bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis
Authors: Chan J , Tanaka K , Carroll D , Flynn J , Bloom B R
Published: January 2020

We have recently demonstrated that the macrophage L-arginine-dependent cytotoxic pathway effectively kills the virulent Erdman strain of Mycobacterium tuberculosis in vitro via the generation of toxic reactive nitrogen intermediates by the enzyme nitric oxide synthase. This report demonstrates that two distinct inhibitors of nitric oxide synthase (aminoguanidine and NG-monomethyl-L-arginine) render similar deleterious effects on tuberculous infection in mice, as assessed by mortality, bacterial burden, and pathological tissue damage, thus confirming the importance of reactive nitrogen intermediates in resistance against M. tuberculosis.
Identification and characterization of a Mycoplasma hyopneumoniae adhesin
Authors: Zhang Q , Young T F , Ross R F
Published: January 2020

An adhesin of Mycoplasma hyopneumoniae was identified and characterized in this study. A monoclonal antibody (MAb), F2G5, and its F(ab')2 fragments inhibited the adherence of M. hyopneumoniae to porcine tracheal cilia, the natural targets to which the mycoplasma binds during infection. MAb F2G5 detected multiple bands, but predominantly recognized a 97-kDa (P97) protein of M. hyopneumoniae on immunoblots. Affinity chromatography, conducted with immobilized MAb F2G5, mainly purified P97. The purified proteins were able to bind to cilia and blocked the adherence of intact M. hyopneumoniae cells to cilia. Immunolabeling of mycoplasmas with MAb F2G5 under electron microscopy demonstrated that the proteins recognized by MAb F2G5 were located at the surface of the mycoplasma, predominantly on a surface fuzzy layer. These results indicate that P97 functions as an adhesin of M. hyopneumoniae. The N-terminal amino acid sequence of P97 did not have significant homology with any known bacterial or mycoplasmal adhesins, suggesting that P97 is a novel protein. The predominant proteins detected by MAb F2G5 in different strains varied in size, indicating that the antigen bearing the epitope for MAb F2G5 undergo intraspecies size variation. Antigenic variation of adhesins may be a pathogenic mechanism utilized by M. hyopneumoniae to evade the porcine immune system.
Altered expression of the platelet aggregation-associated protein from Streptococcus sanguis after growth in the presence of collagen
Authors: Erickson P R , Herzberg M C
Published: January 2020

Certain strains of Streptococcus sanguis adhere selectively to human platelets (Adh+) and, in plasma, induce them to aggregate into in vitro thrombi (Agg+). The induction of aggregation is mediated by the platelet aggregation-associated protein (PAAP) expressed on the cell surface of the streptococcus. In endocarditis, expression of PAAP may be regulated by association with host proteins on damaged heart valves. To begin to test this hypothesis, three strains of S. sanguis were each cultured in the presence or absence of collagens (types I to X), laminin, or PAAP-derived peptide preparations. After harvesting and washing, the platelet-interactive phenotype of strains 133-79 (Adh+ Agg+), L74 (Adh+ Agg-), and 10556 (Adh- Agg-) was unchanged. The cells from each culture were then digested mildly with trypsin to isolate PAAP. PAAP isolated from strain 133-79 (Adh+ Agg+) grown in the absence of added collagen, other proteins, or peptides inhibited platelet aggregation in response to untreated cells of S. sanguis. Platelet aggregation was induced immediately, however, by PAAP from strain 133-79 isolated after growth in the presence of 300 nM type I collagen, while lower concentrations yielded protein fragments that potentiated the response to intact cells. Aggregation-inducing PAAP could be removed by anti-PAAP (PGEQGPK) immunoaffinity chromatography, but only inhibitory activity could be recovered. The agonist effect of PAAP was not associated with collagen itself, since the PAAP preparations did not contain detectable amounts of hydroxyproline. PAAP antigens isolated from cells grown in the presence and absence of collagen had similar apparent molecular weights, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. When electrophoresis was performed under nondenaturing conditions, however, PAAP isolated from cells grown in type I collagen migrated more slowly. Strain L74 grown with type I collagen yielded tryptic fragments of proteins that inhibited aggregation significantly better than control peptides (no collagen in the medium). Strain 10556 was apparently unaffected by growth in type I collagen. The effect of type I collagen was somewhat unique. Growth in the presence of collagen types II to VI (300 nM) yielded protein fragments that potentiated without inducing platelet aggregation, while other collagens, laminin, and PAAP-derived peptides did not affect platelet aggregation. These results suggest that growth in the presence of type I collagen and, perhaps, collagens II to VI alters the expression and conformation of PAAP in certain strains of S. sanguis.
Involvement of 5-hydroxytryptamine and prostaglandin E2 in the intestinal secretory action of Escherichia coli heat-stable enterotoxin B
Authors: Harville B A , Dreyfus L A
Published: January 2020

The intestinal secretory action of Escherichia coli heat-stable enterotoxin B (STb) is poorly defined. Previous work indicates that STb causes loss of intestinal fluid and electrolytes by a mechanism independent of elevated levels of cyclic nucleotides, the hallmark of other E. coli cytotonic enterotoxins. In the work described in this report, we observed that treatment of ligated rat intestinal loops with purified STb of E. coli resulted in a dose-dependent rise in intestinal secretion concomitant with dose-related increases in levels of serotonin (5-hydroxytryptamine [5-HT]) and prostaglandin E2 (PGE2). Treatment of rats with the 5-HT2 receptor antagonist ketanserin prior to STb challenge resulted in significant (P < 0.05) reduction in intestinal secretion. Blockage of 5-HT2 receptors with ketanserin also reduced (P < 0.05) the level of PGE2 observed following STb treatment, indicating that at least a portion of the PGE2 was formed in response to 5-HT2 receptor stimulation. In a similar fashion, indomethacin, an inhibitor of cyclooxygenase activity, significantly reduced the level of secretion (P < 0.05) observed following STb treatment yet had no effect on 5-HT levels. Treatment of rats with both ketanserin and indomethacin further reduced STb-mediated secretion to a level not attained by either drug alone. Taken together, our data suggest that secretion due to STb involves both 5-HT and PGE2 as intestinal secretagogues. Furthermore, PGE2 formation appears to arise through both 5-HT-dependent and 5-HT-independent pathways.
Scavenger receptor pathway for lipopolysaccharide binding to Kupffer and endothelial liver cells in vitro
Authors: Shnyra A , Lindberg A A
Published: January 2020

We have investigated the interaction of Salmonella minnesota R595 lipopolysaccharide (ReLPS) depleted of Ca2+ and Mg2+ with both Kupffer and endothelial liver cells under serum-free conditions. Specific and saturable binding levels of 125I-ReLPS were similar in both types of cells with respect to divalent cation independence, susceptibility to proteases, and concanavalin A inhibition. By using partial structures of ReLPS, it was demonstrated that acidic 3-deoxy-D-manno-octulosonic acid residues and phosphoryl groups on lipid A are of primary importance in ReLPS binding. The role of ionic interactions in LPS recognition by the cells was further confirmed by susceptibility of the binding to competitive inhibition by polyanions. Both ReLPS and ReLPS partial structures inhibited the specific cellular binding of acetylated low-density lipoprotein (Ac-LDL) by Kupffer cells and Ac-LDL- and formaldehyde-treated albumin by endothelial cells whose cellular accumulation is mediated by a different type(s) of scavenger receptor(s). In contrast, 125I-ReLPS binding to Kupffer and endothelial cells was not competed by Ac-LDL or formaldehyde-treated albumin. Our results indicate the scavenger pathway of LPS uptake by Kupffer and endothelial cells and the primary role of LPS anionic properties in this process.
Penetration and damage of endothelial cells by Candida albicans
Authors: Filler S G , Swerdloff J N , Hobbs C , Luckett P M
Published: January 2020

The mechanisms of phagocytosis of Candida albicans by human vascular endothelial cells and subsequent endothelial cell injury were examined in vitro. Both live and killed C. albicans cells were phagocytized by endothelial cells. This organism specifically induced endothelial cell phagocytosis because neither Candida tropicalis nor Torulopsis glabrata was ingested. Endothelial cell microfilaments polymerized around C. albicans as the organisms were phagocytized. Cytochalasin D inhibited this polymerization of microfilaments around C. albicans and blocked phagocytosis. The blocking of actin depolymerization with phalloidin had no effect on microfilament condensation around the organism, indicating that the microfilaments surrounding C. albicans are formed from a pool of G-actin. Intact microtubules were also necessary for the phagocytosis of C. albicans, since the depolymerizing of endothelial cell microtubules with nocodazole prevented the condensation of actin filaments around the organisms and inhibited phagocytosis. In contrast, microtubule depolymerization was not required for microfilament function because the blocking of microtubule depolymerization with taxol had no effect on microfilament condensation around C. albicans. The phagocytosis of C. albicans was pivotal in the induction of endothelial cell damage, since the blocking of candidal internalization significantly reduced endothelial cell injury. Endothelial cells were not damaged by phagocytosis of dead organisms, indicating that injury was caused by a factor associated with viable organisms. Therefore, C. albicans is uniquely able to induce endothelial cell phagocytosis by comparison with non-albicans species of Candida. Furthermore, at least two components of the endothelial cytoskeleton, microfilaments and microtubules, are necessary for the phagocytosis of C. albicans.
Identification and characterization of genes encoding the human transferrin-binding proteins from Haemophilus influenzae
Authors: Gray-Owen S D , Loosmore S , Schryvers A B
Published: January 2020

Haemophilus influenzae, a strict human pathogen, acquires iron in vivo through the direct binding and removal of iron from human transferrin by an as yet uncharacterized process at the bacterial cell surface. In this study, the tbpA and tbpB genes of H. influenzae, encoding the transferrin-binding proteins Tbp1 and Tbp2, respectively, were cloned and sequenced. Alignments of the H. influenzae Tbp1 and Tbp2 protein sequences with those of related proteins from heterologous species were analyzed. On the basis of similarities between these and previously characterized proteins, Tbp1 appears to be a member of the TonB-dependent family of outer membrane proteins while Tbp2 is lipid modified by signal peptidase II. Isogenic mutants deficient in expression of Tbp1 or Tbp2 or both proteins were prepared by insertion of the Tn903 kanamycin resistance cassette into cloned sequences and reintroduction of the interrupted sequences into the wild-type chromosome. Binding assays with the mutants showed that a significant reduction in transferrin-binding ability resulted from the loss of either of the Tbps and a complete loss of binding was evident when neither protein was expressed. Loss of either Tbp2 or both proteins correlated with an inability to grow on media supplemented with transferrin-bound iron as the sole source of iron, whereas the Tbp1+ Tbp2- mutant was able to grow only at high transferrin concentrations.
Breakdown of the round window membrane permeability barrier evoked by streptolysin O: possible etiologic role in development of sensorineural hearing loss in acute otitis media
Authors: Engel F , Blatz R , Kellner J , Palmer M , Weller U , Bhadki S
Published: January 2020

Sensorineural hearing loss is a common sequela of acute and chronic otitis media, and the round window membrane (RWM) is currently being considered as a major route for noxious agents to pass from the middle ear cavity to the cochlea. Streptococcus pneumoniae, a major causative agent of otitis media, and Streptococcus pyogenes A produce molecularly related toxins, pneumolysin and streptolysin O (SLO), that form large pores in target membranes. In this study, we analyzed the effects of SLO on the permeability of the RWM. Resected RWMs from a total of 104 guinea pigs were embedded between two chambers of an in vitro system. One chamber was designated as the tympanal (cis) compartment, and the other was designated as the inner ear (trans) compartment. The permeability of normal and SLO-damaged RWMs towards Na+, [14C]mannitol, and proteins was investigated. SLO evoked permeability defects dose dependently in the RWM with fluxes of both Na+ and [14C]mannitol being demonstrable over a time span of up to 8 h. Serum proteins and radioiodinated SLO were also shown to pass through the damage RWM. Scanning electron microscopy revealed the morphological correlates to these results. We propose that damage to the RWM by potent pore-forming cytolysins leads to leakage of ions from the perilymph. Ionic disequilibrium and passage of noxious macromolecules to the cochlea could contribute to disturbances of the inner ear function.
Involvement of Ras-related Rho proteins in the mechanisms of action of Clostridium difficile toxin A and toxin B
Authors: Dillon S T , Rubin E J , Yakubovich M , Pothoulakis C , LaMont J T , Feig L A , Gilbert R J
Published: January 2020

Toxins A and B of Clostridium difficile are responsible for pseudomembranous colitis, a disease that afflicts a substantial number of hospitalized patients treated with antibiotics. A major effect of these proteins is the disruption of the actin cytoskeleton. Recently, I. Just, G. Fritz, K. Aktories, M. Giry, M. R. Popoff, P. Boquet, S. Hegenbarth, and C. von Eichel-Streiber (J. Biol. Chem. 269:10706-10712, 1994) implicated Rho proteins as cellular targets of C. difficile toxin B, since pretreatment of cells or purified Rho with toxin prevented subsequent ADP-ribosylation of Rho by exoenzyme C3. Moreover, they showed that overexpression of Rho proteins in cells suppressed cell rounding normally associated with exposure of cells to C. difficile toxin B. Here we expand these findings by showing directly that Rho proteins are covalently modified by both C. difficile toxins A and B. In addition, we demonstrate that the stability of toxin-modified Rho in NIH 3T3 cells is dramatically reduced. Finally, we show that C. difficile toxins A and B do not have similar effects on the closely related Rac and CDC42 GTP-binding proteins.
Association with MDCK epithelial cells by Salmonella typhimurium is reduced during utilization of carbohydrates
Authors: Schiemann D A
Published: January 2020

Association of Salmonella typhimurium with MDCK epithelial cells in monolayers, represented primarily by intracellular bacteria after 30 min of contact, with centrifugation followed by vigorous washing, was measured during aerobic and anaerobic growth of the bacteria in brain heart infusion broth. Cell association was greatest during a short period in the late log phase of growth under aerobic conditions. At this time, the pH of the growth medium was changing from acid to alkaline and glucose (0.2% initially) was exhausted. Addition of excess glucose (0.5%) to brain heart infusion broth, which was not exhausted before the bacteria entered the stationary phase of growth, in which cell association dropped sharply, resulted in repression of cell association by the bacteria. The repressive effect of glucose on cell association could not be reversed by exogenous cyclic AMP in the bacterial growth medium. Under anaerobic conditions, the effect of glucose on cell association by the bacteria was not as great and the glucose was not exhausted before the bacteria entered the stationary phase. When S. typhimurium was grown in a rich but carbohydrate-free medium, cell association by the bacteria increased earlier in the growth cycle under both aerobic and anaerobic conditions. The addition of glucose and certain other utilizable carbohydrates to this medium caused a repression of cell association by S. typhimurium that was greater under aerobic growth conditions. These results show that cell association by S. typhimurium, which is accompanied by rapid internalization (cell invasion), is the same under aerobic and anaerobic conditions if the bacteria are grown to the log phase in a carbohydrate-free medium. This suggests that prior reports of greater cell invasion by S. typhimurium during anaerobic growth may have arisen from the use of media containing carbohydrates which were found to be more repressive during aerobic growth of the bacteria.
Bone marrow nitric oxide production and development of anemia in Trypanosoma brucei-infected mice
Authors: Mabbott N , Sternberg J
Published: January 2020

Mice infected with Trypanosoma brucei rapidly develop anemia, with the number of circulating erythrocytes reduced by 50% within a week after infection. The present study investigated the relationship between anemia and bone marrow nitric oxide (NO) production. Bone marrow cell populations from T. brucei-infected mice exhibited elevated levels of NO synthase activity which was inhibitable by NG-nitro-L-arginine methyl ester. NO production was found to coincide with suppressed bone marrow T-cell proliferation in response to stimulation with the mitogen concanavalin A both in vitro and in vivo. As this indicated that NO may inhibit proliferation in other cell types, particularly hemopoietic precursors, we examined the role of NO in anemia during trypanosome infection. NO production correlated directly with the development of anemia, and treatment of infected mice with NG-nitro-L-arginine methyl ester in vivo to systemically inhibit NO synthesis led to a significant reduction in the anemia. Thus, elevated NO production in the bone marrow of T. brucei-infected mice is likely to play a significant role in the anemia resulting from T. brucei infection.
Direct anticryptococcal activity of lymphocytes from Cryptococcus neoformans-immunized mice
Authors: Muth S M , Murphy J W
Published: January 2020

Assessment of the direct anticryptococcal activity of murine lymphocytes from both Cryptococcus neoformans-immunized and control mice was the focus of this investigation. We demonstrate that at a 2:1 effector cell-to-cryptococcal target cell ratio, effector cell populations comprised of alpha beta T-cell receptor-positive T lymphocytes (98 to 99% CD3+) from C. neoformans-immunized mice inhibited the growth of cryptococcal cells better than similar populations of lymphocytes from nonimmunized control mice. Almost immediately after mixing of cryptococci with the effector cells, C. neoformans-lymphocyte conjugates were observed. The percentage of conjugates increased over the first 30 min of incubation and then remained constant over the next 1.5 h. T-lymphocyte-enriched populations from C. neoformans-immunized mice formed significantly greater percentages of conjugates with cryptococci than control T lymphocytes at each time period that assessment was made. For growth inhibition to occur, direct contact between the effector and target cells was necessary, as evidenced by abrogation of cryptococcal growth inhibition when lymphocyte and cryptococcal cell populations were separated by a porous membrane during the growth inhibition assay. Vital staining of cryptococci after incubation with the T-cell-enriched populations showed that the T lymphocytes killed the cryptococcal cells.
Effect of polyclonal and monoclonal antibodies on surface properties of Streptococcus sobrinus
Authors: van Raamsdonk M , van der Mei H C , de Soet J J , Busscher H J , de Graaff J
Published: January 2020

In this study, the effect of antibody adsorption on physicochemical properties of Streptococcus sobrinus was studied. Bacteria were preincubated with polyclonal antibodies or with OMVU10, a monoclonal antibody (MAb) reactive with S. sobrinus. The zeta potentials and the hydrophobicity as determined by microbial adhesion to hydrocarbons were measured in potassium phosphate buffer with a pH ranging from 2 to 9. S. sobrinus preincubated with polyclonal antibodies was positively charged at pH 2, 3, and 4 and had an isoelectric point at pH 4.8. Untreated S. sobrinus cells or cells preincubated with MAbs were negatively charged over the whole pH range. X-ray photoelectron spectroscopy showed a decrease in O/C and P/C ratios for bacteria preincubated with polyclonal antibodies. A combination of the pH-dependent zeta potential and the X-ray photoelectron spectroscopy data of the overall chemical composition of the cell surface suggests that polyclonal antibody adsorption occurs through blocking of surface phosphate. The measurement of hydrophobicity by microbial adhesion to hydrocarbons revealed that S. sobrinus preincubated with polyclonal antibodies was hydrophobic (90% of the bacteria bound to hexadecane), whereas the controls were relatively hydrophilic. S. sobrinus preincubated with OMVU10 was found to be more hydrophobic than the controls at pH 5 and 7. Hydrophobicity as measured by water contact angles showed an increase in hydrophobicity when S. sobrinus was preincubated with polyclonal antibodies. The epitopes to which the antibodies are directed were visualized by immunogold labeling and electron microscopy. The results suggested that OMVU10 is reactive with only a few epitopes of the cell surface, whereas polyclonal antibodies were found to be reactive with many epitopes. In conclusion, adsorption of polyclonal antibodies was found to influence the overall physicochemical surface properties of the organism, probably by forming a coating over the whole cell surface. Adsorption of MAbs was more localized, which could explain their lesser influence on these surface properties.
Extracellular neuraminidase production by a Pasteurella multocida A:3 strain associated with bovine pneumonia
Authors: White D J , Jolley W L , Purdy C W , Straus D C
Published: January 2020

The properties of an extracellular neuraminidase produced by a Pasteurella multocida A:3 strain that was isolated in a case of bovine pneumonia were examined during growth in a defined medium. This enzyme (isolated from concentrated culture supernatants of P. multocida A:3) was active against N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Enzyme elaboration was correlated with the growth of the organism in a defined medium, with maximum quantities produced in the stationary phase. The enzyme was purified by a combination of ammonium sulfate fractionation, ion exchange on DEAE-Sephacel, and gel filtration on Sephadex G-200. The purified neuraminidase possessed a specific activity of 9.36 mumol of sialic acid released per min per mg of protein against fetuin. The enzyme possessed a pH optimum of 6.0 and a Km of 0.03 mg/ml. The P. multocida A:3 neuraminidase had a molecular weight of approximately 500,000 as estimated by gel filtration. The enzyme was stable at 4 and 37 degrees C for 3 h. Approximately 75% of the neuraminidase activity was lost within 30 min at 50 degrees C. Greater than 90% of the enzyme activity was destroyed within 10 min at temperatures of > or = 65 degrees C. The P. multocida neuraminidase does not appear to be serologically related to the Pasteurella haemolytica A1 neuraminidase since antiserum prepared against the purified P. haemolytica enzyme did not neutralize the P. multocida enzyme.
Heparin protects Opa+ Neisseria gonorrhoeae from the bactericidal action of normal human serum
Authors: Chen T , Swanson J , Wilson J , Belland R J
Published: January 2020

The pathobiological significance of lipooligosaccharide (LOS) and outer membrane opacity protein (Opa) changes in gonorrheal disease are poorly understood. We assessed variants of strain MS11mk with different LOS and Opa phenotypes for their liability to killing by normal human sera. LOS differences correlated with strikingly disparate susceptibilities to serum killing; LOSa variants were serum resistant, LOSb variants were serum sensitive, and sialylation of LOSb variants enhanced their survival (as reported previously). Opa phenotype had little influence on the killing of serum-sensitive LOSb cells that were incubated directly in normal human sera, but preincubation of Opa+ LOSb variants in heparin increased their serum resistance whereas Opa- LOSb variants showed no change. Some Opa proteins conferred slightly higher resistance than others, but heparin preincubation increased serum resistance for variants expressing each of seven Opa proteins. These in vitro phenomena may relate to conditions within the male urethra where sulfate-containing proteoglycans are abundant and where antibody and complement may transude from blood plasma. The results suggest that the selective advantage for Opa+ Neisseria gonorrhoeae bacteria observed in vivo may reflect their ability to utilize host cell components to resist killing by host defenses.
Effect of salicylate on expression of flagella by Escherichia coli and Proteus, Providencia, and Pseudomonas spp
Authors: Kunin C M , Hua T H , Bakaletz L O
Published: January 2020

Osmotic stress, salicylate, and Mar (multiple antibiotic resistance) mutation are known to block the expression of the OmpF porin. Since these conditions have also been shown to inhibit the expression of P and CFA fimbriae in Escherichia coli, we speculated that they might affect the expression of flagella as well. Hyperosmotic conditions have been shown to block the synthesis of flagellin and expression of flagella in E. coli (C. Li, C. J. Louise, W. Shi, and J. Adler, J. Bacteriol. 175:2229-2235, 1993). In the current study, sodium salicylate was found to inhibit the motility of E. coli, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, and Providencia stuartii in a reversible, concentration-dependent manner. Swarming did not occur at 20 mM sodium salicylate. Salicylate also blocked the synthesis of flagellin in E. coli. Phenotypic Mar mutants of E. coli derived from motile strains were amotile. Flagella were markedly reduced as determined by scanning electron microscopy when P. mirabilis was grown in broth containing 20 mM salicylate. Salicylate had no apparent effect, however, on expression of a 40-kDa porin protein in P. mirabilis. This finding suggests that the noted effect of salicylate on Proteus spp. may be mediated through a mechanism other than porin production or that the Proteus porin may not be analogous to OmpF in E. coli. Salicylate decreased the motility of Pseudomonas cepacia but had no effect on Pseudomonas aeruginosa. The exact mechanism by which salicylate exerts its effect is not known, but it appears to be related to osmoregulation.
Determination of antigen binding specificities of Cryptococcus neoformans factor sera by enzyme-linked immunosorbent assay
Authors: Belay T , Cherniak R
Published: January 2020

The competitive binding specificities of glucuronoxylomannan (GXM) and its derivatives to factor sera of Cryptococcus neoformans were studied by enzyme-linked immunosorbent assay. An effort was made to determine the epitope specificity of each factor serum. Despite the presence of antigenic factor 1 on all serotypes of C. neoformans, variations in inhibition ability were observed with different GXMs. The panspecific component of factor serum 1 (antibody 1) appeared to be due to the presence of more than one antibody component. The activity was dependent on the 6-O-acetyl substituent. GXMs of serotypes A and D inhibited factor serum 2 equally well, indicating a low titer for the antibody 7 component. Serotype B GXM was a poor inhibitor, and serotype C GXM did not inhibit factor serum 2. The activity of factor serum 2 was 6-O-acetyl dependent. GXMs from typical serotype A and serotype D isolates were excellent inhibitors of factor serum 3. GXMs from serotype B were poorly inhibitory and serotype C did not inhibit factor serum 3. The activity of factor serum 3 was 6-O-acetyl dependent. The activity of factor serum 4 was due predominantly to antibody component 6. The activity of factor 4 was directed mainly against serotype C, and it was independent of 6-O-acetyl substitution Factor serum 5 was specific for serotype B GXMs. The inhibitory effect was independent of 6-O-acetyl substitution, but the effect was diminished by reduction of the glucuronic acid. The GXMs with a typical serotype C structure inhibited antibody 6. O deacetylation of the GXMs did not affect their inhibitory activity. However, reduction of glucuronic acid reduced factor serum 6 binding. Factor serum 8 was specific to serotype D; native GXMs of serotype A were slightly inhibitory. O deacetylation of the serotype D GXMs abrogated the inhibitory effect. O deacetylation alone abrogates the activity of antibody components 1, 2, 3, and 8. Reduction of glucuronic acid reduces the inhibitory activity of the GXM to antibody components 4, 5, and 6. Partial GXM structures and methyl glycosides did not effectively inhibit the activity of any of the factor sera.
Internalization of Staphylococcus aureus by endothelial cells induces cytokine gene expression
Authors: Yao L , Bengualid V , Lowy F D , Gibbons J J , Hatcher V B , Berman J W
Published: January 2020

The ability of the vascular endothelium to elaborate cytokines in response to gram-positive sepsis has received limited attention. This study examined cytokine expression by human umbilical vein endothelial cells (EC) following infection with a gram-positive bacterial pathogen, Staphylococcus aureus. S. aureus infection of EC resulted in the production of interleukin-6 (IL-6) and IL-1 beta. For IL-6, message was detected at 3 h after infection, protein was present at 24 h, and both message and protein persisted for 72 h. IL-1 beta message was detected at 12 h, IL-1 beta protein was detected at 24 h, and both persisted for 72 h. Message for colony-stimulating factor 1 remained unaltered. UV-killed S. aureus also elicited IL-1 beta and IL-6 message and protein expression at 24 and 48 h. Twenty-one clinical isolates of S. aureus were tested, and all induced IL-6 release by 48 h. However, the laboratory strain 8325-4 did not induce cytokine expression at any time point and was internalized by EC 1,000-fold less than other strains were. Internalization of latex beads by EC did not induce IL-6 gene expression. Furthermore, cytochalasin D treatment of the EC prevented IL-1 and IL-6 induction by S. aureus but not by tumor necrosis factor alpha or lipopolysaccharide. These results indicate that S. aureus is a potent inducer of IL-1 and IL-6 in EC and that internalization of S. aureus by EC is necessary for their cytokine expression. Thus, our data suggest that the vascular endothelium may play an important role in the pathogenesis of septicemia caused by gram-positive organisms.
Endotoxin-induced desensitization of mouse macrophages is mediated in part by nitric oxide production
Authors: Fahmi H , Charon D , Mondange M , Chaby R
Published: January 2020

Refractoriness (tolerance) to endotoxin effects, such as induction of tumor necrosis factor alpha (TNF-alpha) secretion, can be elicited in vitro in macrophages by preexposure of cells to endotoxin (lipopolysaccharide [LPS]) itself. The aim of this study was to determine whether this phenomenon is due to negative feedback mediated by the free radical nitric oxide (NO) produced by cells when they are activated by LPS. Among several efficient inhibitors of NO production, NG-monomethyl-L-arginine did not induce concomitant inhibition of TNF-alpha secretion. Mouse macrophages that were exposed to LPS in the presence of NG-monomethyl-L-arginine partially maintained the ability to secrete TNF-alpha in response to a second LPS stimulation, compared with cells preexposed to LPS alone, thus suggesting that NO is involved in part in LPS-induced desensitization of cells. Furthermore, direct exposure of cells to the NO-generating compounds sodium nitroprusside and S-nitroso-N-acetylpenicillamine mimicked LPS-induced desensitization. However, low concentrations of a synthetic lipid (lipid M4) that is structurally analogous to the reducing end of the lipid A moiety of LPS induced desensitization of mouse macrophages without concomitant production of NO. Taken together, these data suggest that although NO actually takes part in LPS-induced desensitization of mouse macrophages, additional and yet unknown mechanisms must also exist.
Intracellular killing of Listeria monocytogenes in the J774.1 macrophage-like cell line and the lipopolysaccharide (LPS)-resistant mutant LPS1916 cell line defective in the generation of reactive oxygen intermediates after LPS treatment
Authors: Inoue S , Itagaki S , Amano F
Published: January 2020

Listeria monocytogenes is a facultative intracellular pathogen and survives within phagocytic cells by escaping from phagosomes into the cytoplasm. It has been reported that, in vivo, L. monocytogenes is effectively eliminated through cell-mediated immunity, especially by macrophages which have been immunologically activated by cytokines such as gamma interferon (IFN-gamma). However, this killing mechanism for L. monocytogenes and the role of macrophage activation in this bacterial killing are unclear. We demonstrated the listericidal effect of oxidative radicals induced by lipopolysaccharide (LPS) and IFN-gamma, using a macrophage-like cell line, J774.1, and a mutant cell line, LPS1916. LPS1916 cells do not exhibit normal generation of O2- and H2O2 after treatment with 0.1 microgram of LPS per ml, although J774.1 cells generate 100 times the normal level of oxidative radicals with the same LPS treatment. The growth of L. monocytogenes was strongly inhibited in J774.1 cells pretreated with 0.1 microgram of LPS per ml or the combination of 0.1 microgram of LPS per ml and 10 U of IFN-gamma per ml. On the other hand, in LPS1916 cells, the growth of L. monocytogenes was not inhibited by treatment with LPS only, although LPS1916 cells pretreated with the combination of LPS and IFN-gamma showed moderate inhibition of listerial growth. This killing was not influenced by treatment with NG-monomethyl-L-arginine, which is a strong inhibitor of nitrite oxide generation. Interestingly, J774.1 cells treated with LPS did not show enhanced intraphagosomal killing of a nonhemolytic strain of avirulent L. monocytogenes that lacks the ability to escape from phagosomes, and this killing was not influenced by treatment with NG-monomethyl-L-arginine either. These results suggest that the reactive oxygen radicals are more important than nitric oxide in the mechanism underlying the intracellular killing of virulent L. monocytogenes and that there seem to be different killing mechanisms for virulent and avirulent strains of L. monocytogenes in activated-macrophage cell lines.
Identification and characterization of a surface-exposed, 66-kilodalton protein from Borrelia burgdorferi
Authors: Probert W S , Allsup K M , LeFebvre R B
Published: January 2020

The surface-exposed antigens of Borrelia burgdorferi represent important targets for the development of a protective immune response. We have identified a proteinase K-accessible, 66-kDa protein from B. burgdorferi and have demonstrated that at least a portion of this protein is surface exposed. The 66-kDa protein was purified by sequential extraction of spirochetes with butanol and Triton X-114 followed by preparative gel electrophoresis. Polyclonal antibodies developed against the purified 66-kDa protein were Borrelia spp. specific, whereas a monoclonal antibody, Route 66, displayed a genospecies-specific pattern of recognition for the 66-kDa protein. N-terminal amino acid sequence was obtained from an internal fragment, a truncated version, and the full-length form of the 66-kDa protein. A search of protein and gene databases for homologous sequences yielded a match with the predicted amino acid sequence from a segment of B. burgdorferi chromosomal DNA (P. A. Rosa, D. Hogan, and T. G. Schwan, J. Clin. Microbiol. 29:524-532, 1991). The construction of primers based on this DNA sequence and the N-terminal amino acid sequence allowed the amplification and cloning of the 66-kDa-protein gene. The identity of the cloned gene was verified by the recognition of the expressed gene product by Route 66. Pulsed-field gel electrophoresis and Southern blot analysis were performed to confirm the chromosomal location of the 66-kDa-protein gene. This study describes the identification and cloning of the first chromosomally encoded, surface-exposed protein from B. burgdoferi.
Role for circulating lipoproteins in protection from endotoxin toxicity
Authors: Feingold K R , Funk J L , Moser A H , Shigenaga J K , Rapp J H , Grunfeld C
Published: January 2020

Previous studies have shown that endotoxin (lipopolysaccharide [LPS])-induced death can be prevented by preincubating LPS with lipoproteins in vitro or by infusing large quantities of lipids into animals prior to LPS administration. In the present study we determined whether physiological levels of lipids also provide protection. Serum lipid levels were decreased by two different mechanisms: administration of 4-aminopyrolo-(3,4-D)pyrimide, which prevents the hepatic secretion of lipoproteins, and administration of pharmacological doses of estradiol, which increases the number of hepatic low-density lipoprotein receptors, leading to increased lipoprotein clearance. In both hypolipidemic models, LPS-induced mortality is markedly increased compared with that of controls with normal serum lipid levels. In both hypolipidemic models, administration of exogenous lipoproteins, which increase levels of serum lipids into the physiological range, reduces the increased mortality to levels similar to that seen in normal animals. In normal lipidemic animals, 63% of 125I-LPS in plasma is associated with lipoproteins, where it would not be capable of stimulating cytokine production. In contrast, in hypolipidemic animals, very little LPS (12 to 17%) is associated with lipoproteins. Rather, more LPS is in the lipoprotein-free plasma compartment, where it could exert biological effects. In both hypolipidemic models, LPS produces a greater increase in serum tumor necrosis factor levels than it does in controls (three- to fivefold increase), and administration of exogenous lipoproteins prevents this increase. Cytokines, in particular tumor necrosis factor, are responsible for most of the toxic effects of LPS. These data provide evidence that physiological levels of serum lipids protect animals from LPS toxicity. Thus, lipoproteins, in addition to playing a role in lipid transport, may have protective functions. Moreover, as part of the immune response, cytokine-induced increases in serum lipid levels may play a role in host defense by decreasing the toxicities of biological and chemical agents.
The genes for the Clostridium botulinum type G toxin complex are on a plasmid
Authors: Zhou Y , Sugiyama H , Nakano H , Johnson E A
Published: January 2020

Clostridium botulinum type G produces a toxin complex that is composed of neurotoxin, hemagglutinin, and nontoxic nonhemagglutinin. The three genes encoding these proteins were closely linked on a plasmid of about 114 kb (76 MDa) but not on chromosomal DNA. In contrast to the genes of other C. botulinum serotypes, the genes encoding type G toxin are on a plasmid.
Biological activities of staphylococcal enterotoxin type A mutants with N-terminal substitutions
Authors: Harris T O , Betley M J
Published: January 2020

The purpose of this study was to examine the importance of certain N-terminal amino acid residues of staphylococcal enterotoxin type A (SEA) for biological activity. The results confirm our previous observation that Asn-25, Phe-47, and Leu-48 are important for SEA's emetic and superantigen activities. Substitutions at six other sites (Leu-12, Lys-14, Ser-16, Asp-45, Gln-46, and Thr-51) did not reveal any additional residues required for biological activity. Mutant SEAs with substitutions at 25, 47, or 48 all had decreased T-cell stimulatory activity, with the mutants at position 47 being the most defective. Results of a competition assay for binding to the major histocompatibility complex (MHC) class II-expressing cell line Raji suggested that the decreased superantigen activities of the mutants with substitutions at positions 47 and 48 are due to poor interactions with MHC class II molecules, whereas the defects of the mutants at position 25 are a consequence of faulty interactions with T-cell receptors. With respect to emetic activity in rhesus monkeys, the mutants at position 25 or 48 exhibited decreased but significant activity. Interestingly, the two mutants at position 47 had different emetic activities; SEA-F47G was nonemetic when administered intragastrically at 500 micrograms per animal, whereas SEA-F47S was emetic at this dosage. Since the mutants at position 47 were equally defective for superantigen activity, this further supports our previous suggestion of an incomplete correlation between SEA's emetic and superantigen activities.
Cross-reacting lipopolysaccharide antigens in Legionella pneumophila serogroups 1 to 14
Authors: Jürgens D , Fehrenbach F J
Published: January 2020

Immunological cross-reactions among Legionella species were investigated with sonicated, proteinase K-digested cell lysates. The antigens separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were either analyzed for lipopolysaccharides (LPSs) by silver staining or transferred to nitrocellulose membranes for serological characterization with rabbit antibodies directed against Legionella pneumophila serogroups 1 and 5. When antiserum prepared against serogroup 5 was used to probe the LPSs from L. pneumophila serogroups 1 to 14, the antibodies recognized a common epitope harbored by all L. pneumophila serogroups but not by other Legionella species or by the gram-negative bacteria tested as controls. Hence, the serogroup 5 antiserum correctly identified all serogroups of L. pneumophila tested in the LPS immunoblot assay. Moreover, the silver-stained profiles of the isolated LPSs revealed characteristic patterns allowing the identification of the individual serogroups of L. pneumophila.
Characterization of a Helicobacter pylori neutrophil-activating protein
Authors: Evans D J , Evans D G , Takemura T , Nakano H , Lampert H C , Graham D Y , Granger D N , Kvietys P R
Published: January 2020

Helicobacter pylori-associated gastritis is mainly an inflammatory cell response. In earlier work we showed that activation of human neutrophils by a cell-free water extract of H. pylori is characterized by increased expression of neutrophil CD11b/CD18 and increased adhesiveness to endothelial cells. The work reported here indicates that the neutrophil-activating factor is a 150,000-molecular-weight protein (150K protein). Neutrophil proadhesive activity copurified with this protein, which is a polymer of identical 15K subunits. Specific antibody, prepared against the purified 15K subunit, neutralized the proadhesive activity of the pure protein and of water extracts obtained from different strains of H. pylori. The gene (napA) for this protein (termed HP-NAP, for H. pylori neutrophil-activating protein) was detected, by PCR amplification, in all of the H. pylori isolates tested; however, there was considerable strain variation in the level of expression of HP-NAP activity in vitro. HP-NAP could play an important role in the gastric inflammatory response to H. pylori infection.
Growth of Mycobacterium tuberculosis in BCG-resistant and -susceptible mice: establishment of latency and reactivation
Authors: Brown D H , Miles B A , Zwilling B S
Published: January 2020

Growth of mycobacterial species is controlled by a gene, Bcg (candidate Nramp). Bcg acts at the macrophage level and is thought to control some aspect of macrophage priming for activation. Infection of Mycobacterium bovis BCG-susceptible (Bcgs) mice with several different mycobacterial species results in the growth of the microorganisms, while the growth of the same organisms is controlled in BCG-resistant (Bcgr) mice. The capacity of Bcg to control the growth of M. tuberculosis has not been extensively explored. The purpose of this investigation, therefore, was to compare the growth of M. tuberculosis in Bcgr and Bcgs mice. We found that the growth of tubercule bacilli was different in the lungs and spleens of Bcgr and Bcgs mice when they were inoculated with fewer then 10(3) CFU of the mycobacterium. The differences in growth were more easily distinguished in the lungs then in the spleens. The growth of the microorganisms in both strains of mice peaked between 35 and 43 days, and a latent infection was established by 65 days after infection. Activation of the hypothalamic-pituitary-adrenal axis resulted in reactivation of the growth of M. tuberculosis in both Bcgr and Bcgs mice. Greater numbers of tubercule bacilli were isolated from lungs than from spleens following reactivation. The utility of this mouse model in the study of the establishment of latency and reactivation of M. tuberculosis is discussed.
Virulence plasmid-encoded YopK is essential for Yersinia pseudotuberculosis to cause systemic infection in mice
Authors: Holmström A , Rosqvist R , Wolf-Watz H , Forsberg A
Published: January 2020

The virulence plasmid common to pathogenic Yersinia species encodes a number of secreted proteins denoted Yops (Yersinia outer proteins). Here, we identify and characterize a novel plasmid-encoded virulence determinant of Yersinia pseudotuberculosis, YopK. The yopK gene was found to be conserved among the three pathogenic Yersinia species and to be homologous to the previously described yopQ and yopK genes of Y. enterocolitica and Y. pestis, respectively. Similar to the other Yops, YopK expression and secretion were shown to be regulated by temperature and by the extracellular Ca2+ concentration; thus, yopK is part of the yop regulon. In addition, YopK secretion was mediated by the specific Yop secretion system. In Y. pseudotuberculosis, YopK was shown neither to have a role in this bacterium's ability to resist phagocytosis by macrophages nor to cause cytotoxicity in HeLa cells. YopK was, however, shown to be required for the bacterium to cause a systemic infection in both intraperitoneally and orally infected mice. Characterization of the infection kinetics showed that, similarly to the wild-type strain, the yopK mutant strain colonized and persisted in the Peyer's patches of orally infected mice. A yopE mutant which is impaired in cytotoxicity and in antiphagocytosis was, however, found to be rapidly cleared from these lymphoid organs. Neither the yopK nor the yopE mutant strain could overcome the primary host defense and reach the spleen. This finding implies that YopK acts at a different level during the infections process than the antiphagocytic YopE cytotoxin does.