bluuebunny/crossref_metadata_2025 huggingface dataset:
Immune suppression induced by Actinobacillus actinomycetemcomitans: effects on immunoglobulin production by human B cells
Authors: Shenker B J , Vitale L A , Welham D A
Published: January 2020

Actinobacillus actinomycetemcomitans produces an immunosuppressive factor (ISF) which has been shown to suppress mitogen- and antigen-induced DNA, RNA, and protein synthesis in human T lymphocytes. In this study, we examined purified A. actinomycetemcomitans ISF for its ability to alter immunoglobulin production by human B cells. The ISF caused a dose-dependent inhibition of pokeweed mitogen (PWM)-induced immunoglobulin G (IgG) and IgM production. Preexposure to ISF was not required to achieve maximal inhibition of immunoglobulin synthesis, as previously observed for its effect on T-cell activation. Nevertheless, the ISF appeared to act by irreversibly affecting the early stages of cell activation. While PWM-induced immunoglobulin production is under the influence of T-regulatory circuits, it appears that the ISF interacts directly with B cells. First, ISF failed to alter either the synthesis of interleukin-2 (IL-2) or the expression of IL-2 receptors on T cells. Second, experiments in which individual purified populations of cells were exposed to ISF, washed, and placed back into tissue culture indicated that when all cells (i.e., T cells, B cells, and monocytes) were exposed to ISF, significant suppression was observed. However, when only one cell population was treated with ISF, suppression of both IgG and IgM synthesis was observed only when the B-cell-enriched population was exposed to ISF. These results in conjunction with our earlier findings suggest that the ISF functions via the activation of a regulatory subpopulation of B lymphocytes, which in turn either directly or indirectly (via suppressor T cells) downregulate both B- and T-cell responsiveness. Furthermore, it is hypothesized that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. This suppression may enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism.
Quantitative relationship between anticapsular antibody measured by enzyme-linked immunosorbent assay or radioimmunoassay and protection of mice against challenge with Streptococcus pneumoniae serotype 4
Authors: Musher D M , Johnson B , Watson D A
Published: January 2020

We have recently shown that a substantial proportion of antibody to pneumococcal polysaccharide as measured by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay is removed by adsorption with pneumococcal cell wall polysaccharide (CWPS). The present study was undertaken to validate the hypothesis that only serotype-specific antibody that remains after adsorption with CWPS provides protection against pneumococcal infection. Serum samples were obtained from human subjects before and after they had been vaccinated with pneumococcal polysaccharide vaccine. Antibody to Streptococcus pneumoniae serotype 4 was measured by ELISA without adsorption or after adsorption of serum with CWPS. Groups of mice were injected with graded doses of serum and then challenged intraperitoneally with 10, 100, or 1,000 50% lethal doses (LD50) of S. pneumoniae serotype 4. Without adsorption, prevaccination sera from five healthy adults appeared to contain up to 33 micrograms of antibody to S. pneumoniae serotype 4 antigen per ml; adsorption with CWPS removed all detectable antibody, and pretreating mice with up to 0.1 ml of these sera (less than or equal to 3.3 micrograms of antibody) failed to protect them against challenge with 100 LD50. In contrast, postvaccination sera contained 2.9 to 30 micrograms of antibody per ml that was not removed by adsorption. Diluting sera to administer desired amounts of serotype-specific immunoglobulin G showed a significant relationship between protection and antibody remaining after adsorption (P less than 0.05 by linear regression analysis); 150 ng was uniformly protective against 1,000 LD50, and 50 ng was protective against 100 LD50. These studies have, for the first time, quantitated the amount of serotype-specific antibody that protects mice against challenge with S. pneumoniae type 4. In light of these observations, it is necessary to reassess current concepts regarding the presence of antipneumococcal antibody in the unvaccinated population, responses to pneumococcal vaccination, and protective levels of immunoglobulin G.
An HlyB-type function is required for expression of the Enterococcus faecalis hemolysin/bacteriocin
Authors: Gilmore M S , Segarra R A , Booth M C
Published: January 2020

The nucleotide sequence of a 3,422-bp internal restriction fragment from the Enterococcus faecalis pAD1 hemolysin/bacteriocin-encoding region was determined. This fragment was associated with expression of hemolysin/bacteriocin component L and contained a 2,142-bp open reading frame. The inferred amino acid sequence revealed a protein which shared extensive similarity with HlyB of the Escherichia coli alpha-hemolysin operon. The inferred protein, CylB, was observed to be independently expressed in E. coli and capable of complementing an insertion mutation in the cloned hemolysin/bacteriocin operon in trans. Despite the extensive similarity to HlyB, CylB was incapable of complementing an insertion mutation in hlyB. Cytolysin determinants possessing an HlyB-type transport function are widely dispersed throughout gram-negative genera. We believe this to be the first example of an HlyB-type protein encoded within a cytolysin determinant from a gram-positive bacterium.
Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species
Authors: Hoehn G T , Clark V L
Published: January 2020

The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus.
In vitro sensitivity of oral, gram-negative, facultative bacteria to the bactericidal activity of human neutrophil defensins
Authors: Miyasaki K T , Bodeau A L , Ganz T , Selsted M E , Lehrer R I
Published: January 2020

Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria.
Interleukin-1-induced promotion of T-cell differentiation in mice immunized with killed Listeria monocytogenes
Authors: Igarashi K , Mitsuyama M , Muramori K , Tsukada H , Nomoto K
Published: January 2020

We studied the effects of administration of recombinant interleukin-1 alpha (rIL-1 alpha) to mice after immunization with killed Listeria monocytogenes cells on the promotion of the functional differentiation of T cells in vivo. Mice immunized with killed L. monocytogenes were unable to express cell-mediated immunity to specific antigen in vivo, as determined by delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR), and splenic T cells obtained from such mice were unable to respond to rIL-2 and specific antigen and to produce IL-2 after antigenic restimulation in vitro. When rIL-1 alpha was given to mice after immunization with killed bacteria. T cells became capable of responding to rIL-2 and specific antigen in vitro. These functions of T cells were similar to those from mice immunized with viable listeriae. Moreover, using a local passive transfer system, it was found that effector T cells mediating DTH but not ACR to L. monocytogenes were generated in mice treated with rIL-1 alpha after immunization with killed bacteria. These T cells were able to produce macrophage chemotactic factor but not macrophage-activating factor or gamma interferon in vitro in response to stimulation with specific antigen. These results suggest that in vivo administration of rIL-1 alpha facilitates the maturation of antigen-specific T cells mediating DTH and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.
pap-2-encoded fimbriae adhere to the P blood group-related glycosphingolipid stage-specific embryonic antigen 4 in the human kidney
Authors: Karr J F , Nowicki B J , Truong L D , Hull R A , Moulds J J , Hull S I
Published: January 2020

A subtype of P fimbriae, encoded by the pap-2 gene cluster, has been analyzed for agglutination of erythrocytes and for binding to cryostat sections of the human kidney. We have demonstrated that pap-2-encoded fimbriae are capable of binding to erythrocytes from some animal species and to human erythrocytes which express globoside and the LKE (stage-specific embryonic antigen 4 [SSEA-4]) antigen. The pap-2 fimbriae bind to Bowman's capsule in the human kidney. Monoclonal antibodies directed against glycosphingolipids were used for the detection of specific P blood group-related antigens in the human kidney and on erythrocytes. Preincubation of kidney sections with monoclonal antibody MC813-70, which binds to the SSEA-4 antigen, inhibited adherence of purified pap-2-encoded fimbriae to Bowman's capsule. We suggest that one receptor for pap-2-encoded fimbriae is the antigen known as LKE (Luke) on human erythrocytes or SSEA-4 in the tissues.
Efficacy of a cell extract from Actinobacillus (Haemophilus) pleuropneumoniae serotype 1 against disease in swine
Authors: Fedorka-Cray P J , Huether M J , Stine D L , Anderson G A
Published: January 2020

We partially characterized a cell extract (CE) from Actinobacillus pleuropneumoniae serotype 1 and used the CE to test the efficacy of secreted proteins against disease. Secreted products from 4-h culture supernatants were precipitated with 20% polyethylene glycol. Analysis of the CE indicated the presence of protein, endotoxin, and carbohydrate. Hemolytic activity to bovine erythrocytes and cytotoxic activity to porcine mononuclear leukocytes was also demonstrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the CE from a 4-h culture showed a major band at 110 kilodaltons (kDa), while a CE of a 26-h culture indicated the presence of a number of additional proteins, including the 110-kDa protein. The 110-kDa protein was also identified as a glycoprotein by periodic acid-Schiff and silver staining. A single band precipitated against convalescent-phase pig antiserum when the polyethylene glycol precipitate was used in an Ouchterlony plate. Vaccination with CE conferred greater protection against challenge with the homologous serotype than either a commercial bacterin or an outer membrane protein vaccine. Hemolysin-neutralizing titers were higher both pre- and postchallenge in the group vaccinated with the CE compared with in all other groups. We believe that this demonstrates the importance of secreted factors in protection against disease and suggests that the 110-kDa protein is an important immunogen.
Passive immunization of chickens against Eimeria maxima infection with a monoclonal antibody developed against a gametocyte antigen
Authors: Wallach M , Pillemer G , Yarus S , Halabi A , Pugatsch T , Mencher D
Published: January 2020

Eimeria maxima gametocytes contain two major antigens with molecular masses of 56 and 82 kilodaltons (kDa) which are recognized by convalescent sera from immune chickens. Preparations enriched in these two antigens were used to immunize mice, and several monoclonal antibodies which specifically reacted with the 56-kDa antigen were produced. One of these monoclonal antibodies of the immunoglobulin M subclass, along with immune chicken sera raised against affinity-purified 56- and 82-kDa antigens, was used to passively immunize chicks. On the basis of the parameter of total oocyst output, it was found that these antibodies provided partial protection (40 to 50% inhibition) against E. maxima challenge infections.
Protein malnutrition alters the distribution of Fc gamma R+ (T gamma) and Fc mu R+ (T mu) T lymphocytes in experimental pulmonary tuberculosis
Authors: McMurray D N , Bartow R A , Mintzer C L
Published: January 2020

Inbred, strain 2 guinea pigs were given isocaloric diets containing either 30% (control diet) or 10% (low-protein diet) ovalbumin and infected 4 weeks later by the respiratory route with virulent Mycobacterium tuberculosis. By using an Fc receptor rosette assay, the proportions of T lymphocytes bearing Fc receptors for immunoglobulin G (T gamma cells) or immunoglobulin M (T mu cells) were quantified in blood and lymphoid tissues taken postinfection. A significant elevation in the proportion of the putative suppressor T subset (T gamma) in the blood of protein-deprived guinea pigs was observed at all intervals postinfection. Conversely, the levels of the putative helper T subset (T mu) in the bronchotracheal lymph nodes draining the site of virulent infection in malnourished animals were significantly reduced. Diet did not influence T gamma or T mu cells in the spleens. Diet-induced loss of purified protein derivative-specific T-cell functions in tuberculosis may be associated with alterations in the proportions of or the balances between T gamma and T mu subsets.
Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH
Authors: Antoine J C , Prina E , Jouanne C , Bongrand P
Published: January 2020

Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h exposure, the tracer was mainly detected in perinuclear lysosomes and parasitophorous vacuoles of uninfected and infected macrophages, respectively. Fluorescence intensities were determined from digitized video images of single cells after processing and automatic subtraction of background. We found statistically different mean pH values of 5.17 to 5.48 for lysosomes and 4.74 to 5.26 for parasitophorous vacuoles. As for lysosomes of monensin-treated cells, the pH gradient of parasitophorous vacuoles collapsed after monensin was added. This very likely indicates that these vacuoles maintain an acidic internal pH by an active process. These results show that L. amazonensis amastigotes are acidophilic and opportunistic organisms and suggest that these intracellular parasites have evolved means for survival under these harsh conditions and have acquired plasma membrane components compatible with the environment.
Size of the Streptococcus mutans GS-5 chromosome as determined by pulsed-field gel electrophoresis
Authors: Tudor J J , Marri L , Piggot P J , Daneo-Moore L
Published: January 2020

Rare cutting restriction endonucleases were used to cut the Streptococcus mutans chromosome into large fragments. Restriction enzymes utilizing recognition sites containing 6-, 7-, or 8-base-pair sequences with only G and C nucleotides produced few fragments, most of which were greater than 100 kilobase pairs in size. Addition of the fragments from digests of SmaI, NotI, ApaI, RsrII, and EagI yielded a molecular size for the S. mutans GS-5 genome of 2,819 +/- 60 kilobase pairs.
Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudotuberculosis harboring the pYV plasmid
Authors: Simonet M , Richard S , Berche P
Published: January 2020

Electron microscopic evidence is presented that bacteria harboring the virulence plasmid pYV from Yersinia pseudotuberculosis are localized in extracellular sites during the course of infection in mice, often unambiguously undergoing active replication. Virulent pYV+ bacteria, often seen adherent to platelets, severely restricted granuloma formation, creating necrotic microabscesses poorly populated with inflammatory cells. This contrasts with granulomas produced by pYV- bacteria, which appear to be composed mainly of polymorphonuclear and mononuclear cells. Our results therefore strongly suggest that active replication of pYV+ bacteria predominantly, if not exclusively, occurs in vivo in extracellular sites.
Cyclic rickettsemia during persistent Anaplasma marginale infection of cattle
Authors: Kieser S T , Eriks I S , Palmer G H
Published: January 2020

Submicroscopic levels of Anaplasma marginale rickettsemia in persistently infected cattle were determined by using nucleic acid hybridization. Within individuals, the rickettsemia levels steadily increased from less than 10(4) infected erythrocytes per ml to a peak of more than 10(6) infected erythrocytes per ml and then rapidly declined. This logarithmic variation parallels the variation of the rickettsemia level seen in acute infection and suggests that cyclic emergence of antigenic variants is a mechanism of rickettsial persistence.
Purification and characterization of an Escherichia coli Shiga-like toxin II variant
Authors: MacLeod D L , Gyles C L
Published: January 2020

A Shiga-like toxin II variant was purified to homogeneity from Escherichia coli TB1(pCG6), which contained the toxin genes cloned in multicopy plasmid pUC18. The purification scheme involved polymyxin B extraction of the toxin from bacterial cells, followed by differential (NH4)2SO4 precipitation, anion- and cation-exchange fast-protein liquid chromatography, and immunoaffinity exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified toxin revealed three protein bands that migrated with calculated molecular weights of 33,000, 27,500, and 7,500. These bands correspond to values for the A, A1, and B subunits, respectively, that would be expected on the basis of the nucleotide sequence and comparison with data for Shiga toxin and other Shiga-like toxins. Electrophoresis under nonreducing conditions resulted in disappearance of the 27,000-molecular-weight band. Western blot (immunoblot) analysis revealed three protein bands with molecular weights of 33,000, 27,500, and 7,500. The purified toxin induced typical signs of edema disease in pigs injected intravenously with doses as small as 3 ng/kg of body weight. The 50% cytotoxic doses for Vero, PK15, and Madin-Darby bovine and canine kidney cells were 0.5, 2.0, 8.0, and 8.0 pg, respectively. The 50% lethal dose of purified toxin for mice was 0.9 pg by the intraperitoneal route. Approximately 75 micrograms of purified toxin was required to induce a 1-ml/cm fluid response in rabbit ileal loops. Antiserum to the Shiga-like toxin II variant neutralized homologous toxin, Shiga-like toxin II, and Verotoxin 2 but not Shiga-like toxin I.
Characterization of genetically inactivated pertussis toxin mutants: candidates for a new vaccine against whooping cough
Authors: Nencioni L , Pizza M , Bugnoli M , De Magistris T , Di Tommaso A , Giovannoni F , Manetti R , Marsili I , Matteucci G , Nucci D
Published: January 2020

the introduction of two amino acid substitutions within the enzymatically active subunit S1 of pertussis toxin (PT) abolishes its ADP-ribosyltransferase activity and toxicity on CHO cells (Pizza et al., Science 246:497-500, 1989). These genetically inactivated molecules are also devoid of other in vivo adverse reactions typical of PT, such as induction of leukocytosis, potentiation of anaphylaxis, stimulation of insulin secretion, and histamine sensitivity. However, the mutant PT molecules are indistinguishable from wild-type PT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maintain all the physical and chemical properties of PT, including affinity for toxin-neutralizing poly- and monoclonal antibodies. Either alone or stabilized with formaldehyde, PT mutants are able to induce high levels of neutralizing antibodies and to protect mice in a dose-dependent fashion against intracerebral challenge with virulent B. pertussis. These results clearly show that these genetically inactivated PT molecules are nontoxic but still immunogenic and justify their development as a component of a new, safer acellular vaccine against whooping cough.
Mucinophilic and chemotactic properties of Pseudomonas aeruginosa in relation to pulmonary colonization in cystic fibrosis
Authors: Nelson J W , Tredgett M W , Sheehan J K , Thornton D J , Notman D , Govan J R
Published: January 2020

Representative isolates of nonmucoid Pseudomonas aeruginosa were studied to investigate the hypothesis that mucinophilic and chemotactic properties in this species act as potential factors in the initial stages of pulmonary colonization in patients with cystic fibrosis (CF). Transmission electron microscopy with a surfactant monolayer technique was used in a novel manner to demonstrate the adhesion of all 10 P. aeruginosa strains examined to porcine gastric mucin and tracheobronchial mucin from a patient with CF. Control experiments showed that Escherichia coli K-12 and single representatives of Proteus mirabilis and Klebsiella aerogenes did not bind to these mucins. The Adler capillary technique, used to measure bacterial chemotactic response, showed that purified CF mucin acted as a chemoattractant for most P. aeruginosa strains, with the exception of the nonmotile mutant M2Fla- and the nonchemotactic mutant WR-5. The ability of the major sugar and amino acid components of mucin to act as chemoattractants was investigated. The degree of chemotaxis was strain specific; optimum chemotaxis was observed toward serine, alanine, glycine, proline, and threonine. No strain showed chemotaxis to N-acetylneuraminic acid, but all strains showed a strain-dependent chemotactic response to the sugars L-fucose, D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine. These results provide new information on the mucinophilic and chemotactic properties of nonmucoid P. aeruginosa and support the hypothesis that these properties could play a role in the initial stages of pulmonary colonization in patients with CF.
A model of sustained gastrointestinal colonization by Candida albicans in healthy adult mice
Authors: Samonis G , Anaissie E J , Rosenbaum B , Bodey G P
Published: January 2020

Three-month-old male Crl:CD1(ICR)BR and C3H/HeJ mice were fed chow containing Candida albicans for 14 days, while similar control mice were fed regular food. Stool cultures were done for all mice before and after administration of the special diet. Stool cultures were repeated 48 h, 1 week, and 1 and 3 months after stopping the diet for Crl:CD1(ICR)BR mice and again 5 months afterward for C3H/HeJ mice. Some animals were sacrificed at the end of the special diet, and cultures and histopathologic examination of various organs were performed. Colonization with C. albicans occurred in the Candida-fed mice, and the fungus was maintained in the gastrointestinal tract at a concentration of 10(3) to 10(4) CFU/g of stool for up to 5 months. There was no histologic evidence of organ infection with Candida spp. The fungus was not found in stool cultures or organs of mice in the control group. The results suggest that persistent gastrointestinal colonization of adult mice by C. albicans can be achieved without immunosuppression. Thus, with additional manipulations, this model could be useful for studying the role of gastrointestinal colonization by C. albicans in the development of systemic infection.
Haemocin, the bacteriocin produced by Haemophilus influenzae: species distribution and role in colonization
Authors: LiPuma J J , Richman H , Stull T L
Published: January 2020

Four hundred thirty-eight strains of Haemophilus influenzae were examined for production of and sensitivity to haemocin, a bacteriocin produced by some members of this species. Whereas 199 of 212 (94%) type b isolates produced haemocin, 131 of 134 (98%) nontypeable and 91 of 92 (99%) encapsulated non-type b isolates were sensitive to haemocin. Among strains previously genetically characterized by multilocus enzyme electrophoresis, haemocin production was detected in type b isolates belonging to 25 of 29 (86%) clonally distinct electrophoretic types. None of 60 clonally distinct nontypeable strains produced this substance, and all were sensitive to it in vitro. The genes encoding haemocin production were transformed independently of the genes necessary for capsule expression from a prototypic type b strain to a nontypeable strain. After intranasal inoculation of infant rats with an equal mixture of a non-haemocin-producing strain and its haemocin-producing transformant, organisms capable of haemocin production predominated in both nasopharyngeal and blood cultures. These data demonstrate that haemocin production is strongly associated with type b encapsulated members of this species and suggest a mechanism by which haemocin might play a role in host nasopharyngeal colonization by this pathogen.
A strong antibody response to the periplasmic C-terminal domain of the OmpA protein of Escherichia coli is produced by immunization with purified OmpA or with whole E. coli or Salmonella typhimurium bacteria
Authors: Puohiniemi R , Karvonen M , Vuopio-Varkila J , Muotiala A , Helander I M , Sarvas M
Published: January 2020

We produced in Bacillus subtilis the complete, as well as the N-terminal two-thirds, OmpA protein of Escherichia coli (called here Bac-OmpA and Bac-OmpA-dN, respectively). These Bac-OmpA proteins were used to examine the immunological properties of different parts of OmpA, free of lipopolysaccharide and other components of the outer membrane. The full-length Bac-OmpA was indistinguishable from the authentic protein isolated from E. coli (Coli-OmpA) both as immunogen and as antigen in enzyme immunoassay (EIA). The N-terminal Bac-OmpA-dN was a poor immunogen which gave rise to significantly lower titers of anti-OmpA antibody than did the full-length OmpA preparations. When used as an antigen in EIA, the Bac-OmpA-dN detected anti-OmpA antibody in serum samples from animals immunized with the full-length OmpA much less efficiently than did either Bac-OmpA or Coli-OmpA. The periplasmic C-terminal domain therefore appears to be an immunodominant epitope of the purified OmpA protein. Also, when rabbits and mice were immunized with intact, live or dead E. coli, the antibody response detected by EIA with the full-length protein, Bac-OmpA, was much stronger than that detected with the N-terminal two-thirds, Bac-OmpA-dN. Similar results were obtained with the OmpA of Salmonella typhimurium. Because the ompA gene of enterobacteria is highly conserved, the Bac-OmpA might be useful as a group-specific EIA antigen to diagnose diseases caused by members of the family Enterobacteriaceae.
Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus
Authors: Wright A C , Simpson L M , Oliver J D , Morris J G
Published: January 2020

Translucent, avirulent spontaneous phase variants of Vibrio vulnificus MO6-24 reverted back to the original opaque, encapsulated phenotype under both in vivo and in vitro conditions. Two translucent, acapsular mutants, which did not show phase variation, were constructed by using the transposon Tn5 IS50L::phoA (TnphoA). Loss of capsule was accompanied by decreases in virulence, hydrophilicity, and serum resistance. The ability to utilize transferrin-bound iron for growth was lost in only one of the two unencapsulated mutants. Our data emphasize the apparent importance of capsule in the virulence of V. vulnificus and indicate that utilization of transferrin-bound iron is independent of encapsulation.
Nonimmune binding of human immunoglobulin A to type II group B streptococci
Authors: Anthony B F , Concepcion N F , Puentes S M , Payne N R
Published: January 2020

The binding of 125I-labeled human myeloma immunoglobulin A (IgA) to four type II strains and one nontypable strain of group B streptococci was measured after streptococcal chains were broken by brief sonication. Some IgA binding was observed with all strains, but specific binding (binding that was inhibited by excess unlabeled IgA, was dose dependent, and was saturable) occurred only with those strains possessing the trypsin-sensitive beta component of the c protein. Similar amounts of binding were observed with myeloma IgA and IgA1 purified from normal serum. Specific binding was more rapid at 25 degrees C than at 0 or 37 degrees C and reached a plateau in 6 to 8 h. Binding was drastically reduced (85 to 90%) when streptococci had been heated (90 degrees C for 1 h). Most radioactivity bound to group B streptococci could be displaced (greater than 60% in 3 days) by the addition of excess unlabeled IgA. The binding capacity of one strain (10(8) streptococci in 1 ml of buffer) was saturated by approximately 24 micrograms of IgA. When transformed for Scatchard analysis, these data indicated that there was a specific binding capacity of 16,000 molecules of monomeric serum IgA per single streptococcal cell. The dissociation constant for IgA binding was 19.3 nM. Since enzyme-linked immunosorbent assay studies showed that the myeloma IgA used for the studies described above was IgA1, our quantitative data apply only to the binding of this subclass to group B streptococci. However, an enzyme-linked immunosorbent-filtration assay showed that both IgA1 and IgA2 bound to a type II group B streptococcus bearing the c protein.
Disparate mechanisms of induction of procoagulant activity by live and inactivated bacteria and viruses
Authors: Sinclair S B , Rotstein O D , Levy G A
Published: January 2020

This study describes the dose response, time course, and lymphocyte requirements of procoagulant activity (PCA) induction following stimulation of thioglycolate-elicited BALB/c peritoneal macrophages with live and inactivated bacteria (Bacteroides fragilis, Escherichia coli, and Staphylococcus aureus) and murine hepatitis virus type 3 (MHV-3). Induction of PCA by MHV-3 was significantly more rapid and the maximal PCA achieved was significantly greater than by the three bacterial species studied. In relation to induction of PCA by bacteria, the PCA response was more rapid and of greater magnitude with S. aureus and E. coli than with B. fragilis. MHV-3 induced an augmented PCA response at all concentrations of virus studied in a dose-dependent fashion, whereas higher titers of live bacteria (greater than 10(7) CFU/ml) inhibited PCA, suggesting the production of an inhibitory factor. Significant PCA induction was observed when macrophages were incubated with bacteria or virus in the absence of lymphocytes. At low titers of B. fragilis (10(3) CFU/ml), addition of lymphocytes greatly augmented PCA production, whereas at higher titers (10(7) CFU/ml), the addition of lymphocytes only slightly augmented the PCA response. In contrast, MHV-3 induction of PCA was enhanced by the addition of lymphocytes at all concentrations of virus studied, suggesting a lymphocyte-dependent process. Heat-inactivated bacteria were as effective as live bacteria in inducing PCA, suggesting that induction of PCA by bacteria requires only a bacterial surface component. In contrast, UV-inactivated MHV-3 did not induce PCA, suggesting that viral replication is a necessary step in PCA induction. These results suggest that the cellular and metabolic requirements for induction of PCA differ among viral and bacterial pathogens and may partly explain their differences in pathogenicity.
Epitope analysis of the F4 (K88) fimbrial antigen complex of enterotoxigenic Escherichia coli by using monoclonal antibodies
Authors: van Zijderveld F G , Anakotta J , Brouwers R A , van Zijderveld A M , Bakker D , de Graaf F K
Published: January 2020

So far, three subtypes of the F4 (K88) fimbrial antigen of porcine enterotoxigenic Escherichia coli, F4ab, F4ac, and F4ad, have been distinguished by using polyclonal antisera in agglutination and precipitation tests. The a factor represents one or more common epitopes, whereas each of the b, c, and d factors represents one or more subtype-specific epitopes. We further characterized the F4 antigen complex by using a panel of 40 F4-specific monoclonal antibodies (MAbs). The specificity of all MAbs was proven by enzyme-linked immunosorbent assays, agglutination and radioimmunoprecipitation tests, and immunoelectron microscopy. The MAbs either reacted with all F4 subtypes, reacted with two subtypes, or were subtype specific. Epitope analysis by competition enzyme-linked immunosorbent assays revealed at least 11 epitope clusters on the F4 antigen complex, designated a1 to a7, b1, b2, c, and d. The following antigenic formulas were found for the F4 subtypes: F4ab, a1a2a3a4a5a6b1b2; F4ac, a1a2a3(a4)a5a6a7c; and F4ad, a1a2a3a4a7d. All MAbs were directed against conformational epitopes located on the 27,500-dalton major fimbrial subunits. Consequences for the replacement of polyclonal antisera by MAbs in diagnostic tests are discussed.
Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor
Authors: Ogunnariwo J A , Schryvers A B
Published: January 2020

Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure.
Aerolysin of Aeromonas sobria: evidence for formation of ion-permeable channels and comparison with alpha-toxin of Staphylococcus aureus
Authors: Chakraborty T , Schmid A , Notermans S , Benz R
Published: January 2020

Aerolysin from Aeromonas sobria AB3 was isolated and purified. The pure toxin formed sodium dodecyl sulfate-insoluble oligomers in a lipidic environment. The addition of aerolysin to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion-permeable channels which had a single-channel conductance of about 70 pS in 0.1 M KCl. This defines the toxin as a channel-forming component similar to other toxins but without any indication for an association-dissociation reaction, since the channels had a long lifetime at low voltages. At voltages higher than 50 mV, the aerolysin channel switched into a closed state with a low residual conductance. The single-channel conductance was a linear function of the total aqueous conductance, which suggested that the toxin oligomers formed aqueous channels with an estimated minimal diameter of about 0.7 nm. The aerolysin pores were found to be slightly anion selective. The pore-forming properties of aerolysin were compared with those of alpha-toxin of Staphylococcus aureus. Both aerolysin and alpha-toxin share secondary structure features, must oligomerize to form pores in lipid bilayer membranes, and form channels with similar properties.
Size and subdomain architecture of the glucan-binding domain of sucrose:3-alpha-D-glucosyltransferase from Streptococcus sobrinus
Authors: Wong C , Hefta S A , Paxton R J , Shively J E , Mooser G
Published: January 2020

Mild trypsin proteolysis of Streptococcus sobrinus sucrose:3-alpha-D-glucosyltransferase (GTF-I) reduced most of the enzyme to small products but left a few large fragments undigested. The digest had no glucosyl transfer activity, but several digestion products had an affinity for glucan equivalent to that of the native enzyme. The glucan-binding fragments ranged in size from 17 to 60 kilodaltons (kDa), with a particularly prominent 42-kDa fragment. The largest of these (60 kDa) appears to be the full extent of the domain since it increases in abundance when the enzyme is protected with glucan during proteolysis. The presence of smaller fragments with glucan-binding function and intact tertiary structure indicates that the full domain must be built on glucan-binding subdomains. Among the range of glucan-binding fragments, only the 42-kDa segment could be satisfactorily purified. It was subjected to N-terminal sequence analysis and, because of some ambiguity, was also subjected to chymotrypsin digestion and sequence of several chymotryptic peptides. The sequence data established that the 42-kDa domain fragment is initiated approximately two-thirds into the 170-kDa enzyme in a region previously identified as a segment of the gene that includes the glucan-binding domain (J. J. Ferretti, M. L. Gilpin, and R. R. B. Russell, J. Bacteriol. 169:4271-4278, 1987). The site is approximately 60 kDa from the C terminus and covers a region characterized by extensive amino acid sequence repeats. The data are discussed in the context of the size range of the glucan-binding fragments and subdomain architecture of the full glucan-binding domain.
T-cell-independent and T-cell-dependent B-cell responses to exposed variant surface glycoprotein epitopes in trypanosome-infected mice
Authors: Reinitz D M , Mansfield J M
Published: January 2020

The T-cell dependency of B-cell responses to variant surface glycoprotein (VSG) epitopes exposed in their native surface conformation on Trypanosoma brucei rhodesiense clone LouTat 1 was investigated. T-cell requirements were examined by analyses of gamma globulin preparations derived from trypanosome-infected BALB/c nude (nu/nu) and thymus-intact (nu/+) mice. A radioimmunoassay was used to selectively quantitate antibody binding to native VSG 1 epitopes present on the surface of viable trypanosomes. Such analyses of VSG-specific antibody in infected mice demonstrated that in the absence of T cells there was a significant B-cell response to exposed VSG epitopes; however, in the presence of T cells these surface epitope-specific responses were greatly enhanced. In contrast to infection, immunization of mice with purified VSG 1 or paraformaldehyde-fixed parasites elicited significant VSG surface epitope-specific responses only in the presence of T cells (i.e., in nu/+ mice only). VSG-specific antibody responses in mice infected with three other clonal T. brucei rhodesiense populations (LouTat 1.2, 1.5, and 1.9) were found to be similar in this pattern, although not identical, to the anti-LouTat 1 responses. An important exception was that mice infected with LouTat 1.8 required T cells to produce VSG surface-specific antibody. Thus, the VSG surface epitope-specific B-cell responses in trypanosome-infected mice represent composite T-cell-independent and T-cell-dependent processes, and a significantly stronger response is made in the presence of T cells. However, immunization with VSG in the absence of infection elicited only T-cell-dependent responses. Since the relative contribution of T-cell-independent and T-cell-dependent processes to the total VSG-specific antibody produced during infection was variable (as seen with the absence of a T-cell-independent response to LouTat 1.8), this may reflect differences in the primary structure or display of VSG molecules on the trypanosome membrane or may represent active parasite interference with some epitope-specific B-cell responses.
Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by Rickettsia conorii- and Rickettsia rickettsii-infected cultured endothelial cells
Authors: Drancourt M , Alessi M C , Levy P Y , Juhan-Vague I , Raoult D
Published: January 2020

Hemostasis abnormalities have been described in patients with Mediterranean spotted fever and Rocky Mountain spotted fever. Evidence of the activation of the fibrinolytic system has been obtained in both diseases. After experimental Rocky Mountain spotted fever, an elevated level of fibrinogen was found in parallel with the activation of the fibrinolytic system and transient elevation of the tissue-type plasminogen activator. Later protein is mainly synthesized by endothelial cells. The ability to culture human endothelial cells in vitro provides a unique system to study the secretion of tissue-type plasminogen activator and of plasminogen activator inhibitor after rickettsial infection. Human vascular endothelial cells derived from the umbilical vein, when infected with Rickettsia conorii or Rickettsia rickettsii, secreted as much tissue-type plasminogen activator as control cells. The activity of plasminogen activator inhibitor however, was higher in the supernatants of infected cells than in those of control cells. This rickettsia-induced imbalance of the tissue-type plasminogen activator-inhibitor pair was a very early event after in vitro infection. The involvement of this system during Mediterranean spotted fever and Rocky Mountain spotted fever remains to be demonstrated.
Yersinia pestis pH 6 antigen: genetic, biochemical, and virulence characterization of a protein involved in the pathogenesis of bubonic plague
Authors: Lindler L E , Klempner M S , Straley S C
Published: January 2020

We studied a protein antigen, designated pH 6 Ag, that has the same regulation of expression as the previously described Yersinia pestis pH 6 Ag. Monospecific antiserum to this antigen recognized several proteins, ranging from 15 to over 75 kilodaltons (kDa), which were strongly expressed when Y. pestis was cultivated at 37 degrees C and pH 6 but were expressed weakly, if at all, at 37 degrees C and pH 8 and at 26 degrees C. The antigen appeared to be composed of aggregates of a 15-kDa subunit. Escherichia coli minicell analysis and Western blotting (immunoblotting) of minicell extracts containing the cloned pH 6 Ag locus revealed that a 1.7-kilobase-pair (kb) region of Y. pestis chromosomal DNA produced 16- and 15-kDa immunoreactive proteins. We used transposon mutagenesis of the pH 6 Ag-coding region to demonstrate that the 16- and 15-kDa polypeptides were produced by the same cistron. The pH 6 Ag structural gene, psaA, was located within a 0.5-kb region of DNA. A Tn10lacZ transposon insertion 1.2 kb upstream of the psaA locus but outside the psaA transcriptional unit caused decreased expression of pH 6 Ag in both E. coli and Y. pestis and defined the psaE locus necessary for maximum pH 6 Ag expression. This locus itself was not regulated by temperature or pH. However, psaA remained responsive to both of these environmental signals in a Y. pestis psaE mutant. Mutation of either psaE or psaA resulted in at least a 100-fold reduction in the intravenous 50% lethal dose of Y. pestis in mice. Accordingly, pH 6 Ag is involved in the pathogenesis of bubonic plague.
Peptide analogs to a fibronectin receptor inhibit attachment of Staphylococcus aureus to fibronectin-containing substrates
Authors: Raja R H , Raucci G , Hook M
Published: January 2020

Binding of cells of Staphylococcus aureus to fibronectin has been proposed as a mechanism of bacterial adhesion to host tissues. In this study, we have attempted to define the role of a recently identified fibronectin receptor in the adhesion of staphylococcal cells to fibronectin-containing substrates by using different receptor analogs as potential inhibitors of bacterial adherence. The results showed that synthetic peptides D1, D2, and D3, corresponding to variations of a repeated unit in the fibronectin-binding domain of the receptor, and ZZ-FR, a chimeric protein containing the fibronectin-binding domain of the receptor with the D1, D2, and D3 sequences, inhibited the attachment of staphylococcal cells to microtiter wells coated with intact fibronectin or with the 29-kilodalton amino-terminal fragment of fibronectin. The chimeric protein ZZ-FR also partially inhibited the adherence of staphylococci to human plasma clots formed in vitro but had no effect on bacterial adhesion to clots formed from fibronectin-depleted plasma. These data confirm previous reports suggesting that fibronectin may serve as a substrate for adhesion of staphylococcal cells and indicate that bacterial adhesion is mediated by the identified fibronectin receptor. Furthermore, analogs to the fibronectin receptor can be used to inhibit the adhesion of bacterial cells to these model substrates, and these analogs may be of clinical use.