bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the selective inhibition of growth of virulent Legionella pneumophila by supplemented Mueller-Hinton medium
Authors: Catrenich C E , Johnson W
Published: January 2020

The phenotypic difference between virulent and avirulent Legionella pneumophila with regard to growth on supplemented Mueller-Hinton (SMH) agar was investigated. Defined populations of virulent and avirulent L. pneumophila were inoculated onto hybrid growth media containing the combination of SMH agar components with potassium phosphate-buffered charcoal-yeast extract agar. The casein acid hydrolysate component of SMH agar was found to be inhibitory to the growth of virulent but not avirulent cells. The selectively inhibitory component of the casein acid hydrolysate was identified as NaCl.
Effects of eliminating a disulfide bridge within domain II of Pseudomonas aeruginosa exotoxin A
Authors: Madshus I H , Collier R J
Published: January 2020

Cysteines 265 and 287 of Pseudomonas aeruginosa exotoxin A (ETA) were substituted by serine, thereby eliminating a disulfide bridge within domain II, the putative membrane insertion-translocation domain. Purified mutant toxin was 80-fold less toxic for mouse L cells than was wild-type ETA while retaining the same specific activity in the ADP-ribosyltransferase reaction as did wild-type toxin. Binding of the nonionic detergent Triton X-114 by mutant ETA occurred at a slightly higher pH than did binding by wild-type ETA, suggesting that the mutant protein more readily undergoes a conformational change exposing hydrophobic regions. Data are presented supporting the notion that the mutant and wild-type toxins enter from the same intracellular compartment. The lower cytotoxicity of the mutant protein could be due to accelerated intracellular degradation or abortive, premature membrane insertion.
Monoclonal antibody-mediated protection and neutralization of motility in experimental Proteus mirabilis infection
Authors: Harmon R C , Rutherford R L , Wu H M , Collins M S
Published: January 2020

A panel of monoclonal antibodies with specificity for a wound isolate of Proteus mirabilis was established. Of nine antibodies studied in detail, three were broadly reactive with various Proteus isolates, while six reacted in a serotype-specific fashion with the strain used for immunization. Five of the six serotype-specific antibodies were reactive with lipopolysaccharide. The sixth serotype-specific antibody, 4-F (immunoglobulin G1 [IgG1]), was potently protective in a burn wound sepsis model and recognized a protein antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis were used to determine that 4-F was reactive with flagellar protein. Approximately 1.3 micrograms of the antibody was sufficient to provide protection against 8 50% lethal doses of wound isolate, and approximately 26 micrograms provided full protection against challenge with 333 50% lethal doses. In vitro test results indicated that 4-F inhibited the motility of the wound isolate, and in vivo testing showed that it inhibited dissemination of the inoculum from the burn site to the liver and spleen. Whereas the antibody was highly effective in preventing the death of mice subsequent to challenge at a burn site, no protection was seen following an intraperitoneal challenge. These results may therefore indicate that the protection observed in the burn model is solely a reflection of the capacity of 4-F to neutralize bacterial motility.
T-cell recognition of the 18-kilodalton antigen of Mycobacterium leprae
Authors: Dockrell H M , Stoker N G , Lee S P , Jackson M , Grant K A , Jouy N F , Lucas S B , Hasan R , Hussain R , McAdam K P
Published: January 2020

The 18-kilodalton (kDa) antigen of Mycobacterium leprae was expressed as a fusion protein with a 2-kDa leader peptide and used in proliferation assays with peripheral blood cells. Fifty percent of untreated tuberculoid leprosy patients and 93% of long-term leprosy contacts responded to the recombinant protein in lymphocyte transformation tests. Comparison of the stimulation indices in the two groups showed that the contacts responded more strongly than the tuberculoid leprosy patients. Seventy percent of Mycobacterium bovis BCG-vaccinated European donors responded, although with low stimulation indices. The isolation of 18-kDa antigen-responsive T-cell lines from a BCG-vaccinated British donor confirmed that the 18-kDa antigen contains at least one cross-reactive epitope. These results indicate that the 18-kDa protein is an important antigen in the immune response to leprosy.
Effect of Streptococcus pneumoniae on human respiratory epithelium in vitro
Authors: Steinfort C , Wilson R , Mitchell T , Feldman C , Rutman A , Todd H , Sykes D , Walker J , Saunders K , Andrew P W
Published: January 2020

A total of 11 of 15 Streptococcus pneumoniae culture filtrates and all five bacterial autolysates produced by cell death in the stationary phase caused slowed ciliary beating and disruption of the surface integrity of human respiratory epithelium in organ culture. This effect was inhibited by cholesterol and was heat labile and reduced by standing at room temperature but was stable at -40 degrees C. The activity was detected at the late stationary phase of culture and was associated with the presence of hemolytic activity. Gel filtration of a concentrated culture filtrate and autolysate both yielded a single fraction of approximately 50 kilodaltons which slowed ciliary beating and were the only fractions with hemolytic activity. Rabbit antiserum to pneumolysin, a sulfhydryl-activated hemolytic cytotoxin released by S. pneumoniae during autolysis, neutralized the effect of the culture filtrate on respiratory epithelium. Both native and recombinant pneumolysin caused ciliary slowing and epithelial disruption. Electron microscopy showed a toxic effect of pneumolysin on epithelial cells: cytoplasmic blebs, mitochondrial swelling, cellular extrusion, and cell death, but no change in ciliary ultrastructure. Recombinant pneumolysin (10 micrograms/ml) caused ciliary slowing in the absence of changes in cell ultrastructure. Release of pneumolysin in the respiratory tract during infection may perturb host defenses, allowing bacterial proliferation and spread.
General method for site-directed mutagenesis in Escherichia coli O18ac:K1:H7: deletion of the inducible superoxide dismutase gene, sodA, does not diminish bacteremia in neonatal rats
Authors: Bloch C A , Thorne G M , Ausubel F M
Published: January 2020

A defined deletion in the Escherichia coli K-12 sodA gene (encoding manganese-superoxide dismutase) linked to a nontransposable selectable marker was generated by transposon Tn5 insertion in combination with in vitro mutagenesis. This mutant allele was used to replace the wild-type sodA gene in an E. coli clinical isolate of serotype O18ac:K1:H7 by bacteriophage P1 transduction. The O18ac:K1:H7 sodA mutant contained no manganese-superoxide dismutase and no hybrid manganese-iron-superoxide dismutase. The sodA mutant was more sensitive to paraquat toxicity than were the parental strain and an isogenic mutant bearing an analogously constructed sodA+ Tn5 insertion allele. In a suckling rat model for bacteremia following oral inoculation of E. coli K1, the sodA mutant was undiminished in its capabilities both to colonize the gastrointestinal tract and, surprisingly, to cause bacteremia. In conjunction with the rat model for E. coli K1 pathogenesis, the method for site-directed mutagenesis described in this paper permits determination of the role played in colonization and bacteremia by any K1 gene which either has a homolog in E. coli K-12 or can be cloned and manipulated therein.
Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells
Authors: Corbeil L B , Hodgson J L , Jones D W , Corbeil R R , Widders P R , Stephens L R
Published: January 2020

Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective.
Oxidant stress in Neisseria gonorrhoeae: adaptation and effects on L-(+)-lactate dehydrogenase activity
Authors: Fu H S , Hassett D J , Cohen M S
Published: January 2020

Neisseria gonorrhoeae, an obligate human pathogen, is subjected to oxidant stress when attacked by O2 reduction products formed by neutrophils. In this study, exposure of gonococci to sublethal concentrations of superoxide and hydrogen peroxide (and related O-centered radicals) resulted in phenotypic resistance to oxidant stress. Adaptation required new protein formation but was not related to increases in superoxide dismutase or catalase. We have previously demonstrated that gonococci use phagocyte-derived L-(+)-lactate. Oxidant stress of greater magnitude than that required for adaptation led to a generalized increase in bacterial metabolism, particularly in L-(+)- and D-(-)-lactate utilization and lactate dehydrogenase activity. Increased lactate utilization required new protein synthesis. These results suggest the possibility that lactate metabolism is of importance to N. gonorrhoeae subjected to oxidant stress. Use of lct mutant organisms unable to use L-(+)-lactate should allow examination of this hypothesis.
Cellular events and intracellular survival of Campylobacter jejuni during infection of HEp-2 cells
Authors: De Melo M A , Gabbiani G , Pechère J C
Published: January 2020

Invasion and intracellular survival of Campylobacter jejuni in HEp-2 cells were analyzed by transmission electron microscopy and by viable counts after killing of extracellular bacteria by gentamicin. During the first 30 min after challenge, no bacteria were seen in association with the host cell. After 1 h, campylobacters apparently attached to the cell membrane, with areas of close appositions. In these areas, an intracellular network of actin-like filaments was seen beneath the plasma membrane. Other bacteria were included into endocytic vacuoles. After 3 h, an intense lysosomal response was observed in the host cells, as determined by the presence of myelinic forms and acid phosphatase activity. After 9 h, bacteria still contained in vacuoles showed signs of degradation with a change from spiral to coccal forms. Morphological evidence of phagosome-lysosome fusion was also seen, and these observations by transmission electron microscopy correlated well with a decrease in bacteria viability 9 h after challenge, as determined from separate kinetics studies. Inhibitors of phagocytosis were observed to reduce markedly the entry of C. jejuni into the cells at concentrations which apparently did not affect bacterial viability. These results suggest that the campylobacters were successively attached to the HEp-2 cell membrane, internalized by a phagocytic-like mechanism, and digested after phagosome-lysosome fusion.
Identification of functional antigenic segments of toxic shock syndrome toxin 1 by differential immunoreactivity and by differential mitogenic responses of human peripheral blood mononuclear cells, using active toxin fragments
Authors: Edwin C , Kass E H
Published: January 2020

When toxic shock syndrome toxin 1 was subjected to papain hydrolysis, two serologically active fragments of 16.3 kilodaltons (16K fragment) and 12.4 kilodaltons (12K fragment) were generated, whereas a third fragment of 9.7 kilodaltons (10K fragment) was inactive. The biologic activities of the fragments were evaluated in vitro by determining their ability to promote nonspecific proliferation of human peripheral blood mononuclear cells. The 12K fragment was significantly (P less than or equal to 0.013) more stimulatory than the 16K fragment. When human peripheral blood mononuclear cells were preincubated for a period of 24 h with various concentrations of the 16K fragment, followed by incubation with a constant amount (2 x 10(-2) ng/ml) of whole toxin, the level of DNA synthesis induced by the holotoxin was reduced by approximately 60% when compared with that of controls exposed to whole toxin alone. The 12K fragment did not demonstrate a similar blocking effect. Immunoblots of the toxic shock syndrome toxin 1 digest, which were exposed to monoclonal antibodies (MAbs) developed against native toxin, depicted the presence of two different antigenic regions (epitopes). One MAb, 8-5-7, which has been shown previously to inhibit the biologic activity of the holotoxin in vitro and in vivo, reacted primarily with the 12K fragment. A second MAb, 10-6-1, that did not neutralize interleukin-1 production reacted primarily with the 16K fragment. On the basis of the differential mitogenic responses and the identification of heterologous epitopes, it was concluded that the functional region of the holotoxin can be partitioned into at least two functional segments encompassed between amino acid residues 53 and 87 and between amino acid residues 88 and 194 on the polypeptide chain.
Binding of Escherichia coli S fimbriae to cultured human endothelial cells
Authors: Parkkinen J , Ristimäki A , Westerlund B
Published: January 2020

The binding of Escherichia coli adhesins to human umbilical vein endothelial cells was studied by a cell monolayer enzyme-linked immunosorbent assay. S fimbriae displayed a concentration-dependent and saturable binding to the endothelial cells which was mediated by their sialylgalactoside-specific lectin activity. P fimbriae exhibited only low binding, and type 1 fimbriae exhibited no binding to these cells.
In vitro and in vivo toxicity of T-2 toxin, a Fusarium mycotoxin, to mouse peritoneal macrophages
Authors: Vidal D , Mavet S
Published: January 2020

The effects of T-2 toxin on mouse peritoneal macrophages were investigated. Scanning electron microscopy of macrophages treated in vitro with T-2 toxin revealed retraction of pseudopodia. Protein synthesis was inhibited after in vitro contact with T-2 toxin but was not affected 24 h after injection of a sublethal dose of toxin into mice. There was reduction in the phagocytosis of Pseudomonas aeruginosa when macrophages were exposed in vitro to T-2 toxin and when mice were injected with T-2 toxin. Clearance of colloidal carbon was not modified after T-2 toxin injection, whereas spleen weight was decreased 24 h after T-2 injection. T-2 toxin enhanced the mortality of mice infected with Salmonella typhimurium C5S when it was administered 24 h prior to oral challenge with the bacterium.
Congo red-mediated regulation of levels of Shigella flexneri 2a membrane proteins
Authors: Sankaran K , Ramachandran V , Subrahmanyam Y V , Rajarathnam S , Elango S , Roy R K
Published: January 2020

The ability of Shigella spp. to bind Congo red from agar medium is generally correlated with their virulence properties. We used a metabolically active culture of Shigella flexneri 2a to determine the effect of Congo red on its membrane protein profiles. Virulent S. flexneri grown in the presence of Congo red at 37 degrees C showed increased levels of three proteins with Mrs of 43,000, 58,000, and 63,000 (43K, 58K, and 63K proteins) in the Sarkosyl-soluble membrane fractions. The observed phenomenon was temperature dependent. At 30 or 42 degrees C the protein levels remained unaffected by the presence of Congo red. Similar regulation of the levels of the 43K, 58K, and 63K membrane proteins was also observed with Shigella dysenteriae 1 and enteroinvasive Escherichia coli, but not with enteropathogenic E. coli. The cellular uptake of Congo red seemed to be essential, but not sufficient, for regulation. All three proteins reacted with human convalescent-phase sera in immunoblots of S. flexneri 2a Sarkosyl-soluble membrane fractions. Using the 43K-specific antiserum as the primary antibody, by indirect immunofluorescence studies, we detected an increase in the level of the 43K protein in S. flexneri which had invaded epithelial cells. These observations strongly indicate that the 43K, 58K, and 63K proteins are virulence associated. We propose that the observed regulatory effect of Congo red on membrane proteins of S. flexneri is mediated through induction. Since the same regulatory effect was also observed during the invasion of epithelial cells by S. flexneri, it is suggested that Congo red mimics some host tissue factor in vitro.
Purification, primary structure, and biological activity of guinea pig neutrophil cationic peptides
Authors: Yamashita T , Saito K
Published: January 2020

The guinea pig neutrophil cationic peptides GNCP-1 and GNCP-2 were purified from a granule-rich subcellular fraction of peritoneal exudate neutrophils by acid-gel electrophoresis and reversed-phase high-performance liquid chromatography. Both peptides not only released histamine from rat mast cells to the same extent but also were equally active against Staphylococcus aureus and Escherichia coli. The peptides were rich in arginine and cystine and lacked free sulfhydryl groups. Composition and sequence determinations revealed that GNCP-1 and GNCP-2 are each single polypeptides containing 31 amino acid residues and three intramolecular disulfide bonds. The complete amino acid sequence of GNCP-1 is Arg-Arg-Cys-Ile-Cys-Thr-Thr-Arg-Thr-Cys-Arg-Phe-Pro-Tyr-Arg-Arg-Leu-Gly- Thr-Cys - Ile-Phe-Gln-Asn-Arg-Val-Tyr-Thr-Phe-Cys-Cys. The sequence of GNCP-2 is identical except for the substitution of isoleucine for leucine at residue 21.
Immune response to Campylobacter jejuni and Campylobacter coli in a cohort of children from birth to 2 years of age
Authors: Martin P M , Mathiot J , Ipero J , Kirimat M , Georges A J , Georges-Courbot M C
Published: January 2020

A cohort of 111 children from Bangui, Central African Republic, was surveyed for enteric Campylobacter infections from birth to the age of 2 years; stools were examined biweekly in these children until 6 months of age and at least four times per year thereafter until 2 years of age and after each diarrheal episode. Blood samples were obtained at birth and at 3, 6, 9, 12, 18, and 24 months of age. Antibodies against glycine-extracted membrane antigens, purified flagella, and cholera toxin (CT) were assayed by an enzyme-linked immunosorbent assay. The results showed that titers of antibody against the three tested antigens increased in children between 6 and 12 months of age and that nearly all children were immunized by the age of 2 years. A significant fall in anti-flagellum (P less than 0.001) and anti-glycine extract antibodies (P less than 0.001) occurred between birth and age 3 months, and children who had Campylobacter infections during the first 6 months of life had significantly (P less than 0.02) less anti-flagellum antibodies at birth than did those who did not have Campylobacter infections during that time. Three-month-interval stratification showed that CT antibody titers at birth were significantly lower in children who developed Campylobacter infection than in controls (P = 0.05). Comparison of the immune response to a single Campylobacter episode showed that 46.6% of children with asymptomatic carriage did not respond to CT while only 5% of children with diarrhea-producing infection did not respond to CT (P less than 0.01), compared with 30% (P = 0.065) and 56% (P less than 0.01), respectively, of the age-matched controls. Antibodies to flagella seem to protect against enteric colonization by Campylobacter jejuni and Campylobacter coli.
Effects of pyocyanine, a phenazine dye from Pseudomonas aeruginosa, on oxidative burst and bacterial killing in human neutrophils
Authors: Müller P K , Krohn K , Mühlradt P F
Published: January 2020

The effects of pyocyanine (phenazinium, 1-hydroxy-5-methyl-hydroxide, inner salt) on oxidative burst in human polymorphonuclear leukocytes were studied by several different approaches. In a cell- and enzyme-free system, pyocyanine oxidized NADPH. The reduced pyocyanine could be measured by its reaction with ferricytochrome c. It was shown by this assay that resting as well as phorbol myristate acetate- or zymosan-stimulated granulocytes reduced pyocyanine. The effect was independent of mitochondria, as cytoplasts were similarly active. Measurement of the hexose monophosphate shunt in intact granulocytes in the presence of pyocyanine indicated a concentration-dependent activation of the shunt without the generation of O2-, suggesting that pyocyanine oxidizes NADPH to NADP+ when it enters granulocytes. Intracellular NADPH in granulocytes was indeed lowered by almost 40% after incubation with pyocyanine. It is by this shuttling of reduction equivalents, leading to the partial depletion of NADPH, that pyocyanine affects the observed concentration-dependent partial inhibition of the phorbol myristate acetate- and zymosan-stimulated generation of O2-. A further consequence was that the intracellular killing of Staphylococcus aureus was also partially suppressed, particularly at higher loads of granulocytes with bacteria. Phagocytosis was not inhibited by pyocyanine concentrations as high as 500 microM. Pyocyanine did not affect the intracellular killing of Pseudomonas aeruginosa. The possible relevance of these findings to the course of mixed hospital infections in immunocompromised patients is discussed.
Effects of magainins and cecropins on the sporogonic development of malaria parasites in mosquitoes
Authors: Gwadz R W , Kaslow D , Lee J Y , Maloy W L , Zasloff M , Miller L H
Published: January 2020

Magainins and cecropins are families of peptides with broad antimicrobial and antiparasitic activities derived respectively from the skin of frogs or from giant silk moths. In insects, cecropins function as part of an inducible immune system against a number of bacterial infections. When injected into anopheline mosquitoes previously infected with a variety of Plasmodium species, both magainins and cecropins disrupt sporogonic development by aborting the normal development of oocysts; sporozoites are not formed and the vector cannot transmit the parasite to another host. It may be possible to induce effective transmission-blocking immunity in the mosquito vector by the introduction and expression of genes coding for magainins, cecropins, or similarly acting parasiticidal peptides into the mosquito genome.
Parasite exposure elicits a preferential T-cell response involved in protective immunity against Eimeria species in chickens primed by an internal-image anti-idiotypic antibody
Authors: Bhogal B S , Jacobson E B , Tse H Y , Schmatz D M , Ravino O J
Published: January 2020

Polyclonal anti-idiotype 1073 (anti-Id 1073), raised against a monoclonal antibody specific for the protective epitope(s) of Eimeria tenella sporozoites, induced cell-mediated immune (CMI) responses in bursectomized chickens. Whereas alhydrogel-adsorbed anti-Id 1073 was sufficient to engender the CMI response at 4 h after injection, induction of the CMI response at 24 h required both alhydrogel and muramyl dipeptide sterol. Exposure of immunized chickens to live parasites prompted a dichotomous effect on the CMI response engendered by anti-Id in that the 4-h CMI response was preferentially stimulated and the 24-h CMI response was down regulated. Both types of CMI response were transferable to naive chickens by T cells from anti-Id 1073 immune donors or by parasite-specific T cells from clones 21 and 27. These T-cell clones were generated from chickens immunized by repeated infections with E. tenella and showed in vitro proliferative responses to anti-Id 1073. The abilities of T cells from clone 21 to selectively transfer the 4-h CMI response and to generate gamma interferon to activate macrophages for their cytotoxic effects on Eimeria sporozoites correlate with the preferential stimulation by parasites of the 4-h CMI response in chickens immunized with anti-Id 1073. These data show that anti-Id 1073 mimics the protective epitope(s) of the parasite and primes chickens for protective CMI responses. Cytotoxic T cells, equivalent to the mammalian T-cell subset of the Lyt2+ phenotype, appear to be the primary effector T cells in the CMI response engendered by anti-Id 1073 against Eimeria parasites.
Salmonella typhimurium mutants lacking flagella or motility remain virulent in BALB/c mice
Authors: Lockman H A , Curtiss R
Published: January 2020

Nonmotile flagellated (mot) and nonflagellated (fla) mutants of Salmonella typhimurium LT-2 were isolated from a collection of mutants with random Tn10-insertion mutations. Both classes of mutants were resistant to infection by the flagellotropic bacteriophage chi. The nonflagellated (fla::Tn10) mutants did not react with H antigen-specific antisera and did not possess flagella when examined by electron microscopy, and sheared-cell extracts were devoid of flagellin. The nonmotile (mot::Tn10) mutants reacted with H-specific antisera and expressed paralyzed flagella that were indistinguishable from wild-type flagella. The Tn10 insertions in strain LT-2 were mapped to loci in regions II (flh and mot) and III (fli) of the flagellar genes, and the mutations were transduced into the mouse-virulent S. typhimurium strains SR-11 and SL1344. Lack of motility reduced the ability of S. typhimurium to invade Henle cells in vitro, yet the virulence in mice of the nonmotile mutants of SR-11 and SL1344 was unaffected by the inactivity or loss of flagella. Wild-type SR-11 had a 50% lethal dose (LD50) in BALB/c mice following oral (p.o.) challenge of 2.4 x 10(4) CFU. The p.o. LD50 of the SR-11 fli-8007::Tn10 mutant was 4.5 x 10(4) CFU. The mot-8008::Tn10 mutation in SR-11 conferred paralyzed flagella and increased the p.o. LD50 in mice to 2.2 x 10(5) CFU, but this was not statistically significant. A similar increase in the p.o. LD50 was observed when the SL1344 mot-8008::Tn10 mutant was tested in mice. Wild-type SR-11 and the isogenic nonflagellated and nonmotile mutants were equally virulent in mice challenged via intraperitoneal injection.
Prostaglandin E2 enhances and gamma interferon inhibits germ tube formation in Candida albicans
Authors: Kalo-Klein A , Witkin S S
Published: January 2020

Prostaglandin E2 (PGE2), an immunosuppressive monokine that increases intracellular cyclic AMP (cAMP) levels, stimulated Candida albicans germ tube formation. Dibutyryl cAMP (dB-cAMP) and isoproterenol, other compounds that increase cAMP levels, also stimulated germination. Gamma interferon (IFN-gamma), a product of cellular immune system activation, inhibited Candida germ tube formation, even in the presence of PGE2, dB-cAMP, and isoproterenol. Thus, PGE2 and IFN-gamma as well as having opposing roles in the suppression or activation of cell-mediated immunity, are also antagonists for the yeast-to-hyphal transition of C. albicans.
Cloning and complete nucleotide sequence of the gene for the main component of hemagglutinin produced by Clostridium botulinum type C
Authors: Tsuzuki K , Kimura K , Fujii N , Yokosawa N , Indoh T , Murakami T , Oguma K
Published: January 2020

In Clostridium botulinum types C and D, phage conversion to toxin and hemagglutinin (HA) production has been reported. DNA was extracted from a converting type C Stockholm phage, c-st, and a fragment (7.8 kilobase pairs) coding for the parts of both toxin and HA was cloned. The gene for HA was recloned, and the complete nucleotide sequence was determined. The molecular mass of this gene product was 33 kilodaltons, and it showed HA activity. The HA preparation partially purified from a type C Stockholm culture demonstrated two major bands (33 and 53 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reducing agent. The amino acid sequence of the N terminus of the 33-kilodalton component of the native HA preparation, which was determined by a direct protein microsequencing procedure, was identical to that deduced from the nucleotide sequence of cloned HA gene. These data indicate that the cloned gene product (33 kilodaltons) is an important component of HA.
Ingestion of Giardia lamblia trophozoites by murine Peyer's patch macrophages
Authors: Hill D R , Pohl R
Published: January 2020

Macrophages in Peyer's patches are important in the initiation of gastrointestinal immune responses to enteric pathogens. To examine their potential role in giardiasis, murine mononuclear cells were isolated from collagenase-treated Peyer's patches by their adherence to glass. These cells were incubated with Giardia lamblia trophozoites in the presence of nonimmune or immune (anti-Giardia antibody titer, greater than or equal to 1:1,024) mouse serum. Macrophages ingested trophozoites at low levels when they were incubated with nonimmune mouse serum. Ingestion was significantly increased at all time points (P less than 0.01) when cells and parasites were incubated in 5% immune mouse serum; the number of Giardia trophozoites ingested per 100 macrophages was 21.6 +/- 7.1 (standard error of the mean) at 1 h and increased to 59.0 +/- 16.4 at 8 h. Electron microscopy documented trophozoite destruction within macrophages. Association of G. lamblia with macrophages elicited an oxidative response; 50.9 +/- 3.6% of macrophages with trophozoites attached or ingested reduced the dye Nitro Blue Tetrazolium, compared with 13.0 +/- 5.2% for cells without associated trophozoites (P less than 0.04). These results demonstrate that macrophages are capable of ingesting G. lamblia in vitro and may play an important role in host defense in giardiasis.
Ingestion of Legionella micdadei inhibits human neutrophil function
Authors: Donowitz G R , Reardon I , Dowling J , Rubin L , Focht D
Published: January 2020

Legionella micdadei is a human pathogen which survives within leukocytes. To determine how this organism escapes intracellular destruction, we examined its effect on human neutrophil activity. Neutrophils were allowed to ingest L. micdadei prior to evaluation of functional activity. Compared with control cells which did not ingest organisms, cells ingesting L. micdadei showed significantly depressed production of superoxide anion (24.5 +/- 9.0 nmol/10(6) cells per 15 min versus 6.9 +/- 3.2 nmol/10(6) cells per 15 min, respectively; P = 0.002), chemotaxis (43.9 +/- 0.8 mm versus 0.9 +/- 1.3 mm of directed migration, respectively; P = 0.001) and bactericidal activity against Staphylococcus aureus (97.9% versus 37.6% of ingested organisms killed, respectively; P = 0.001). Similar degrees of inhibition could not be demonstrated when either Staphylococcus aureus or Escherichia coli was ingested by cells prior to evaluation. Inhibition of neutrophil function did not occur when phagocytosis of L. micdadei was prevented. However, inhibition occurred with heat-killed as well as with viable organisms. The inhibition of neutrophil function by ingested L. micdadei may help explain the bacterium's ability to survive intracellularly and may begin to explain the pathogenesis of this disease.
Characterization of a mutant of Haemophilus influenzae type b lacking the P2 major outer membrane protein
Authors: Cope L D , Pelzel S E , Latimer J L , Hansen E J
Published: January 2020

An isogenic mutant of Haemophilus influenzae type b (Hib) lacking the ability to express the P2 major outer membrane (porin) protein was constructed and characterized in various model systems. Linker insertion mutagenesis of a cloned Hib DNA insert containing the P2 structural gene was used in conjunction with a genetic transformation system to obtain a transformant unreactive with a P2-specific monoclonal antibody. This transformant was shown to lack detectable P2 protein by both protein staining and immunoblot methods. The P2 mutant exhibited a generation time in complex broth medium that was significantly longer than that of the wild-type parent strain. The P2 mutant was also unable to produce detectable bacteremia in infant rats after intraperitoneal challenge, while the wild-type parent strain produced bacteremia in all animals challenged with this strain. Reintroduction of a wild-type copy of the P2 gene into this mutant yielded a transformant strain that had a generation time in vitro identical to that of the wild-type parent strain and that was also fully virulent in the infant rat model. These findings suggest that the ability to synthesize the P2 protein may be necessary but not sufficient for full expression of virulence by this pathogen.
Vibrio cholerae HlyA hemolysin is processed by proteolysis
Authors: Hall R H , Drasar B S
Published: January 2020

The leukocidal activity of the Vibrio cholerae hemolysin (HlyA) was utilized to detect, enrich, and clone hybridoma cells expressing neutralizing monoclonal antibody in a new survivor selection protocol. A bank of 550 hybridoma clones was obtained from a mouse immunized with hemolysin by using standard techniques. The hybridoma bank was treated with a dose of HlyA hemolysin lethal to nonimmune clones. Five surviving hybridoma clones (X1 through X5) which possessed anti-HlyA activity were obtained. Western immunoblot analysis of V. cholerae culture supernatants with monoclonal antibody from clone X1 identified proteins with Mrs of 83,200, 71,600, and 60,300. Amino-terminal sequence analysis of the 71,600-Mr and 60,300-Mr forms showed homology with the published predicted sequence of HlyA. Our data indicate that proteolytic cleavage occurs between residues 120 and 121 (Glu-Leu) of the 83,200-Mr form, producing the 71,600-Mr form with the terminus NH2-L-L-F-T-P-F-D-Q-A-E-E-. Cleavage between residues 150 and 151 (Gly-Phe) releases the 60,300-Mr form with the terminus NH2-F-A-S-P-A-P-A-N-S-E-. Calculations based on the DNA sequence and the N termini indicated that the actual molecular masses of the 83,200-, 71,600-, and 60,300-Mr forms were, respectively, 79.4 kilodaltons (kDa), 68.6 kDa, and 65.3 kDa. Survivor selection and amino-terminal microsequencing offer powerful tools for the analysis of leukotoxic agents.
Staphylococcus aureus enterotoxin B challenge of monkeys: correlation of plasma levels of arachidonic acid cascade products with occurrence of illness
Authors: Jett M , Brinkley W , Neill R , Gemski P , Hunt R
Published: January 2020

Arachidonic acid cascade products have been shown to be increased in vitro in Staphylococcus aureus enterotoxin B (SEB)-treated epithelial cell cultures in our laboratory. In order to confirm that these products were clinically related to SEB intoxication, monkeys were administered SEB by nasogastric intubation. It caused emesis in five of six monkeys (less than 4 h), and the sixth monkey showed signs of mild illness. The monkeys which vomited continued to display signs of gastrointestinal illness beyond 8 h but were without any apparent signs of illness by 24 h. Blood samples were collected prior to SEB administration, upon first indication of illness, and at twice that time interval. One week prior to SEB treatment, the same monkeys were administered saline by nasogastric intubation and in every way handled similarly in order to serve as their own controls. Blood samples were taken from the control animals at 0, 4, and 8 h. The plasma concentrations of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5-hydroxyeicosatetraenoic acid (5-HETE) did not vary significantly throughout the 8-h experiment for saline-treated controls, nor did they differ from the concentrations found in the plasma of monkeys just before administration of SEB. When the SEB-treated monkeys showed the first indication of illness (less than 4 h), the mean of the concentration in plasma of PGE2 increased 1.44-fold, that of LTB4 increased 2.23-fold, and that of 5-HETE was essentially unchanged. At twice the time interval of the first display of illness (less than 8 h), PGE2 was still elevated (1.48-fold), LTB4 had decreased slightly to 1.66-fold, and 5-HETE had soared (3,45-fold), suggesting a divergence in the enzymatic utilization of the parent compound of the latter two metabolites, 5-hydroperoxyeicosatetraenoic acid. These studies suggest that arachidonic acid cascade metabolites were a consequence of SEB intoxication and may provide a logical site for metabolic interference in SEB-induced toxicity.
Mycoplasmas induce transcription and production of tumor necrosis factor in a monocytic cell line, THP-1, by a protein kinase C-independent pathway
Authors: Sugama K , Kuwano K , Furukawa M , Himeno Y , Satoh T , Arai S
Published: January 2020

We demonstrated that mycoplasmas (MP), previously shown to augment the antitumor activity of murine peritoneal macrophages, also induce cytotoxic activity in a human monocytic cell line, THP-1. THP-1 cells were induced to produce cytotoxic activity by MP in a time- and dose-dependent manner. By using neutralization by antibody against tumor necrosis factor (TNF), the cytotoxic activity was shown to be due to TNF released from the MP-stimulated cells. Studies with inhibitors of second-messenger pathways and Northern RNA blot analysis indicated that a Ca2(+)-dependent, but not protein kinase C-dependent, biochemical pathway is involved in MP-induced TNF production by THP-1 cells and that MP induce TNF production in the cells at the level of transcription. MP, unlike other bacteria, lack cell walls and lipopolysaccharide. The possible involvement of a TNF production mechanism distinct from that triggered by lipopolysaccharide is discussed.
Opsonic effect of jacalin and human immunoglobulin A on type II group B streptococci
Authors: Payne N R , Concepcion N F , Anthony B F
Published: January 2020

This study examined the effect of immunoglobulin A (IgA) and the IgA-binding lectin jacalin on the phagocytosis of type II group B streptococci (GBS). Strains possessing the trypsin-sensitive and trypsin-resistant components of the c protein (II/c) and type II GBS lacking the c protein (II) were examined by radiolabeled bacterial uptake, bactericidal assays, and electron microscopy. Type II/c GBS resisted phagocytosis by monocytes (4.9% +/- 0.8% uptake, mean +/- SE, n = 25) compared with type II GBS (8.5% +/- 1.4% uptake, n = 14, P = 0.03). Phagocytic killing by polymorphonuclear leukocytes was also less for the type II/c strain 78-471 than for the type II strain 79-176 (68% +/- 5% versus 86% +/- 4% reduction in CFU at 45 min, P = 0.03). IgA binding did not explain the resistance of type II/c GBS to phagocytosis. The uptake of type II/c GBS was not significantly different after opsonization in cord sera lacking endogenous IgA (5.93% +/- 1.4%) than in the same cord sera after addition of exogenous IgA (5.48% +/- 1.4%, P = 0.69, n = 9). Attempts to remove serum IgA with the IgA-binding lectin jacalin resulted in the binding of IgA-jacalin complexes to II/c GBS. This combination of nonspecific IgA and jacalin increased uptake of II/c GBS from 4.9% +/- 0.8% to 11.8% +/- 1.9% (P = 0.002). Jacalin also combined with specific, immune, monoclonal IgA bound to the surface of Haemophilus influenzae and promoted the uptake of these bacteria. Jacalin and IgA mediated phagocytosis of II/c GBS via receptors that were not dependent on divalent cations and that were not modulated by plating monocytes on antigen-antibody complexes.
Bordetella pertussis adenylate cyclase toxin: intoxication of host cells by bacterial invasion
Authors: Mouallem M , Farfel Z , Hanski E
Published: January 2020

Bordetella pertussis produces extracytoplasmic adenylate cyclase toxin (AC toxin) which penetrates target cells and, upon activation by host calmodulin, generates high levels of intracellular cyclic AMP (cAMP). As a result, bactericidal functions of immune effector cells are impaired. Since a considerable amount of AC toxin is associated with the bacterium, it was proposed that the toxin may be delivered by direct interaction of the organism with the target cells (E. L. Hewlett, M. C. Gray, and R. D. Pearson, Clin. Res. 35:477A, 1987). Incubation of CHO cells with intact B. pertussis led to formation of intracellular cAMP at levels comparable to those produced in CHO cells by equivalent activities of isolated AC toxin. cAMP accumulation induced by the whole bacteria appeared after a lag of 40 to 60 min and reached high levels within 2 to 3 h, whereas adherence of the bacteria proceeded rapidly and reached a maximal level within 80 min. Sera of pertussis patients completely blocked cAMP accumulation induced by the whole bacteria without having a major effect on either bacterial adherence or cAMP production by the AC toxin. Cytochalasins B and D, inhibitors of bacterial invasion, abrogated the cAMP response to the whole bacteria but not the response to the AC toxin. These agents did not affect bacterial adherence. Transmission electron micrographs revealed that B. pertussis, within the time course of cAMP induction, invaded CHO cells. We suggest that cAMP induction by B. pertussis is caused by the entry of the whole bacteria into CHO cells rather than by delivery of AC toxin during bacterial adherence. This route of cell intoxication may be relevant to the pathogenesis of whooping cough.
Cloning and nucleotide sequence of the gene encoding arginine deiminase of Mycoplasma arginini
Authors: Ohno T , Ando O , Sugimura K , Taniai M , Suzuki M , Fukuda S , Nagase Y , Yamamoto K , Azuma I
Published: January 2020

The existence of a mycoplasmal arginine deiminase which catalyzes the conversion of L-arginine to L-citrulline has been postulated. Here we show the partial amino acid sequence of arginine deiminase of Mycoplasma arginini and the complete nucleotide sequence of the arginine deiminase gene of M. arginini. The open reading frame deduced from this sequence consists of 1,230 bp encoding 410 amino acids. The mature form of this enzyme contains 409 amino acids after the deletion of the first methionine. In this open reading frame, TGA nonsense codons are used as tryptophan codons; this usage was verified by determination of the amino acid sequence. The molecular weight of the enzyme calculated from the deduced amino acid sequence is 46,372. Recently, the nucleotide sequence of the arginine deiminase gene of M. arginini was reported by Kondo et al. (K. Kondo, H. Sone, H. Yoshida, T. Toida, K. Kanatani, Y.-M. Hong N. Nishino, and J. Tanaka, Mol. Gen. Genet. 221:81-86, 1990). However, their sequence differed from ours in several places and especially at the C terminus.
Patterns of cytokine secretion in murine leishmaniasis: correlation with disease progression or resolution
Authors: Boom W H , Liebster L , Abbas A K , Titus R G
Published: January 2020

Susceptibility or resistance to infection with Leishmania major correlates with the ability of mice to produce characteristic panels of lymphokines in response to the parasite. To investigate the role of antigen-presenting cells in this phenomenon, we developed a model system which used congenic (H-2d) susceptible and resistant mice. L. major-specific T cells were isolated from infected BALB/c and B10.D2 mice, and the cells were restimulated in vitro on syngenic or congenic antigen-presenting cells. BALB/c L. major-reactive T cells restimulated with either antigen-presenting cell produced high levels of interleukin-4 and low levels of gamma interferon. In contrast, T cells from B10.D2 mice produced gamma interferon. Radiation-induced chimeras reconstituted with BALB/c bone marrow also produced more interleukin-4 in response to L. major than did chimeras reconstituted with B10.D2 bone marrow. To test whether this pattern of cytokine secretion was unique to infection with L. major, we infected the mice with a second intracellular pathogen, Mycobacterium bovis BCG. Mycobacterium-specific T cells from both BALB/c and B10.D2 mice produced interleukin-2 and no interleukin-4. Finally, when BALB/c mice were vaccinated with avirulent L. major, the induced resistance correlated with reduced production of interleukin-4 but no increase in gamma interferon production. Instead, T cells from the vaccinated mice produced high levels of tumor necrosis factor. This suggests that tumor necrosis factor, in addition to gamma interferon, may be involved in resistance to L. major and that interleukin-4 may inhibit the leishmanicidal activity of tumor necrosis factor and/or gamma interferon.
Relative activities of distinct isotypes of murine and human major histocompatibility complex class II molecules in binding toxic shock syndrome toxin 1 and determination of CD antigens expressed on T cells generated upon stimulation by the toxin
Authors: Uchiyama T , Saito S , Inoko H , Yan X J , Imanishi K , Araake M , Igarashi H
Published: January 2020

Relative abilities of murine and human major histocompatibility complex class II molecules to bind toxic shock syndrome toxin 1 (TSST-1) and T-cell subsets activated by the toxin were investigated. TSST-1 binding was observed in L cells transfected with I-Ab, I-Ak, DR2, and DQw1 genes. Scatchard plot analysis showed similar Kd values (1 x 10(-8) to 3 x 10(-8) M) for these cells. By comparison, binding was not detected in L cells transfected with I-Ek, DPw4, and DP(Cp63) genes. All of the transfectants supported TSST-1-induced proliferative response and interleukin-2 production by murine and human T cells. Levels of accessory activity were lower in the I-Ek transfectants and the DPw4 and DP(Cp63) transfectants than in the I-Ab and I-Ak transfectants and the DR2 and DQw1 transfectants, respectively. The results indicate that I-A, DR, and DQ molecules bind TSST-1 with similar affinities, whereas I-E and DP molecules bind it with fairly low affinity. TSST-1-activated T cells consisted of both CD4+ and CD8+ T cells, indicating that TSST-1 activates these two T-cell subsets.
Tip-oriented adherence of Treponema denticola to fibronectin
Authors: Dawson J R , Ellen R P
Published: January 2020

The adherence of Treponema denticola to ligands on cell surfaces or in basement membranes of periodontal tissues might play an important role in its pathogenicity. A direct microscopic assay was used to examine the binding of T. denticola to fibronectin and other protein substrates adsorbed on plastic cover slips. All strains of T. denticola that were tested adhered to fibronectin but to different degrees. The strains which bound in high numbers frequently bound by their tips. Type strain ATCC 33520 bound to fibronectin in high numbers (149 +/- 11.3 bacteria per microscopic field), with 60% bound by the tips. Strain e' bound in high numbers (140 +/- 10.2) and had the highest percentage of tip binding (98%); strain e bound in lowest numbers (39 +/- 8.2) and had the lowest percentage of tip binding (15%). Laminin supported binding at a level similar to that of fibronectin, as did fibronectin fragments which contained the cell binding domain peptides, RGDS. Type IV collagen and non-RGDS peptides did not support binding. Binding to fibronectin and laminin was inhibited by the addition of antifibronectin and antilaminin antibodies. By lowering the incubation temperature from 37 to 4 degrees C, the number of cells that attached decreased by 60% and tip binding was reduced by 50%. Pretreatment of the cells with collagen did not affect binding, whereas fibronectin pretreatment enhanced binding by 50% and laminin pretreatment resulted in a decrease of 60%. T. denticola adheres by its tips to fibronectin-coated surfaces, which suggests that fibronectin-specific adhesins cluster at the tips.