bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of a spiral-shaped bacterium from the cat stomach
Authors: Lee A , Hazell S L , O'Rourke J , Kouprach S
Published: January 2020

A spiral- or helix-shaped bacterium that colonizes the stomachs of cats has been isolated in pure culture for the first time. The organism is tightly coiled with tufts of 10 to 17 polar flagella positioned slightly off center at the end of the cell. The body of the cell is entwined with unique periplasmic fibrils that usually occur in pairs, although groupings of one and three fibrils were also seen. The organism is strongly urease, catalase, and oxidase positive and is likely to belong to an as yet unclassified group of bacteria that are specifically adapted to the ecological niche provided by gastrointestinal mucus. Isolation of this organism will allow study of the factors influencing colonization of gastric mucosae, information relevant to the association of another mucus colonizer, Campylobacter pylori, with the human stomach. Recent reports of the isolation of other bacteria with the characteristic periplasmic surface structures suggests that the group may be more widespread than was hitherto thought. Bacteria with the morphology of the organisms seen in the cat stomach have been seen in gastric biopsies from humans. The organism whose isolation is reported here has been used in previous serological studies to support the hypothesis that spiral bacteria from animals can colonize the human stomach.
In vivo activation of neutrophil function in hamsters by recombinant human granulocyte colony-stimulating factor
Authors: Cohen A M , Hines D K , Korach E S , Ratzkin B J
Published: January 2020

The in vivo effect of Escherichia coli-derived recombinant human granulocyte colony-stimulating factor on neutrophil function was studied in golden Syrian hamsters. Significant increases in superoxide generation and specific binding of N-formylmethionyl-leucyl-phenylalanine were observed in neutrophils isolated 4 h following a single subcutaneous injection of the factor (30 micrograms/kg). However, phagocytotic activity was not significantly stimulated in hamsters treated with the factor. Recombinant human granulocyte colony-stimulating factor hastened the recovery of peripheral neutrophil counts in animals made leukopenic by prior treatment with cyclophosphamide. Beginning several hours after infection, resistance to lethal infection following intraperitoneal injection of Staphylococcus aureus was increased when neutropenic animals were treated daily with the factor. This protective effect was associated with increased peritoneal neutrophil counts and a decreased incidence of positive peritoneal bacterial cultures at 24 h after the start of treatment. These results suggest that recombinant human granulocyte colony-stimulating factor may be a useful adjunct in the treatment of bacterial infections in neutropenic patients.
Protection against Salmonella typhi infection in mice after immunization with outer membrane proteins isolated from Salmonella typhi 9,12,d, Vi
Authors: Isibasi A , Ortiz V , Vargas M , Paniagua J , González C , Moreno J , Kumate J
Published: January 2020

The current studies were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce protection against challenge with the bacteria in mucin. OMPs were isolated as described by Schnaitman (J. Bacteriol. 108:553-556, 1971) and were found to be contaminated with approximately 4% lipopolysaccharide (LPS). Immunization with as little as 30 micrograms of OMPs conferred 100% protection to mice challenged with up to 1,000 50% lethal doses (LD50) of two strains of S. typhi (9,12,d, Vi and Ty2). In addition, 30% protection against challenge with up to 500 LD50 of Salmonella typhimurium was achieved. Immunization with LPS at doses equivalent to those found in the OMPs was considerably inferior to the OMPs in the induction of an immune status. Moreover, LPS was effective only when the challenge was performed with S. typhi 9,12,d, Vi (40% protection to 100 LD50). An antiserum raised in rabbits reacted mainly against the bands of the molecular weights corresponding to the so-called porins contained in the OMP preparation as shown by Western blotting (immunoblotting). This rabbit antiserum protected 100% of mice against challenge with 100 LD50 of either strain of S. typhi and 80% of mice against challenge with the same LD50 of S. typhimurium. These results indicate the usefulness of OMPs in the induction of active immunity against S. typhi in mice.
Ability of monophosphoryl lipid A to augment the antibody response of young mice
Authors: Baker P J , Hiernaux J R , Fauntleroy M B , Stashak P W , Prescott B , Cantrell J L , Rudbach J A
Published: January 2020

Treatment with nontoxic monophosphoryl lipid A increased the magnitude of the immunoglobulin M (IgM) antibody response to type III pneumococcal polysaccharide in young (2- to 4-week-old) mice. This was accompanied by the appearance of significant numbers of IgG1- and IgG3- secreting antibody-forming cells in 4-week-old mice. These findings indicate that monophosphoryl lipid A can be used as an adjuvant to improve the immunogenicity of poorly immunogenic antigens in young, immunologically immature animals.
Effect of mycoplasmas on natural cytotoxic activity and release of tumor necrosis factor alpha by spleen cells
Authors: Lin Y , Collins J L , Case P G , Patek P Q
Published: January 2020

It has been reported that mycoplasma-infected cells are more sensitive to lysis by natural cytotoxic (NC) effector cells and that splenic NC cells release tumor necrosis factor (TNF-alpha) when they lyse sensitive target cells. Here we showed that spleen cells released TNF-alpha when they were incubated with NC-sensitive cells that were infected with mycoplasmas or when they were incubated with mycoplasmas alone, but did not release TNF-alpha when incubated with NC-sensitive cells that were not infected with mycoplasmas. Thus, in the presence of mycoplasmas, spleen cell cultures contain both NC effector cells and free TNF-alpha. Because NC-sensitive cells are also sensitive to free TNF-alpha, when mycoplasma-infected cells were incubated with spleen cells, they were lysed by the combination of NC cells and free TNF-alpha. When NC-sensitive cells that were not infected with mycoplasmas were incubated with spleen cells, they were lysed only by NC effector cells and thus appeared to be less sensitive than mycoplasma-infected cells. These results also suggested that the release of TNF-alpha may be part of a host protective response to mycoplasmas.
Lack of lysosomal fusion with phagosomes containing Ehrlichia risticii in P388D1 cells: abrogation of inhibition with oxytetracycline
Authors: Wells M Y , Rikihisa Y
Published: January 2020

Fusion of lysosomes with phagosomes containing Ehrlichia risticii, an obligate intracellular parasite, was evaluated in P388D1 murine macrophagelike cells. Lysosomes in cells ranging in infectivity from 30 to 70% were labeled cytochemically with acid phosphatase or via endocytosis of thorium dioxide or cationized ferritin to document phagosome-lysosome (P-L) fusion in untreated cells and cells treated with oxytetracycline. Regardless of the marker used, P-L fusion was generally not observed in E. risticii-containing vacuoles in untreated cells, while significantly greater P-L fusion with ehrlichia-containing vacuoles was observed after oxytetracycline treatment. When latex beads were introduced into uninfected cell cultures, P-L fusion was observed with vacuoles containing latex. Fusion of lysosomes with latex-containing vacuoles in cells was significantly greater than fusion of lysosomes with ehrlichia-containing vacuoles in the same infected cells. These findings indicate that E. risticii is able to inhibit P-L fusion, whereas oxytetracycline deprives organisms of this ability.
Cutaneous host defense in leishmaniasis: interaction of isolated dermal macrophages and epidermal Langerhans cells with the insect-stage promastigote
Authors: Locksley R M , Heinzel F P , Fankhauser J E , Nelson C S , Sadick M D
Published: January 2020

Leishmania species are obligate intracellular pathogens of mononuclear phagocytes. Successful infection depends on sequestration of the promastigote (insect form) within host cells, allowing transformation into the relatively hardy amastigote stage. Promastigotes are killed readily by circulating phagocytes and nonimmune serum, suggesting that cutaneous infection is initiated within a permissive cell in the epidermis or dermis. From large sections of primate skin dermal macrophages and epidermal Langerhans cells were isolated, and their interaction with promastigotes of Leishmania major was investigated in vitro. Dermal macrophages were readily infected with promastigotes, and successful transformation to and replication of amastigotes was observed. Ingestion of promastigotes by dermal macrophages was not associated with a significant respiratory burst, in contrast to that by other macrophage populations, and was associated with significantly greater survival of parasites. Stimulation of these cells with phorbol myristate acetate or opsonized zymosan revealed that those cells were generally oxidatively deficient. Langerhans cells could not be successfully infected by promastigotes under similar conditions. Examination of these cells for expression of CR3, which has been identified as a potential Leishmania receptor, revealed that Langerhans cells did not express the alpha M subunit of CR3, whereas dermal macrophages were CR3 positive. These data support the concept that dermal macrophages are the site of initiation of Leishmania infection.
Characterization of aromatic- and purine-dependent Salmonella typhimurium: attention, persistence, and ability to induce protective immunity in BALB/c mice
Authors: O'Callaghan D , Maskell D , Liew F Y , Easmon C S , Dougan G
Published: January 2020

Stable transposon-generated auxotrophic mutations in aroA, purA, and purE or aroA and purA together were introduced into Salmonella typhimurium strains which were virulent in mice. Strains harboring any of these mutations were attenuated when tested in BALB/c mice. purE strains were less attenuated than aroA or purA strains. Both aroA and purA mutants persisted for several weeks in the livers and spleens of the mice after intravenous infection, although the numbers of viable cells detected at various times after infection differed. aroA strains persisted at a higher level than purA strains and were effective live vaccines given intravenously or orally. purA strains were ineffective as oral vaccines and were poor intravenous vaccines. Strains harboring both aroA and purA mutations together were ineffective vaccines when administered orally or intravenously even though they persisted in the livers and spleens of the mice for long periods after intravenous infection.
Bacterial antigen immunolabeling in macrophages after phagocytosis and degradation of Bacillus subtilis
Authors: Lang T , Tassin M T , Ryter A
Published: January 2020

After phagocytosis of Bacillus subtilis 168 by bone marrow-derived macrophages, the intracellular pathway followed by different antigens was studied by immunofluorescence and immunoelectron microscopy. Three different rabbit antisera were used: (i) an antiserum to B. subtilis whole cells mainly recognizing the cell wall constituents, (ii) an antiserum to teichoic acid, and (iii) an antiserum to peptidoglycan recognizing the disaccharide tetrapeptide molecules resulting from peptidoglycan degradation. During the first 3 h after phagocytosis of B. subtilis, the three antisera were confined to the same vacuolar compartments, as follows. They were first found in phagosomes gathered in the perinuclear region. Upon bacterial degradation, the three antisera colocalized in an increasing number of small dense vesicles, located in the perinuclear region, that seemed to result from the fragmentation of phagolysosomes. These vesicles correspond to an acidic compartment since they also stained for 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine, a drug known to accumulate in the acidic compartments of cells. At later time points, the antigens recognized by the three antisera followed different pathways. After 18 h, teichoic acid and peptidoglycan were no longer detectable in macrophages whereas an antigen(s) labeled with antiserum to B. subtilis whole cells remained stocked for several days in small acidic vesicles randomly distributed throughout the macrophage. This compartment appeared to be different from the one labeled during the first 3 h after ingestion of bacteria. These results suggest that the transport rate and the compartments implicated in antigen processing differ according to the antigen.
Relationship among cell wall composition, stage of growth, and virulence of Nocardia asteroides GUH-2
Authors: Beaman B L , Moring S E
Published: January 2020

The clearance and organ distribution of virulent Nocardia asteroides GUH-2P and the avirulent mutant GUH-2AI at different stages of growth was determined after intravenous inoculation into BALB/c mice. The mutant differed significantly from the parent strain in its ability to survive and grow within the murine host. Since the mutant GUH-2AI had a very different colonial morphology compared with GUH-2P, it was believed that cell surface components might be affected by the mutation that resulted in the loss of virulence. Therefore, cell walls of both GUH-2P and GUH-2AI at different stages of growth were prepared and their composition determined. There were growth-stage-dependent changes in the composition of the cell walls that appeared to correlate with concurrent alterations in virulence; however, the overall chemical composition of the cell wall of the mutant (GUH-2AI) was not significantly different from that of the parent strain (GUH-2P). Both strains demonstrated significant modifications in fatty and mycolic acid composition at different stages of growth. Furthermore, the specific composition of C54 mycolic acids was very different in virulent log-phase cells compared with less-virulent stationary-phase cells, and the avirulent mutant lacked a C54:3 mycolate that was prominent in the virulent log-phase GUH-2P. Thus, C54:3 mycolic acid represented 2.5% of the cell wall (dry weight) in log-phase GUH-2P, but it was undetectable in the walls of GUH-2AI at the stationary phase of growth. These results suggest that certain mycolic acids are associated with virulence.
Characterization of efferent T suppressor cells induced by Paracoccidioides brasiliensis-specific afferent T suppressor cells
Authors: Jimenez-Finkel B E , Murphy J W
Published: January 2020

Previously, we reported that Paracoccidioides brasiliensis culture filtrate antigen (Pb.Ag) when injected i.v. into mice induces antigen-specific suppressor cells which down-regulate the anti-P. brasiliensis delayed-type hypersensitivity (DTH) response. The suppressor cells are present in both spleens and lymph nodes of Pb.Ag-treated animals and suppress the afferent limb but not the efferent limb of the DTH response to P. brasiliensis. The suppressor cells induced by Pb.Ag are L3T4+ Lyt-1+2- I-J+ T cells and are considered to be equivalent to the Ts1 cells described for other antigen-specific suppressor cell pathways. This report provides data which show that Ts1 cells induced by Pb.Ag or a soluble factor derived from Ts1 cells (TsF1) stimulates the production of second-order or efferent suppressor cells. The second-order suppressor cells are detectable in spleens and lymph nodes of mice 7 days after injection of Ts1 cells or TsF1 and are specific in suppressing the paracoccidioidal DTH response. In addition, the second-order suppressor cells are T cells with an L3T4- Lyt-2+ I-J+ phenotype and are effective in suppressing only the efferent limb of the P. brasiliensis DTH response. On the basis of the characteristics defined in this study, the paracoccidioidal second-order suppressor cells are equivalent to the Ts2 cells described for other antigen-specific suppressor-cell pathways. Thus, the suppressive circuit induced by Pb.Ag is similar to the suppressor-cell pathways that regulate the DTH responses to azobenzenearsonate, 4-hydroxy-3-nitrophenyl acetyl, lysozyme, and Cryptococcus neoformans antigen. We propose that such a suppressor-cell circuit as defined here with the murine model could be responsible for the depressed cell-mediated immune responses observed in paracoccidioidomycosis patients who have antigen circulating in their sera.
Cleavage of immunoglobulin G (IgG) and IgA around the hinge region by proteases from Serratia marcescens
Authors: Molla A , Kagimoto T , Maeda H
Published: January 2020

Seven clinical and two nonclinical isolates of Serratia marcescens were examined for their ability to produce extracellular enzymes that cleave immunoglobulin G (IgG) and IgA molecules. All seven clinical isolates excreted a large amount of a 56-kilodalton (kDa) protease (56K protease) and small amounts of a 60-kDa and a 73-kDa protease (60K and 73K proteases, respectively) in culture medium during growth. All purified proteases cleaved IgG and IgA effectively if the level of protease production exceeded 2 to 5 micrograms/ml. The proteolytic activity in the culture supernatant was inhibited by about 85% by a chelating agent (EDTA), which indicated that the major immunoglobulin-cleaving enzyme is the metalloprotease(s) reported previously. Immunological quantification of proteases by single radial immunodiffusion showed similar results: the amount of 56K protease was about 65% and those of the 60K and 73K proteases were about 20 and 5%, respectively. Incubation for 3 h at 37 degrees C was required to generate immunoreactive Fab and Fc fragments. Further analysis of the cleavage products of IgG or IgA demonstrated that the 56K protease, as well as the 60K and 73K proteases, cleaves only the heavy chain of these immunoglobulins near the hinge region to generate Fab and Fc fragments. The susceptibilities of the subclasses of IgG and IgA to the 56K protease were as follows: IgG3 greater than IgG1 greater than IgG2 greater than IgG4 and IgA1 greater than IgA2. IgG2, IgG4, and IgA2 were relatively resistant to the 56K protease.
Comparison of toxins of Clostridium butyricum and Clostridium botulinum type E
Authors: Giménez J A , Sugiyama H
Published: January 2020

The toxin of Clostridium butyricum strains isolated from two infants with botulism is neutralized by antitoxin for type E botulinum toxin. This toxin and that of a C. botulinum type E strain were purified by the same protocol. Both toxins were Mr 145,000 proteins which, when activated with trypsin, were composed of an H subunit of Mr 105,000 and an L subunit of Mr 50,000. The activated specific toxicity of purified butyricum toxin based on an intravenous assay was 2 X 10(8) mouse 50% lethal doses (LD50s)/mg of protein, but that based on an intraperitoneal assay was 7 X 10(7) LD50s/mg, compared with 6 X 10(7) LD50s/mg for type E toxin as determined by both methods. Immunodiffusion tests with antitoxin raised with type E toxin indicated that the two toxins were serologically very similar except for a spur formed by type E toxin. The close similarities of the two toxins suggest that toxigenic C. butyricum could arise when a wild-type strain, which is normally nontoxigenic, acquires the toxin gene of a C. botulinum type E strain.
Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin
Authors: Gordon V M , Leppla S H , Hewlett E L
Published: January 2020

Bordetella pertussis and Bacillus anthracis produce extracytoplasmic adenylate cyclase toxins (AC toxins) with shared features including activation by calmodulin and the ability to enter target cells and catalyze intracellular cyclic AMP (cAMP) production from host ATP. The two AC toxins were evaluated for sensitivities to a series of inhibitors of known uptake mechanisms. Cytochalasin D, an inhibitor of microfilament function, abrogated the cAMP response to B. anthracis AC toxin (93%) but not the cAMP response elicited by B. pertussis AC toxin. B. anthracis-mediated intoxication of CHO cells was completely inhibited by ammonium chloride (30 mM) and chloroquine (0.1 mM), whereas the cAMP accumulation produced by B. pertussis AC toxin remained unchanged. The block of target cell intoxication by cytochalasin D could be bypassed when cells were first treated with anthrax AC toxin and then exposed to an acidic medium. These data indicate that despite enzymatic similarities, these two AC toxins intoxicate target cells by different mechanisms, with anthrax AC toxin entering by means of receptor-mediated endocytosis into acidic compartments and B. pertussis AC toxin using a separate, and as yet undefined, mechanism.
Differences in Lsh gene control over systemic Leishmania major and Leishmania donovani or Leishmania mexicana mexicana infections are caused by differential targeting to infiltrating and resident liver macrophage populations
Authors: Davies E V , Singleton A M , Blackwell J M
Published: January 2020

Earlier studies had shown that the viscerotropic NIH 173 strain of cutaneous Leishmania major fails to come under Lsh gene control. Visceral Leishmania donovani LV9 and another viscerotropic cutaneous strain, Leishmania mexicana mexicana LV4, are controlled by Lsh. The results of double-infection experiments presented here show that expression of Lsh resistance against L. mexicana mexicana was enhanced in the presence of L. donovani, whereas L. major still failed to come under Lsh gene control, even in the presence of L. donovani. Prior irradiation (850 rads) of mice showed that in the absence of infiltrating monocytes, Lsh did exert some influence over L. major. The presence of a higher infiltrate of fresh monocytes after L. major infection was confirmed in liver macrophage populations isolated from mice after infection in vivo and in liver cryosections immunostained with monoclonal antibody M1/70 directed against the type 3 complement receptor CR3. The results support the hypothesis that Lsh is expressed maximally in the resident tissue macrophages and poorly in the immature macrophages preferentially infected by L. major amastigotes.
Identification and characterization of the human lactoferrin-binding protein from Neisseria meningitidis
Authors: Schryvers A B , Morris L J
Published: January 2020

Lactoferrin-binding activity in Neisseria meningitidis was detected by a solid-phase binding assay with horseradish peroxidase-conjugated human lactoferrin (HRP-lactoferrin). Expression of lactoferrin-binding activity was regulated by the level of iron in the medium, so that growth in the presence of the iron chelator EDDA (ethylenediamine di-ortho-hydroxyphenylacetic acid) resulted in a greater than 350-fold increase in binding activity, which was reversed by addition of excess iron. A maximal level of expression could be obtained at reasonable culture densities by using either intermediate levels of EDDA or high levels of EDDA and moderate levels of complexed iron sources such as hemoglobin and transferrin. Competition binding assays demonstrated that the binding of lactoferrin was specific for human lactoferrin in that neither bovine lactoferrin, human transferrin, nor human hemoglobin was able to block binding of HRP-lactoferrin. The binding specificity for human lactoferrin correlated with growth studies in which human but not bovine lactoferrin could support the growth of iron-starved cells. Binding of lactoferrin was not dependent on its level of iron saturation, since iron-saturated lactoferrin and apolactoferrin were equally effective at blocking binding of HRP-lactoferrin in competitive binding assays. The lactoferrin-binding protein was identified as a 105,000-molecular-weight iron-regulated outer membrane protein in three different meningococcal strains by a batch affinity method with biotinylated human lactoferrin and streptavidin-agarose.
Cloning of a Streptococcus sanguis adhesin which mediates binding to saliva-coated hydroxyapatite
Authors: Ganeshkumar N , Song M , McBride B C
Published: January 2020

Chromosomal DNA from a salivary aggregating strain of Streptococcus sanguis 12 was partially digested with PstI and ligated into the plasmid vector pUC18 and transformed into Escherichia coli JM83. A total of 1,700 recombinant clones of E. coli were examined by a colony immunoassay with antisera raised against either S. sanguis 12 whole cells or S. sanguis 12 surface fibrils. Five clones which reacted with one or the other antiserum were shown to be unique by Western blotting (immunoblotting) and restriction endonuclease digestion. One recombinant plasmid pSA2 expressed two proteins with Mrs of 20,000 and 36,000. The 36,000-Mr protein has been designated SsaB. Both proteins were purified to homogeneity by Sephadex G-75 and ion-exchange chromatography. The proteins were present in mutanolysin digests of whole-cell lysates of S. sanguis 12 and in the non-saliva-aggregating variant 12na and the hydrophilic variant 12L. Polyclonal antiserum raised against the SsaB protein reacted strongly with the cell surfaces of S. sanguis 12 and 12na but not with that of 12L. SsaB inhibited the adhesion of S. sanguis 12na to saliva-coated hydroxyapatite, indicating that the adhesin mediates the binding to the pH-sensitive receptor.
Identification of monomeric and oligomeric forms of a major Leishmania infantum antigen by using monoclonal antibodies
Authors: Soteriadou K P , Tzinia A K , Hadziantoniou M G , Tzartos S J
Published: January 2020

Ten monoclonal antibodies (MAbs) produced against isolated Leishmania infantum membranes were used as probes of L. infantum membrane antigens. Western blots of L. infantum membranes, sodium dodecyl sulfate solubilized and heated at 100 degrees C before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that all 10 MAbs recognized a band at 58 kilodaltons (kDa). However, when solubilized membranes were not heated, 2 of the 10 MAbs recognized, in addition to the 58-kDa band, bands of higher molecular weight. Limited digestion of heated or nonheated membranes showed that both groups of MAbs (i.e., not capable or capable of binding to the high-molecular-weight bands) recognized the same proteolytic digests. Hydrophilic forms of the above proteins, possessing proteolytic activity, were detected and isolated by gel filtration. Protein staining of the isolated monomer analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing and heating conditions, revealed incomplete reduction of the 58-kDa protein. The reduced form of the 58-kDa protein migrated at 63 to 65 kDa and was not recognized by the MAbs. These results suggest the existence of a monomeric and an oligomeric form of the 58-kDa antigen. The observed inhibition of Leishmania promastigote-macrophage binding caused by MAbs representative of the two groups (capable of oligomeric and/or monomeric antigen recognition) suggest that the 58-kDa monomer and oligomer play an important role in promastigote-macrophage interaction. We suggest that the 58-kDa L. infantum antigen is the major surface Leishmania antigen (p63) identified by others.
Purification and characterization of Clostridium sordellii hemorrhagic toxin and cross-reactivity with Clostridium difficile toxin A (enterotoxin)
Authors: Martinez R D , Wilkins T D
Published: January 2020

Hemorrhagic toxin (toxin HT) was purified from Clostridium sordellii culture filtrate. The purification steps included ultrafiltration through an XM-100 membrane filter and immunoaffinity chromatography, using a monoclonal antibody to toxin A of Clostridium difficile as the ligand. Toxin HT migrated as a major band with a molecular weight of 525,000 and a minor band at 450,000 on nondenaturing gradient polyacrylamide gel electrophoresis. The molecular weight was estimated at 300,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing indicated an apparent pI of 6.1. Toxin HT was cytotoxic for cultured cells and lethal for mice by intraperitoneal injection, and it elicited an accumulation of hemorrhagic fluid in rabbit ileal loops. Immunodiffusion analysis revealed a reaction of partial identity between toxins A and HT. Immunological cross-reactivity between these toxins was further demonstrated by immunoblotting and by neutralization of toxin HT biological activity with antibodies to toxin A. A sensitive indirect enzyme-linked immunosorbent assay was used to examine the affinity involved in homologous and heterologous antigen-antibody interactions. Our findings show that toxin HT has biological activities and immunological properties similar to those of toxin A; however, the toxins are not identical.
A galE via (Vi antigen-negative) mutant of Salmonella typhi Ty2 retains virulence in humans
Authors: Hone D M , Attridge S R , Forrest B , Morona R , Daniels D , LaBrooy J T , Bartholomeusz R C , Shearman D J , Hackett J
Published: January 2020

We have recently described the construction of a galE derivative of Salmonella typhi Ty2 (Ty2H1) which had a 0.4-kilobase deletion in the galE gene and was sensitive to galactose-induced lysis when cultured with greater than or equal to 0.06 mM galactose (D. M. Hone, R. Morona, S. Attridge, and J. Hackett, J. Infect. Dis. 156:167-174, 1987). We now report the selection of a rifampin-resistant, via derivative of Ty2H1, EX462. Compared with the Ty2 parent strain, EX462 was serum sensitive and highly attenuated in the mouse mucin virulence assay. When four human volunteers ingested 7 X 10(8) viable EX462, two became ill and developed a typhoidlike disease with fever and bacteremia. Blood isolates from these individuals were indistinguishable from the vaccine strain by a variety of criteria. We concluded that, even in a via background, the galE mutation was not attenuating for S. typhi in humans.
Model for invasion of human tissue culture cells by Neisseria gonorrhoeae
Authors: Shaw J H , Falkow S
Published: January 2020

A tissue culture model has been developed for studying the ability of Neisseria gonorrhoeae to invade eucaryotic cells. The cell line HecIB, a human adenocarcinoma endometrial cell line, was found to support gonococcal invasion. The bactericidal antibiotic gentamicin was used to kill those bacteria that had not entered the HecIB cells, allowing us to quantitate internalized bacteria. Kinetic studies showed an increase in the titer of gentamicin-protected gonococci at 4 h postinfection followed by a decrease; a second increase occurred after 6 h. The state of piliation did not affect the degree of invasion when the bacteria were spun down onto the monolayer. Gonococcal invasion was inhibited when the HecIB cells were preincubated with cytochalasin D before bacterial infection. N. lactamica was used as a negative control. No internalized N. lactamica cells were observed by electron microscopy. Electron microscopy documented the intracellular location of the gonococci in HecIB cells and the eventual destruction of the invaded HecIB cells. After 24 h, clusters of gonococci encased in a matrix of cell debris were observed.
Cytokine regulation of the intracellular growth of Mycobacterium paratuberculosis in bovine monocytes
Authors: Zurbrick B G , Follett D M , Czuprynski C J
Published: January 2020

In this study we examined the influence of various crude and recombinant cytokines on the ingestion and intracellular survival of Mycobacterium paratuberculosis within bovine monocytes and monocyte-derived macrophages. Cytokine pretreatment had little effect on the ingestion of M. paratuberculosis by bovine monocytes and macrophages. Monocytes that were continuously incubated with virus-induced crude bovine interferon (100 U) or recombinant bovine alpha interferon (100 U) significantly restricted the subsequent intracellular growth of M. paratuberculosis, as determined by microscopic counts of acid-fast bacilli and by recovery of CFU from lysed monocyte monolayers. In contrast to their effects on freshly adherent monocytes, these cytokines had little effect on the growth of M. paratuberculosis within monocyte-derived macrophages. In two separate experiments, we also observed inhibition of bacillary growth in monocytes treated with unpurified recombinant human granulocyte-macrophage colony-stimulating factor. Conversely, intracellular growth of M. paratuberculosis was enhanced in monocytes that were pretreated with culture supernatants from M. paratuberculosis-stimulated peripheral blood mononuclear cells obtained from an immunized calf. The growth-enhancing activity of these supernatants was labile at pH 2.0, suggesting a role for gamma interferon; however, subsequent experiments indicated that recombinant gamma interferon alone neither enhanced nor restricted intracellular bacillary growth. To determine the possible contributions of monocyte oxidative activity to cytokine-induced bacteriostasis, we compared the release of superoxide anion from cytokine-treated and control monocytes. No obvious relationship was observed between the release of superoxide anion and the subsequent intracellular fate of the bacilli.
Heparin-inhibitable basement membrane-binding protein of Streptococcus pyogenes
Authors: Bergey E J , Stinson M W
Published: January 2020

Solubilized surface proteins of Streptococcus pyogenes serotype M6 were found by indirect immunofluorescence assays to bind selectively to proteoglycan-containing regions of basement membranes of kidney and cardiac muscle in vitro. Epithelial, endothelial, and interstitial cells were unstained. Binding of streptococcal protein to basement membranes was competitively inhibited by heparin and, to a lesser extent, by heparan sulfate. Weak inhibition was also observed with other glycosaminoglycans, including dermatan sulfate, chondroitin sulfate, and hyaluronic acid. Type IV collagen, gelatin, serum fibronectin, glucuronic acid, and a selection of monosaccharides had no significant effects on binding. The heparin-inhibitable basement membrane-binding protein was purified by affinity chromatography on heparin-Sepharose 6-B. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea dissociated the affinity-purified protein into two polypeptides of 9,000 and 15,000 mrs. Chemical analyses revealed that the purified protein was devoid of cysteine, amino and neutral sugars, and phosphate. Thus, the polypeptides are not glycosylated or complexed with trace amounts of lipoteichoic acid or polysaccharide. Binding of purified protein to tissue was determined by direct radioassay and indirect immunofluorescence and was inhibitable by heparin. Although the in vivo effects of this streptococcal component remain to be determined, its deposition on basement membranes in vitro supports the hypothesis that it contributes to the pathogenesis of poststreptococcal glomerulonephritis or acute rheumatic fever.
Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb
Authors: Fukuta S , Magnani J L , Twiddy E M , Holmes R K , Ginsburg V
Published: January 2020

The heat-labile enterotoxins of Vibrio cholerae and Escherichia coli are related in structure and function. They are oligomers consisting of A and B polypeptide subunits. They bind to gangliosides, and they activate adenylate cyclase. The toxins form two antigenically distinct groups; members of each group cross-react but are not necessarily identical. Serogroup I includes cholera toxin (CT) and type I heat-labile enterotoxin (LT-I) of E. coli. LTh-I and LTp-I are antigenic variants of LT-I produced by strains of E. coli from humans and pigs, respectively. Serogroup II contains the type II heat-labile enterotoxin (LT-II) of E. coli. Two antigenic variants designated LT-IIa and LT-IIb have been described. The binding of CT, LTh-I, LT-IIa, and LT-IIb to gangliosides was analyzed by immunostaining thin-layer chromatograms and by solid-phase radioimmunoassay. The four toxins have different glycolipid-binding specificities. LTh-I and CT bind strongly to ganglioside GM1 and less strongly to ganglioside GD1b. However, LTh-I, unlike CT, also binds weakly to GM2 and asialo GM1. LTh-I, like CT, probably binds to the terminal sugar sequence Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal . . ., where GalNAc is N-acetylgalactosamine and NeuAc is N-acetylneuraminic acid. LT-IIa probably binds to the same sugar sequence to which CT and LTh-I bind, with the additional contribution to binding of a second NeuAc as in GD1b and GD2. Also, LT-IIa must bind the Gal beta 1-3GalNAc . . . sequence in such a way that its binding is relatively unaffected by attachment of NeuAc to the terminal galactose residue as in GD1a, GT1b, and GQ1b. LT-IIb probably binds to the terminal sugar sequence NeuAc alpha 2-3Gal beta 1-4GalNAc . . ., as it binds to gangliosides GD1a and GT1b but not to GM1.
Mediation of immunity to Eimeria vermiformis in mice by L3T4+ T cells
Authors: Rose M E , Joysey H S , Hesketh P , Grencis R K , Wakelin D
Published: January 2020

Immunity to infection with Eimeria vermiformis was transferred in NIH mice by both the nylon wool-adherent (B-cell-enriched) and nonadherent (T-cell-enriched) fractions of lymphocytes (spleen and mesenteric lymph node) taken from infected donors. Transfer was more variable with the adherent fraction, and when contaminating T cells were removed by treatment with anti-Thy1 monoclonal antibody (MAb) and complement, this fraction lost all protective activity. The protective effect of T-cell-enriched populations of mesenteric lymphocytes was abrogated by treatment with anti-L3T4 MAb and complement in vitro before transfer or by opsonization with this MAb in vitro before intravenous inoculation into recipients. Similar treatments of cells with anti-Lyt2 MAb did not have this effect, confirming that Thy1+ L3T4+ cells mediate the adoptive transfer of immunity to E. vermiformis. Thy1+ L3T4+ cells were also shown to limit the replication of E. vermiformis in primary infections: mice depleted of this subset (by thymectomy followed by intravenous injection of anti-L3T4 MAb) passed greater numbers of oocysts over a longer period of time than did mice similarly depleted of Lyt2+ cells.
Immunization with a Pseudomonas aeruginosa immunotype 5 O polysaccharide-toxin A conjugate vaccine: effect of a booster dose on antibody levels in humans
Authors: Cryz S J , Sadoff J C , Fürer E
Published: January 2020

Healthy adult volunteers were vaccinated on day 0 and 28 and at 15 months with a Pseudomonas aeruginosa immunotype 5 O polysaccharide-toxin A conjugate vaccine. Immunization resulted in mild, transient local reactions in less than 20% of the subjects. Maximal immunoglobulin G (IgG) antibody titers to both toxin A and lipopolysaccharide (LPS) as determined by enzyme-linked immunosorbent assay were seen at day 42, at which time 50% of the vaccinees showed a fourfold or greater rise in toxin A-neutralizing titers. By 15 months postvaccination, both antitoxin A and anti-LPS IgG antibodies had markedly declined. A booster dose of vaccine administered at 15 months evoked a vigorous anti-toxin A IgG antibody response with 100% of the volunteers showing a fourfold or greater rise in neutralizing antibody titer compared with preimmunization levels. In contrast, there was no significant elevation of anti-LPS IgG antibody levels. At 24 months postimmunization, only anti-toxin A antibody levels were significantly higher than preimmunization levels.
Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation
Authors: Schwan T G , Burgdorfer W , Garon C F
Published: January 2020

In vitro cultivation of Borrelia burgdorferi, the etiologic agent of Lyme spirochetosis, allows for the isolation and growth of this bacterium from infected tissues. However, continuous cultivation in modified Kelly medium causes a reduction in the number of detectable plasmids and the loss of infectivity in the white-footed mouse, Peromyscus leucopus. In an unpassaged culture of B. burgdorferi, nine plasmids were present, including seven linear plasmids ranging in size from 49 to 16 kilobases (kb) and two circular plasmids of 27 and 7.6 kb. The 7.6-kb circular and 22-kb linear plasmids were no longer detectable in spirochetes noninfective in white-footed mice, suggesting that a gene(s) encoding for factors responsible for infection may be present on one or more of these extrachromosomal elements. Furthermore, changes in spirochetal proteins and lipopolysaccharide-like material were observed also during early cultivation and may be related to loss of infectivity.
Two distinct forms of Chlamydia psittaci associated with disease and infertility in Phascolarctos cinereus (koala)
Authors: Girjes A A , Hugall A F , Timms P , Lavin M F
Published: January 2020

While several diseases associated with Chlamydia psittaci infection have been reported in Phascolarctos cinereus (koala), it is still unclear whether one or more chlamydial strains are responsible. In this study, we provide evidence, obtained by restriction enzyme and gene probe analysis, that two quite distinct strains of C. psittaci infect koalas; one strain was isolated from the conjunctivae, and the other was isolated from the urogenital tract and the rectum. A gene probe, pFEN207, containing the coding sequence for an enzyme involved in the biosynthesis of the chlamydial genus-specific lipopolysaccharide antigen, and a separate probe, pCPML-4N, prepared from a DNA fragment of a koala-infecting strain of C. psittaci, were used to determine the patterns of hybridization in the koala-infecting strains; these patterns were found to be quite distinct from those observed with C. psittaci isolates from other animals. We also demonstrated by hybridization analysis with an avian strain plasmid that all three koala urogenital isolates contain a plasmid and that there is no evidence for the presence of a homologous plasmid in any of the ocular isolates.
Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies
Authors: Downes F P , Barrett T J , Green J H , Aloisio C H , Spika J S , Strockbine N A , Wachsmuth I K
Published: January 2020

Shiga-like toxin (SLT-II) was purified to apparent homogeneity from Escherichia coli K-12 strain NM522 containing the cloned toxin genes on recombinant plasmid pEB1. Purification was accomplished by a series of column chromatography techniques: anion-exchange, chromatofocusing, cation-exchange, and monoclonal antibody affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the pure toxin showed that SLT-II consisted of A and B subunits with apparent molecular weights of 32,000 and 10,200 +/- 800, respectively. A band of molecular weight 25,000 was also observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as the A1 subunit by Western immunoblot analysis with toxin-specific monoclonal antibodies (MAbs). The pI of the purified toxin was 5.2. Approximately 1 pg of pure SLT-II, but was not neutralized by polyclonal antibodies or MAbs to SLT-I. Five hybridomas against SLT-II were produced (BC5 BB12, DC1 EH5, EA5 BA3, ED5 DF3, and GB6 BA4). Culture supernatant fluids containing MAbs from these hybridomas did not neutralize the cytotoxicity of SLT-I or Shiga toxin. Western blot analysis showed that two MAbs (MAb DC1 EH5 and MAb GB6 BA4) recognized the A and A1 subunits of SLT-II and three MAbs (MAb BC5 BB12, MAb EA5 BA3, and MAb ED5 DF3) recognized the B subunit of SLT-II. MAb BC5 BB12 was used to prepare an affinity column for toxin purification.
Inhibition of phagocytosis in Yersinia pseudotuberculosis: a virulence plasmid-encoded ability involving the Yop2b protein
Authors: Rosqvist R , Bölin I , Wolf-Watz H
Published: January 2020

Virulence plasmid-containing cells of Yersinia pseudotuberculosis had the ability to inhibit phagocytosis by mouse peritoneal macrophages cultured in vitro, but cells of its plasmid-cured derivative did not. Inhibition was most pronounced when the pathogen was incubated under Ca2+-deficient conditions, which allowed a high level of expression of outer membrane proteins (Yops). The addition of 2.5 mM Ca2+ to the growth medium reduced the degree of inhibition by the pathogen, but it was still significantly higher than that of the plasmid-cured strain. An avirulent mutant strain, from which the entire yopH gene was deleted, was impaired in its phagocytosis inhibition ability. This mutant could be trans-complemented by the yopH+ gene back to the wild-type phenotype with respect to virulence, as well as the ability to inhibit phagocytosis, demonstrating that the ability to inhibit phagocytosis is an important virulence function. The mutant strain was still cytotoxic for HeLa cells, indicating that inhibition of phagocytosis can be genetically separated from the ability to cause a cytotoxic effect.
Clearance of different strains of Mycoplasma pulmonis from the respiratory tract of C3H/HeN mice
Authors: Davidson M K , Davis J K , Lindsey J R , Cassell G H
Published: January 2020

Pathogen-free C3H/HeN mice were exposed by aerosol to Mycoplasma pulmonis PG34(ASH), UAB 5782C, M1, UAB T, or UAB CT, and clearance of mycoplasmas from the nasal passages, trachea, and lungs was determined during the first 72 h postinoculation (PI). There were differences among strains of mycoplasmas in physical removal of organisms and in killing by nonspecific factors in the nasal passages and trachea. The avirulent strain, PG34(ASH), was quickly removed from the nasal passages and trachea. Physical removal of the other mycoplasmal strains occurred slowly, with 60 to 89% of the radioactive label remaining in the nasal passages and trachea even after 72 h. There were significant differences in killing among mycoplasmal strains by nonspecific host mechanisms in the nasal passages, trachea, and lungs. Strain UAB T was quickly killed at all levels of the respiratory tract. Strains UAB 5782C and M1 were killed at all three sites by 2 to 4 h PI. The most virulent strain, UAB CT, was killed much more slowly than the other strains. However, there was no statistical difference in the relative numbers of mycoplasmas present in the lungs at 72 h PI among strains UAB CT, UAB 5782C, and M1. These studies showed that the different mycoplasmal strains were cleared from the respiratory tract by different mechanisms and suggest that the differences in virulence among the mycoplasma strains can be explained, in part, by the differences in elimination of the organisms from the respiratory tract by nonspecific host defense mechanisms.
Small, antibacterial and large, inactive peptides of neutrophil granules share immunoreactivity to a monoclonal antibody
Authors: Marzari R , Scaggiante B , Skerlavaj B , Bittolo M , Gennaro R , Romeo D
Published: January 2020

Monoclonal antibodies were raised against a bactericidal protein fraction that was purified from an extract of bovine neutrophil granules and that was previously shown (A. Savoini, R. Marzari, L. Dolzani, D. Serranò, G. Graziosi, R. Gennaro, and D. Romeo, Antimicrob. Agents Chemother. 26:405-407, 1984) to inhibit bacterial DNA synthesis. One of these antibodies, BP97, was covalently linked to Affi-Gel 10 and was used for immunoaffinity chromatography of granule extracts. Elution of the bound proteins, followed by reversed-phase high-performance liquid chromatography, generated several peptides whose molecular weights fell in the range of 1,600 to 64,000 and which reacted to BP97 but not to other mouse monoclonal or polyclonal antibodies. The reaction to BP97 appeared to be specific, inasmuch as a full panel of cationic oligo- or polypeptides was not recognized by this monoclonal antibody. Among the purified granule polypeptides, the more cationic ones (with molecular weights of 4,300 to 8,000) inhibited the growth of Escherichia coli at a MIC of 12 to 50 micrograms/ml. In addition, a 1,600-molecular-weight, highly cationic peptide also inhibited the growth of Staphylococcus aureus and Staphylococcus epidermidis at MICs of 3 to 8 micrograms/ml.