Molecular cloning and expression of Campylobacter pylori species-specific antigens in Escherichia coli K-12
Authors: Clayton C L , Wren B W , Mullany P , Topping A , Tabaqchali S
Published: January 2020

A gene bank of Campylobacter pylori DNA in Escherichia coli was constructed by cloning Sau3A-cleaved DNA fragments into the bacteriophage vector lambda EMBL3. The expression of C. pylori antigens was determined by screening the gene library with adsorbed C. pylori whole-cell rabbit antisera. One recombinant clone which reacted positively (lambda CP2) was studied further. Immunoblot analysis with lambda CP2 showed a polypeptide band of 66 kilodaltons (kDa) reacting antigenically with the adsorbed antiserum. Extraction of DNA from lambda CP2 and digestion with SalI revealed a DNA insert of 17 kilobases (kb). Subcloning with SalI and the E. coli vector pUC18 showed that the DNA also encoded a 31-kDa antigen. The cloned antigens were shown by immunoblotting to have the same molecular weight in E. coli as in C. pylori and to be present in all C. pylori strains. Antiserum was raised against the cloned polypeptides and found to react only with C. pylori when analyzed by dot blotting and indirect immunofluorescence. The cloned antigens were determined to be expressed from the pUC18 lac promoter. The DNA encoding these antigens was radiolabeled with 32P and found to hybridize only to C. pylori strains. Immunoblotting with affinity-purified polyclonal antibody to the urease enzyme of C. pylori revealed that the cloned antigens may be part of the urease enzyme.
Immunoglobulin-binding activity among pathogenic and carrier isolates of Haemophilus somnus
Authors: Widders P R , Dorrance L A , Yarnall M , Corbeil L B
Published: January 2020

Nonimmune binding of immunoglobulin to whole bacteria was quantitated for North American isolates of Haemophilus somnus recovered from cattle with pneumonia, reproductive failure (abortion), or thromboembolic meningoencephalitis or from the vagina or prepuce of carrier cattle. Quantitative binding activity covered a wide range, with most pathogenic and carrier isolates demonstrating significant immunoglobulin-Fc binding. Isolates for which Fc binding was not detectable were recovered only from the prepuces of asymptomatic bulls. Expression of Fc-binding activity correlated with the presence of the 41,000-molecular-weight protein (41K protein) and 270K protein. Isolates that lacked Fc-binding activity did not possess 41K or 270K protein. A 33K protein was detected in isolates that lacked Fc-binding activity but not in isolates that bound Fc.
Antagonism exerted by an association of a Bacteroides thetaiotaomicron strain and a Fusobacterium necrogenes strain against Clostridium perfringens in gnotobiotic mice and in fecal suspensions incubated in vitro
Authors: Yurdusev N , Ladire M , Ducluzeau R , Raibaud P
Published: January 2020

Antagonism between an association of Bacteroides thetaiotaomicron and Fusobacterium necrogenes strains and two strains of Clostridium perfringens was evidenced both in vivo in gnotobiotic mice and ex vivo in fecal suspensions incubated for 22 h at 37 degrees C. Several features of this antagonism were similar in and ex vivo. (i) An obligate and continuous synergy between B. thetaiotaomicron and F. necrogenes was required; (ii) the two C. perfringens strains did not respond to the same extent to this antagonism; and (iii) expression of the antagonism was host and diet dependent. Neither diffusible nor soluble inhibitory substances were detectable in feces of gnotobiotic mice, nor could depletion of nutrients be identified as causing antagonism in both in and ex vivo experiments. Our findings support the hypothesis that a reversible bacteriostasis induced by the inhibitory strains acting together continuously, and hindering the target strain from utilizing available nutrients, was responsible for this antagonism.
Energy is required for maximal adherence of Neisseria gonorrhoeae to phagocytic and nonphagocytic cells
Authors: Weber R D , Britigan B E , Svendsen T , Cohen M S
Published: January 2020

The possibility that gonococcal energy might play a role in the interaction of Neisseria gonorrhoeae with both phagocytic and nonphagocytic cells was examined. Respiratory chain inhibitors including KCN and amobarbital resulted in reduction in gonococcal association with human neutrophils. Similar results were seen with HeLa cells and the human promyelocytic (HL-60) cell line. Identical conditions did not affect the opsonin-dependent association of Staphylococcus aureus with the same cell types. New protein synthesis by gonococci did not account for the observed reduction in association. These results suggest that energy is needed for maximal opsonin-independent association of gonococci with mammalian cells.
Adjuvant effects of trehalose dimycolate on the antibody response to type III pneumococcal polysaccharide
Authors: Baker P J , Fauntleroy M B , Stashak P W , Hiernaux J R , Cantrell J L , Rudbach J A
Published: January 2020

Treatment with trehalose dimycolate (TDM) increases the magnitude of the immunoglobulin M (IgM) antibody response of mice to type III pneumococcal polysaccharide (SSS-III). Such enhancement is demonstrable over a wide range of immunizing doses and does not require thymus-derived (T) cells to be elicited. Although young adult mice immunized with SSS-III do not usually make anti-SSS-III antibodies of the IgG1 and IgG3 classes, antibodies of one or both isotypes were produced after immunization and treatment with TDM and/or monophosphoryl lipid A (MPL); the additive nature of the effect produced by both TDM and MPL suggests that the two immunomodulators act by different mechanisms. TDM and MPL have different effects on the induction and expression of low-dose immunological paralysis, a form of unresponsiveness known to be mediated by suppressor T cells. The relevance of these findings to the modes of action of TDM and MPL is discussed.
Antigenic analysis of Campylobacter species and an intracellular Campylobacter-like organism associated with porcine proliferative enteropathies
Authors: McOrist S , Boid R , Lawson G H
Published: January 2020

Whole-cell and outer membrane preparations of Campylobacter mucosalis, C. hyointestinalis, C. jejuni, and C. coli isolated from porcine intestines were compared with preparations of intracellular Campylobacter-like organisms extracted directly from the lesions of pigs with proliferative enteropathy. By gradient polyacrylamide gel electrophoresis, outer membrane and total protein profiles of C. mucosalis, C. hyointestinalis, C. jejuni, and C. coli were significantly different from each other and from those of the Campylobacter-like organisms. Immunoblotting of these preparations with rabbit antisera or monoclonal antibodies prepared against the intracellular Campylobacter-like organisms showed strong reactions only with a 25,000- to 27,000-molecular-weight component of preparations of the intracellular organisms. Antisera to cultivable Campylobacter species isolates did not react with preparations of intracellular organisms. Isoelectric focusing of sonicated preparations showed protein profile differences and an immune-reactive component in the intracellular organisms with a pI of 4.5. This study suggests that the intracellular Campylobacter-like organism associated with proliferative enteropathy may be a novel bacterium with significant antigenic differences from the Campylobacter species previously associated with the disease.
Adherence of Pseudomonas aeruginosa to tracheal epithelium
Authors: Marcus H , Austria A , Baker N R
Published: January 2020

Adherence of mucoid and nonmucoid strains of Pseudomonas aeruginosa to tracheal epithelium was studied with a perfused-trachea model. The species specificity of adherence was studied by infecting tracheas from hamsters, guinea pigs, or mice. Perfused tracheas from hamsters were infected with strains of P. aeruginosa in the presence of various sugars, lectins, cations, or charged polymers. Adherence of mucoid strains of P. aeruginosa was greatest for guinea pigs; that for hamsters and mice was approximately the same. Nonmucoid strains did not adhere well to epithelium from any of the species tested. N-Acetylglucosamine, galactose, and N-acetylneuraminic acid were the best inhibitors of adherence of mucoid strains of P. aeruginosa. Phaseolus vulgaris agglutinin and Arachis hypogaea agglutinin enhanced adherence of mucoid strains. Adherence of mucoid strains was also enhanced by the presence of Ca2+ in the incubation medium. Poly-L-lysine, poly-L-aspartic acid, and polyglycine inhibited adherence of a mucoid strain by 96, 86, and 52%, respectively. In general, the adherence of nonmucoid strains was not affected. The results indicate that carbohydrates are involved in the interaction of mucoid strains of P. aeruginosa with tracheal cells and that divalent cations may enhance this interaction. The lectin data show that lectins can interact with the mucoid organisms and the host and suggest that lectins may play a role in the adhesion process.
Modulation of the immune response to pneumococcal type 14 capsular polysaccharide-protein conjugates by the adjuvant Quil A depends on the properties of the conjugates
Authors: Verheul A F , Versteeg A A , De Reuver M J , Jansze M , Snippe H
Published: January 2020

Streptococcus pneumoniae type 14 capsular polysaccharide-bovine serum albumin (S14PS-BSA) conjugates were prepared by water-soluble-carbodiimide-mediated condensation with or without the use of N-hydroxy-sulfosuccinimide. The immunogenicities of the capsular polysaccharide (S14PS) and of the conjugates were studied in (CBA/N x BALB/c)F1 mice and in female BALB/c mice. The response in these mice indicates that S14PS could be classified as a thymus-independent type 2 antigen. Coupling of S14PS to BSA improved the immunogenicity of this polysaccharide, and an immunoglobulin G memory response was evoked. Conjugation with N-hydroxysulfosuccinimide resulted in a product with a higher polysaccharide/protein ratio. This conjugate induced a greater immune response than did the classical conjugate. Quil A enhanced the immune response to S14PS and to most S14PS-BSA conjugates. The enhancement of the immune response to the conjugates seemed to depend on the coupling procedure. Our results indicate that for the construction of immunostimulating complexes based on polysaccharide or oligosaccharide-protein conjugates, attention should be paid to the degree of cross-linking of the antigens involved.
Production of a monoclonal antibody specific for Mycobacterium avium and immunological activity of the affinity-purified antigen
Authors: Abe C , Saito H , Tomioka H , Fukasawa Y
Published: January 2020

Three hybridomas which secrete antibodies to Mycobacterium avium were obtained by the fusion of p3u1 myeloma cells with spleen cells of mice immunized with M. avium culture sonicate. The reactivity of these monoclonal antibodies was determined in 16 species of mycobacteria by an enzyme-linked immunosorbent assay. An antibody, designated Avi-3, reacted only with M. avium and not with the reference strains of the other 15 species of mycobacteria tested, including M. intracellulare, M. paratuberculosis, and M. lepraemurium. Specificity of the antibody was confirmed by assay, using a specific DNA probe of M. avium complex in 29 M. avium complex isolates. An antigen was purified from M. avium culture sonicate on a monoclonal antibody Avi-3-coupled affinity column. The purified antigen gave a single band (molecular size, about 27 kilodaltons) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen Avi-3 showed strong skin test activity in guinea pig preimmunized with heat-killed M. avium but not in those sensitized with heat-killed M. intracellulare or M. bovis BCG. Purified protein derivative elicited positive skin reactions in all the immunized guinea pigs. When heat-killed M. avium was used as the immunogen, strong lymphoproliferative responses were observed in cultures stimulated with the antigen Avi-3. These results suggest that M. avium-specific antigen Avi-3 may facilitate the diagnosis of mycobacterial infections.
Molecular cloning and nucleotide sequence of the colonization factor antigen I gene of Escherichia coli
Authors: Karjalainen T K , Evans D G , So M , Lee C H
Published: January 2020

The colonization factor antigen I (CFA/I) gene has been isolated and sequenced. The amino acid sequence of CFA/I deduced from the nucleotide sequence is composed of 170 amino acids. The first 23 amino acids are considered to be the signal peptide of the CFA/I protein since they are not present in the protein sequence. Among the remaining amino acids, only two are different from the protein sequence: amino acid position 76 is an aspartic acid instead of an asparagine, and position 97 is a serine instead of an alanine. The CFA/I gene has a typical Shine-Dalgarno sequence located 10 base pairs (bp) upstream from the initiation codon. The sequence TACAAT located 48 bp upstream from the initiation codon was tentatively designated the -10 sequence of the CFA/I gene promoter. No sequences homologous to the consensus -35 promoter sequence was found. A pair of inverted repeat sequences followed by a stretch of eight A's are located 45 bp downstream from the termination codon of the CFA/I gene; this region may be a rho-independent transcriptional terminator.
Failure of pertussis toxin to inhibit activation of guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) in the T84 cell line
Authors: Crane J K , Hewlett E L , Weikel C S
Published: January 2020

The heat-stable enterotoxin (STa) of Escherichia coli causes intestinal secretion by stimulating guanylate cyclase, an enzyme believed to be distinct from the STa receptor. Pertussis toxin (PT) has been reported to block the ability of STa to stimulate guanylate cyclase in rat intestinal mucosa (S. A. Epstein, R. A. Giannella, and H. J. Brandwein, FEBS Lett. 203:44-48, 1986). This suggested that a guanine nucleotide regulatory protein (G protein) coupled the STa receptor to guanylate cyclase, a function not previously recognized for G proteins. We sought to explore this phenomenon and, if possible, to identify this G protein. Initial experiments with the human colon carcinoma cell line T84 revealed that higher-than-expected concentrations (1 micrograms/ml) of PT were needed to intoxicate cells, as assessed by ADP-ribosylation of endogenous PT substrate, but that 99 to 100% intoxication could be achieved. Homogenates made from fully intoxicated cells did not differ from controls in basal or STa-stimulated guanylate cyclase activity, and cyclic GMP accumulation in intact T84 cells was not changed by PT treatment. We conclude that a PT-sensitive G protein is not involved in the stimulation of cyclic GMP production by the enterotoxin STa.
The gene coding for the 190,000-dalton iron-regulated protein of Yersinia species is present only in the highly pathogenic strains
Authors: Carniel E , Mercereau-Puijalon O , Bonnefoy S
Published: January 2020

A genomic library containing DNA fragments of 0.5 to 2 kilobase pairs in length from Yersinia enterocolitica serovar O:8 was constructed in a bacteriophage lambda gt11 expression vector. Mouse antibodies specific for the iron-regulated high-molecular-weight proteins (HMWPs) were used to screen the library. Two positive clones of 1 and 0.5 kilobase pairs, designated A13 and D7, respectively, were detected and isolated. They coded for beta-galactosidase fusion proteins of 151,000 and 138,000 daltons (Da). Antibodies affinity purified on the two recombinant lambda gt11 vectors specifically recognized the smaller HMWP (190,000 Da) and not the larger (240,000 Da). The two cloned DNA fragments were used to construct recombinant amplification plasmid pUC13 and to obtain large amounts of purified A13 and D7 inserts. Southern hybridizations performed with the inserts used as probes revealed that: (i) the two cloned DNA fragments overlap; (ii) only one gene hybridizes with the A13 and D7 inserts; (iii) the gene coding for the HMWP is conserved among all highly pathogenic Yersinia species studied; (iv) this gene is missing in the low-virulence and nonvirulent strains; and (v) transcription of the HMWP gene is induced by iron starvation.
Sialo-oligosaccharide receptors for Mycoplasma pneumoniae and related oligosaccharides of poly-N-acetyllactosamine series are polarized at the cilia and apical-microvillar domains of the ciliated cells in human bronchial epithelium
Authors: Loveless R W , Feizi T
Published: January 2020

The occurrence and distribution of the sialo-oligosaccharide receptors (sialosyl-I and sialosyl-i) for Mycoplasma pneumoniae as well as other related oligosaccharide structures of poly-N-acetyllactosamine type, and their short-chain analogs based on galactose linked beta 1-4 or beta 1-3 to N-acetylglucosamine (Gal beta 1-4GlcNAc or Gal beta 1-3GlcNAc, respectively) were investigated in the human bronchial epithelium by histochemistry by using sequence-specific monoclonal antibodies and lectins. Among the mature epithelial cells, only ciliated cells were found to express the long-chain antigens, whereas mucus-secreting cells contained the short-chain antigens associated with mucus globules. The long-chain oligosaccharides were found to be highly polarized at the luminal aspects of the ciliated cells where the branched structures (I and sialosyl-I antigens) were detected both at the apical-microvillar border and on the cilia, but the linear structures (i, sialosyl-i, and VIM-2 antigens) were detected exclusively at the apical-microvillar border. These observations provide the first in situ visualization of the receptor structures for M. pneumoniae at the primary site of infection. The lack of sialo-oligosaccharide receptors in secretory cells and the mucus they produce provides a biochemical basis for evasion by this microorganism of the secreted mucus barrier.
Construction of a plasmid for expression of foreign epitopes as fusion proteins with subunit B of Escherichia coli heat-labile enterotoxin
Authors: Schödel F , Will H
Published: January 2020

A novel vector (pFS2.2) for high-level expression of fusion polypeptides with the nontoxic subunit B (LT-B) of Escherichia coli heat-labile enterotoxin in Escherichia coli and salmonellae is presented. It carries the complete coding sequence of LT-B under lac promoter control and a universal polylinker site for the in-frame insertion of foreign genes at the LT-B gene 3' end. By using this vector, fusion proteins comprising parts of the human or woodchuck hepatitis B virus surface and nucleocapsid antigens are expressed in E. coli and salmonella.
Campylobacter-Wolinella group organisms are the only oral bacteria that form arylsulfatase-active colonies on a synthetic indicator medium
Authors: Wyss C
Published: January 2020

Most oral bacteria tested formed colonies on a chemically defined medium with a chromogenic arylsulfatase substrate. Arylsulfatase activity was, however, restricted to Campylobacter-Wolinella group organisms, including Wolinella recta, a possible periodontopathogen. W. recta was the only arylsulfatase-active species against which consistently high levels of antibody were detected in human sera.
Cloning of regions required for contact hemolysis and entry into LLC-MK2 cells from Shigella sonnei form I plasmid: virF is a positive regulator gene for these phenotypes
Authors: Kato J , Ito K , Nakamura A , Watanabe H
Published: January 2020

Two distinct regions required for both contact hemolysis and entry into LLC-MK2 cells were cloned into Escherichia coli from the Shigella sonnei form I plasmid, pSS120. The first region was cloned into an E. coli HB101 strain containing noninvasive Tn1 insertion mutants of the form I plasmid, and expression of ipa (invasion plasmid antigen) gene products was restored. The plasmid carrying the first region was then transformed into E. coli lacking the form I plasmid, and additional DNA fragments from the form I plasmid were cloned into the same recipient on compatible vectors. Five of these double transformants were found to be positive for contact hemolysis activity. Restriction analysis of these five clones indicated that the previously reported ipa locus and the invA locus were present on the second plasmid region. Only the strains carrying both of these regions were active in contact hemolysis and cell invasion assays. Several proteins, including the a, b, c, and d proteins encoded by the ipa genes, were detected in the double transformants by Western blot (immunoblot) analysis with serum of a monkey convalescing from shigellosis. A positive regulator was suggested to exist in the first region, since the amounts of most of these proteins were simultaneously increased in the presence of this region. Subcloning and nucleotide sequencing indicated that this positive regulator gene was virF. Product analysis of the virF gene with minicells showed that two peptides (30 and 21 kilodaltons) were synthesized and that at least the 30-kilodalton protein was essential for regulation of the ipa genes.
Saliva-mediated aggregation of Enterococcus faecalis transformed with a Streptococcus sanguis gene encoding the SSP-5 surface antigen
Authors: Demuth D R , Berthold P , Leboy P S , Golub E E , Davis C A , Malamud D
Published: January 2020

The interaction of a high-molecular-weight salivary glycoprotein (agglutinin) with Streptococcus sanguis M5 leads to the formation of bacterial aggregates. We have previously shown that the SSP-5 surface antigen from S. sanguis M5 binds the salivary agglutinin and therefore may be involved in the aggregation process. Here we report the transformation of a nonaggregating Enterococcus faecalis strain with the SSP-5 gene and show that the protein is expressed on the cell surface and confers an aggregation-positive phenotype. E. faecalis S161 protoplasts were transformed with pAM401 EB-5, a shuttle vector containing the S. sanguis SSP-5 gene, resulting in the isolation of E. faecalis S161EB-5. Crude cell extracts from this transformant and from S. sanguis M5 were analyzed by Western blotting. Extracts from S. sanguis M5 possessed peptides of 190 and 205 kilodaltons that reacted strongly with polyclonal antibodies against the recombinant SSP-5 antigen. E. faecalis S161EB-5 contained only the 190-kilodalton immunoreactive protein, suggesting that the antigen may be processed differently in E. faecalis S161EB-5. The parent strain, E. faecalis S161, did not react with this antibody preparation. Immunogold labeling of intact E. faecalis S161EB-5 and S. sanguis M5 with anti-SSP-5 immunoglobulin G showed that both organisms expressed similar levels of the antigen. Both organisms formed visible aggregates upon incubation with salivary agglutinin. These results suggest that the SSP-5 antigen may mediate both the binding of agglutinin to S. sanguis M5 and the subsequent formation of bacterial aggregates.
Immunomodulatory activity of monophosphoryl lipid A in C3H/HeJ and C3H/HeSnJ mice
Authors: Hiernaux J R , Stashak P W , Cantrell J L , Rudbach J A , Baker P J
Published: January 2020

Treatment with nontoxic monophosphoryl lipid A (MPL) derived from a polysaccharide-deficient, heptoseless Re mutant of either Salmonella typhimurium or Salmonella minnesota R595 enhanced the immunoglobulin M (IgM) anti-type III pneumococcal polysaccharide (SSS-III) antibody response of C3H/HeSnJ mice. Such an adjuvant effect was not observed in lipopolysaccharide-nonresponder C3H/HeJ mice. Nevertheless, C3H/HeJ spleen cells produced a weak mitogenic response to both preparations of MPL in vitro, and C3H/HeJ mice showed a significant increase in serum IgM levels without an increase in numbers of splenic IgM-secreting plaque-forming cells after in vivo treatment with MPL. A significant increase in serum IgG3 levels was accompanied by a transient decrease in serum IgG1 levels in C3H/HeSnJ mice given MPL; such non-antigen-specific polyclonal effects were not observed in C3H/HeJ or in athymic nu/nu mice. Since the enhanced antibody response to SSS-III has been attributed to the inactivation of suppressor T cells by MPL and since suppressor-T-cell activity is demonstrable in both C3H/HeSnJ and C3H/HeJ mice, these findings imply that (i) the suppressor T cells of C3H/HeJ mice are refractory to inactivation by MPL and (ii) some of the polyclonal and mitogenic effects produced in C3H/HeJ mice are due to the direct action of MPL on B lymphocytes.
Neuraminidase-enhanced attachment of Bacteroides intermedius to human erythrocytes and buccal epithelial cells
Authors: Okuda K , Ono M , Kato T
Published: January 2020

Bacteroides intermedius strains strongly agglutinated only neuraminidase-treated erythrocytes. The neuraminidase-dependent hemagglutinating activity of B. intermedius was abolished by heating or treating with protease. The adherence of these microorganisms to human buccal epithelial cells was enhanced by neuraminidase pretreatment of the cells (P less than 0.01).
Mechanisms of recrudescence of Mycobacterium bovis BCG infection in mice
Authors: Cox J H , Knight B C , Ivanyi J
Published: January 2020

The capacity of various immunosuppressive agents to cause a recrudescence of the replication of Mycobacterium bovis BCG in the spleens of chronically infected mice was investigated. The actions of three corticosteroid preparations, cyclosporin A, and anti-T-cell subset monoclonal antibodies were compared. Treatment of mice with hydrocortisone acetate, which depressed the number of splenic lymphocytes and suppressed T-cell responses, most effectively exacerbated the stationary BCG counts, at 4 to 6 months after infection. The magnitude of reactivation was more pronounced in innately resistant CBA/Ca mice than in the susceptible C57BL/6 strain of mice. Splenic bacterial counts were also amplified by anti-L3T4 antibody when the antibody was injected at the chronic phase, whereas cyclosporin A had an effect only during the initial 6 weeks after BCG infection. Cultures of spleen cells from chronically infected mice showed a significant increase in the numbers of viable BCG recovered after 7 days of incubation in the presence of dexamethasone but not with cyclosporin A. The observed differences between the tested immunosuppressive agents indicate that the stationary bacterial counts during chronic BCG infection are maintained by discrete T-cell actions on the infected macrophages.
Dissociation of catalytic and invasive activities of Bordetella pertussis adenylate cyclase
Authors: Raptis A , Knipling L , Wolff J
Published: January 2020

Bordetella pertussis organisms secrete adenylate cyclase, at least one form of which can invade host cells and appears to be a virulence factor. Treatment of urea extracts containing invasive cyclase of B. pertussis with trypsin, chymotrypsin, or subtilisin abolishes the ability to increase intracellular cyclic AMP levels in CHO cells (invasiveness) at concentrations that have minimal or no effects on adenylate cyclase activity. Higher protease concentrations can inhibit catalytic activity, and 1 microM calmodulin protects this catalytic activity, but not invasiveness, against proteolytic inhibition. Rabbit immunoglobulin G (IgG) fractions from antisera prepared against urea extracts inhibited invasiveness at 10-fold-lower concentrations than inhibited catalytic activity. One IgG from a rabbit immunized against a partially purified, noninvasive form of the B. pertussis adenylate cyclase inhibited catalytic activity but was ineffective against invasiveness. We conclude that these two properties of the adenylate cyclase are independent functions that reside on different domains of the same protein or on different proteins.
Retention of bacterial lipopolysaccharide at the site of subcutaneous injection
Authors: Yokochi T , Inoue Y , Yokoo J , Kimura Y , Kato N
Published: January 2020

The tissue distribution of Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) was studied in mice injected subcutaneously (s.c.) or intraperitoneally (i.p.) with 125I-labeled KO3 LPS. Marked retention of KO3 LPS radioactivity could be found at the site of s.c. injection for several weeks. On the other hand, about 85% of the radioactivity rapidly disappeared from the peritoneal cavity within 6 h after i.p. injection. The long-term presence of KO3 LPS at the injection site was also supported by experiments with 51Cr-labeled KO3 LPS and immunoblotting and immunofluorescence staining methods. The R-form LPS lacking the O-specific polysaccharide chain of KO3 LPS and the lipid A fraction of KO3 LPS seemed to remain at the site in larger amounts and for longer times than KO3 LPS. There were no marked differences in the retention pattern at the injection site among KO3 LPS, Escherichia coli LPS, Salmonella typhosa LPS, and Salmonella enteritidis LPS. However, much less radioactivity accumulated in the livers and spleens of mice injected with either KO3 LPS or S. typhosa LPS compared with the other LPS preparations. It was suggested that retention of LPS at the site of s.c. injection may play an important role in the development of various biological actions of s.c. injected LPS.
Phenotypes of infiltrating cells in trehalose dimycolate-induced interstitial pneumonitis
Authors: Sakamoto Y , Goren M B , Kirkpatrick C H
Published: January 2020

Trehalose dimycolate is a glycolipid component of the cell walls of mycobacteria, nocardia, and corynebacteria. When trehalose dimycolate is injected into certain strains of mice, they develop interstitial pneumonitis that is characterized by mononuclear cell infiltration of the alveolar walls, intra-alveolar hemorrhages, and in some animals, granuloma formation. The disorder is seldom fatal, and in approximately 4 weeks, the lungs are normal. There is strong evidence that T lymphocytes are essential for production of interstitial pneumonitis by trehalose dimycolate, but little is known about the mechanisms of lung injury in this model. The experiments described in this report were conducted to identify the roles of the various cells that accumulate in the lungs of mice with this form of interstitial pneumonitis. We found that Mac3+ macrophages were the first cells to appear in the alveolar walls. Increases in the number of L3T4+ T lymphocytes, Lyt2+ T lymphocytes, and surface-immunoglobulin-positive lymphocytes followed, but significant increases in the number of lymphoid cells were not observed until day 7, when the pulmonary lesions were well developed. Treatment of the mice with cyclophosphamide or anti-T-cell sera significantly reduced the number of lymphoid cells in the alveolar walls but did not affect the number of Mac3+ cells and did not affect development of intra-alveolar hemorrhages. Treatment with poly(I.C) significantly decreased the number of Mac3+ cells in the lungs, and these mice did not develop pulmonary hemorrhages. We conclude that although development of pulmonary lesions in trehalose dimycolate-treated mice is a T-cell-dependent process, macrophages are also essential and are more directly involved in production of the lung injury. We postulate that the lung lesions are the direct effect of macrophage-produced cytokines, such as tumor necrosis factor.