bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo activation of peripheral blood polymorphonuclear neutrophils by gamma interferon results in enhanced fungal killing
Authors: Morrison C J , Brummer E , Stevens D A
Published: January 2020

The effect of in vivo administration of murine recombinant gamma interferon (IFN) on the fungicidal activity of murine peripheral blood polymorphonuclear neutrophils (PB-PMNs) was studied. Mice were injected intramuscularly with 250, 2,500, 25,000 or 250,000 U of IFN 5 h before collection of peripheral blood. Purified PB-PMNs were cocultured in vitro with Blastomyces dermatitidis yeast cells for 2 h. PB-PMNs from untreated mice killed 44.5 +/- 12.5% of the fungal inoculum, whereas PB-PMNs from mice treated with 25,000 or 250,000 U of IFN showed significantly enhanced in vitro killing (68.0 +/- 9.4% [P less than 0.005] and 72.3 +/- 1.1% [P less than 0.001], respectively). Treatment with 250 or 2,500 U of IFN or 25,000 U of heated (100 degrees C, 15 min) IFN had no effect. The IFN-induced activation of PB-PMNs was transitory. Significant enhancement of PB-PMN killing activity occurred 1, 2, or 5 h after in vivo IFN administration, but no enhancement was observed 16 or 24 h after IFN treatment. Enhanced fungicidal activity by PB-PMNs from mice treated for 5 h with 25,000 U of IFN correlated with an increased release of superoxide anion (O2-) in vitro after stimulation of PB-PMNs with phorbol ester; normal PB-PMNs and IFN-activated PB-PMNs, respectively, produced 2.2 +/- 2.5 and 23.5 +/- 4.8 nmol of O2- per 10(6) PB-PMNs per 30 min (P less than 0.005). The exogenous addition of compounds that antagonize or inhibit the formation of oxygen radicals (superoxide dismutase, catalase, dimethyl sulfoxide, or sodium azide) significantly inhibited fungal killing by both normal and IFN-activated PB-PMNs. In addition to the enhanced microbicidal activity and superoxide generation demonstrated in vitro with constant cell numbers, there was a transient leukocytosis (particularly neutrophilia) in peripheral blood at doses of IFN and at times after IFN administration where enhanced activity was also demonstrated. In summary, our results indicate that PB-PMNs can be activated in vivo for enhanced killing of a fungal target. The enhanced killing capacity of IFN-activated PB-PMNs is due at least in part to the enhancement of oxidative killing mechanisms.
Purification, composition, and activity of two bactenecins, antibacterial peptides of bovine neutrophils
Authors: Gennaro R , Skerlavaj B , Romeo D
Published: January 2020

Extracts of granules of bovine neutrophils are known to exhibit a marked antibacterial activity in vitro. By a simple, two-step chromatographic procedure, we have resolved two peptide components of the antibacterial system. They were named Bac-5 and Bac-7 from the general term bactenecin and had molecular masses of about 5 and 7 kilodaltons, respectively. Over 45 and 20% of the amino acid residues in the two bactenecins are proline and arginine, respectively. The remaining amino acids are mainly hydrophobic (isoleucine, leucine, and phenylalanine). Both Bac-5 and Bac-7 efficiently kill Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae. They also arrest the growth of Enterobacter cloacae (MICs, 25 to 200 micrograms/ml) but not of Proteus vulgaris, Staphylococcus aureus, and Streptococcus agalactiae (MIC, greater than 200 micrograms/ml). Finally, Bac-7 but not Bac-5 has MICs of less than or equal to 200 micrograms/ml for Pseudomonas aeruginosa and Staphylococcus epidermidis. From the comparison between the efficient bactericidal concentrations in vitro and the estimated content of bactenecins in neutrophils (125 ng of Bac-5 and Bac-7 each per 10(6) cells), it is reasonable to conclude that the two cationic peptides may exert a major role in host defense against at least some microorganisms.
Coaggregation of Fusobacterium nucleatum, Selenomonas flueggei, Selenomonas infelix, Selenomonas noxia, and Selenomonas sputigena with strains from 11 genera of oral bacteria
Authors: Kolenbrander P E , Andersen R N , Moore L V
Published: January 2020

Twenty-eight strains of Fusobacterium nucleatum and 41 Selenomonas strains, including S. sputigena (24 strains), S. flueggei (10 strains), S. infelix (5 strains), and S. noxia (2 strains), were tested for their ability to coaggregate with each other and with 49 other strains of oral bacteria representing Actinobacillus, Actinomyces, Bacteroides, Capnocytophaga, Gemella, Peptostreptococcus, Porphyromonas, Propionibacterium, Rothia, Streptococcus, and Veillonella species. Selenomonads coaggregated with fusobacteria and with Actinomyces naeslundii PK984 but not with any of the other bacteria, including other selenomonads. In contrast, fusobacteria coaggregated with members of all genera, although not with all strains of each species tested. Each fusobacterium strain appeared to have its own set of partners and coaggregation properties, unlike their partners, whose coaggregation properties in earlier surveys delineated distinct coaggregation groups. Coaggregations of fusobacteria with the 63 gram-negative strains were usually inhibited by EDTA, whereas those with the 27 gram-positive strains were usually not inhibited. Likewise, lactose-inhibitable coaggregations were common among some strains of fusobacteria and some strains from each of the genera containing gram-negative partners but were rarely observed with gram-positive partners. Heating the fusobacteria at 85 degrees C for 30 min completely prevented coaggregation with most partners, suggesting the involvement of a protein on the fusobacteria. Heat treatment of many of the gram-negative partners not only enhanced their coaggregation with the fusobacteria but also changed lactose-sensitive coaggregations to lactose-insensitive coaggregations. Although fusobacteria coaggregated with a broader variety of oral partner strains than any other group of oral bacteria tested to date, each fusobacterium exhibited coaggregation with only a certain set of partner strains, and none of the fusobacteria adhered to other strains of fusobacteria, indicating that recognition of partner cell surfaces is selective. The strains of F. nucleatum are heterogeneous and cannot be clustered into distinct coaggregation groups. Collectively, these results indicate that coaggregation between fusobacteria and many gram-negative partners is significantly different from their coaggregation with gram-positive partners. The contrasting variety of partners for fusobacteria and selenomonads supports the concept of coaggregation partner specificity that has been observed with every genus of oral bacteria so far examined.
A Salmonella typhimurium virulence gene linked to flg
Authors: Carsiotis M , Stocker B A , Weinstein D L , O'Brien A D
Published: January 2020

Isogenic pairs of strains of Salmonella typhimurium which differed only in whether or not they were flagellate were found to be equally virulent in C57BL/6J mice infected orally, intravenously, or intraperitoneally. Therefore, we investigated the genetic basis for our previous observation that in this mouse model, nonflagellate delta flagABCDE25 strains were reduced in virulence compared with isogenic wild-type flagellate strains. The recombinant plasmid pMH6, which contains several flg+ genes and a segment of the S. typhimurium chromosome adjacent to the flg genes, was introduced into a delta flgABCDE25 mutant. This restored virulence in mice challenged intraperitoneally, which suggested that a virulence gene occurs adjacent to the flg genes. When plasmid pMH64, which lacks the chromosomal segment adjacent to the flg genes, was introduced into the same delta flgABCDE25 mutant, virulence was not restored. In contrast, the introduction of pMH71, a plasmid which retains the chromosomal segment adjacent to the flg genes, restored virulence. We concluded that a hitherto unknown virulence gene, which we have named mviS, occurs adjacent to the flg genes and that its absence in delta flgABCDE25 mutants, rather than the nonflagellate phenotype of the delta flgABCDE25 mutants, caused the previously reported attenuation of such mutants.
Stable expression of lipooligosaccharide antigens during attachment, internalization, and intracellular processing of Neisseria gonorrhoeae in infected epithelial cells
Authors: Weel J F , Hopman C T , van Putten J P
Published: January 2020

Immunoelectron microscopy enables the detection and localization of bacterial antigens during in vitro infection (J.F.L. Weel and J.P.M. van Putten, Microb. Pathog. 4:213-222, 1988). In this study, we have used this method to get information on the role of lipooligosaccharides (LOS) in the pathogenesis of neisserial infections at the mucosal level. Ultrathin cryosections of Chang conjunctive epithelial cells infected with Neisseria gonorrhoeae (3 to 18 h) were incubated with LOS-specific monoclonal antibodies and gold-labeled protein A and viewed in the electron microscope. Our results demonstrate that the probed LOS determinants are stably expressed during the adherence, internalization, and intracellular processing of the bacteria. There was no indication of an adaptation of the gonococcal LOS expression to the host cell environment or of a degradation of the probed epitopes. The gold particles, representing LOS molecules, were predominantly located at the bacterial membranes, but sometimes the host cell plasma membrane was labeled as well, suggesting that LOS or LOS-containing membrane fragments interacted with the eucaryotic cells. This was confirmed when purified LOS was added to the cells. Two hours after LOS exposure, gold particles were observed at the plasma membrane of a subpopulation of the cells. After 18 h of LOS exposure, gold particles were also found in large vacuoles inside the cells, suggesting that LOS molecules were internalized by the cells. The function of observed LOS binding and endocytosis in the pathogenesis of neisserial infections remains to be defined.
Capacity of passively administered antibody to prevent establishment of Brucella abortus infection in mice
Authors: Winter A J , Duncan J R , Santisteban C G , Douglas J T , Adams L G
Published: January 2020

In contrast to immunity against some other facultative intracellular parasites, protective immunity against Brucella abortus is mediated in mice by antibodies as well as by cell-mediated immune responses. It was the purpose of this study to determine whether antibody alone would prevent infection with B. abortus. The majority (82%) of CD-1 outbred mice infected with 100 CFU of virulent B. abortus 2308 preincubated with graded quantities of an O polysaccharide-specific IgG2a monoclonal antibody (MAb) were free of infection 1. 2, 4, and 6 weeks later, based on detection limits of 13 brucellae per spleen and 39 per liver. Infection was present in 95% of control animals. Similar results were obtained with a challenge dose of 500 CFU, but with a challenge dose of 5,000 CFU, infection became established even with the highest concentration of MAb used (50 micrograms of MAb per 5,000 brucellae). Pretreatment with an O polysaccharide-specific IgG1 MAb or with convalescent-phase serum diminished but did not prevent establishment of infection by 100 CFU of B. abortus. A majority of culture-negative mice tested 6 weeks after infection were serologically negative, which could have signified either the absence of previous infection or the early elimination of infection. In an in vitro test system, all of the antibody preparations were efficient in opsonizing B. abortus. Effective killing of the organism by unelicited mouse peritoneal macrophages occurred in conventional but not in endotoxin-free medium, suggesting that activated macrophages were required for killing of opsonized B. abortus. These results emphasize the potential importance of antibodies in the immunoprophylaxis of brucellosis and suggest that the design of a successful vaccine will require the induction of antibodies not only of appropriate specificity but also of the optimal isotype for mediating protective functions.
Biochemical characterization and evaluation of virulence of a fructosyltransferase-deficient mutant of Streptococcus mutans V403
Authors: Schroeder V A , Michalek S M , Macrina F L
Published: January 2020

The Streptococcus mutans extracellular fructosyltransferase (FTF) enzyme may play a role in the formation of dental caries by synthesizing a fructan polymer that serves as an extracellular storage polysaccharide. We sought to determine if an FTF-deficient strain of S. mutans was less virulent than wild-type cells in a rat animal model system. Cloned ftf gene sequences from S. mutans GS5 were used to generate a defective copy of the ftf gene by inserting into the ftf coding region a DNA fragment which encoded erythromycin resistance. The plasmid which carried the defective ftf construct was introduced into S. mutans V403 by using genetic transformation. This defective construct replaced, by allelic exchange, the wild-type copy of the ftf gene carried on the V403 chromosome. FTF activity assays indicated that the recombinant strain, V1741, was deficient in fructan synthesis. However, extracellular protein preparations from this strain displayed an increased ability to generate glucose polymers (glucans) compared with V403 preparations. Levels of adherence to glass and rat tooth surfaces by strain V1741 were similar to those of the V403 strain. Both strains caused moderate decay on rat tooth surfaces; however, the FTF-deficient strain was less pathogenic compared with the wild-type strain. These results suggest that FTF activity contributes to the pathogenicity of S. mutans V403, possibly by generating extracellular fructans which serve as storage compounds.
Characterization of the Pseudomonas aeruginosa pilus adhesin: confirmation that the pilin structural protein subunit contains a human epithelial cell-binding domain
Authors: Irvin R T , Doig P , Lee K K , Sastry P A , Paranchych W , Todd T , Hodges R S
Published: January 2020

Previous studies have suggested that the Pseudomonas aeruginosa PAK pilus adhesin moiety resides in an epithelial cell-binding domain located in the C-terminal region of the PAK pilin structural protein. Synthetic peptides Ac17red (a synthetic peptide with a sequence identical to that of PAK pilin residues 128 to 144, with the Cys-129 and Cys-142 residues being in the reduced state) and Ac17ox (a synthetic peptide with a sequence identical to that of PAK pilin residues 128 to 144, with a formed disulfide bridge between the amino acid residues Cys-129 and Cys-142), which should contain the epithelial cell-binding domain, were synthesized. Ac17red and Ac17ox both bound to buccal epithelial cells (BECs) and to ciliated tracheal epithelial cells (TECs). Ac17ox had a Km of 6.40 microM for binding to BECs, while Ac17red had a Km of 9.87 microM. Ac17red bound to the same receptor sites that purified pili did and competitively inhibited the binding of purified PAK pili to BECs. BEC glycoproteins with molecular masses of 82, 55 to 51, and 40 kilodaltons immobilized on nitrocellulose exhibited periodate-sensitive receptor activity for Ac17red; similar activity has been found for PAK pili. Ac17red, Ac17ox, and PAK pili bound to the cilia and luminal portions of the cytoplasmic membrane of human TECs, the same regions to which P. aeruginosa whole cells bind. PAK pilin has an epithelial cell-binding domain that resides in the C-terminal region of the protein.
Lethal infection by Bordetella pertussis mutants in the infant mouse model
Authors: Weiss A A , Goodwin M S
Published: January 2020

Different aspects of lethal infection of infant mice with Bordetella pertussis were examined. Mutants deficient in vir-regulated genes were tested for the ability to cause a lethal infection in the infant mouse model. Adenylate cyclase toxin-hemolysin and pertussis toxin were required to cause a lethal infection at low doses. Mixed infection caused by challenging the mice with an equal number of pertussis toxin and adenylate cyclase toxin-hemolysin mutants at a dose at which neither alone was lethal was also unable to cause a lethal infection. Production of the filamentous hemagglutinin and the dermonecrotic toxin was not required to cause a lethal infection. Nine other mutants in vir-regulated genes whose phenotypes have yet to be determined were also tested. Only two of these mutants were impaired in the ability to cause a lethal infection. Expression of fimbriae does not appear to affect the dose required to cause a lethal infection; however, fimbrial expression was correlated with the later stages of a nonlethal, persistent infection. Growth of the bacteria in MgSO4, a condition which reversibly suppresses expression of the genes required for virulence, did not alter the ability of the bacteria to cause a lethal infection. Auxotrophic mutants deficient in leucine biosynthesis were as virulent as the parental strain; however, mutants deficient in methionine biosynthesis were less virulent. A B. parapertussis strain was much less effective in promoting a lethal infection than any of the wild-type B. pertussis strains examined. A persistent infection in the lungs was observed for weeks after challenge for mice given a sublethal dose of B. pertussis, and transmission from infected infants to the mother was never observed.
Inhibition of pertussis toxin binding to model receptors by antipeptide antibodies directed at an antigenic domain of the S2 subunit
Authors: Schmidt M A , Schmidt W
Published: January 2020

Receptor recognition of pertussis toxin is mediated by the B-oligomer consisting of subunits S2, S3, S4 (two), and S5. To understand the structure-function relationships of the receptor-binding oligomer and to elucidate the immunological structure of pertussis toxin, we assayed antisera generated against each of 10 synthetic peptides corresponding to segments of the pertussis toxin S2 subunit for their ability to recognize the native toxin. Only antisera raised against peptides R1-7, R35-50, and R91-106 recognized pertussis toxin in an enzyme-linked immunosorbent assay and Western blotting (immunoblotting). These segments thus correspond to linear antigenic epitopes. The highly homologous S3 subunit was only weakly recognized by antibodies to R91-106 in Western blotting. The ability of affinity-purified antipeptide antibodies to interfere with the binding of pertussis toxin was investigated by pertussis toxin-mediated hemagglutination of goose erythrocytes, the binding of pertussis toxin to fetuin, and the toxin-induced clustered growth pattern of CHO cells as model receptor systems. Antibodies directed against synthetic peptides R1-7, R35-50, and R91-106 inhibited the binding of pertussis toxin in the two model receptor systems that solely depend on the interactions of the S2 subunit. The toxin-mediated clustered growth pattern of CHO cells could not be inhibited. The results point to a second binding site with distinct specificity involving the S3 subunit of pertussis toxin. The regions identified in this work contribute to the definition of receptor-binding sites of pertussis toxin and should thus improve the development of an acellular-component pertussis vaccine.
Resistance of highly pathogenic Naegleria fowleri amoebae to complement-mediated lysis
Authors: Whiteman L Y , Marciano-Cabral F
Published: January 2020

Weakly pathogenic and nonpathogenic Naegleria spp. are readily lysed by human and guinea pig complement. Highly pathogenic Naegleria fowleri are resistant to complement-mediated lysis. Electrophoretic analysis of normal human serum (NHS) incubated with pathogenic or nonpathogenic Naegleria spp. demonstrates that amoebae activate the complement cascade, resulting in the production of C3 and C5 complement cleavage products. To determine whether surface constituents play a role in resistance to complement lysis, trophozoites of Naegleria spp. were subjected to enzymatic treatments prior to incubation in NHS. Treatment of trophozoites with papain or trypsin for 1 h, but not with neuraminidase, increased susceptibility of highly pathogenic Naegleria fowleri to complement lysis. Treatment of trophozoites with actinomycin D or cycloheximide during incubation with NHS or pretreatment with various protease inhibitors for 4 h did not increase the susceptibility of N. fowleri amoebae to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appears to account for the increased resistance of N. fowleri amoebae to complement-mediated lysis.
Mapping of linear B-cell epitopes of the S2 subunit of pertussis toxin
Authors: Schmidt W , Schmidt M A
Published: January 2020

The linear immunogenic and antigenic structure of the S2 subunit of pertussis toxin was investigated with synthetic peptides corresponding to regions of the protein sequence predicted to contain surface-exposed hydrophilic beta turns. Five peptides as peptide-bovine serum albumin conjugates were recognized by anti-pertussis toxin antiserum and were thus designated "immunogenic epitopes." Two prominent immunogenic epitopes were specified by peptides corresponding to sequences spanning R107-120 and R186-199, whereas peptides corresponding to residues R35-50 and R91-106 were only bound in low titer. Three peptides as thyroglobulin conjugates elicited antisera in rabbits that bound intact pertussis toxin by enzyme-linked immunosorbent assay and immunoblot. These peptides were designated "antigenic epitopes." The most prominent antigenic determinant was localized to the N-terminal end of the S2 sequence encompassing residue R1-7. Peptides R35-50 and R91-106 represented two minor antigenic epitopes. Antisera to two additional peptides corresponding to residues R134-149 and R186-199 recognized the S2 subunit only by Western blotting (immunoblotting). Only antiserum raised against peptide R91-106 also recognized the S3 subunit by Western blotting, indicating a marked antigenic and probably also structural difference between the two highly homologous subunits.
Chemotactic properties of rat immunoglobulins and immune complexes
Authors: Arashi M , Sibille Y , Merrill W W , Rits M , Bazin H , Vaerman J P
Published: January 2020

The effect of rat immunoglobulins and immune complexes on the locomotor function of rat polymorphonuclear leukocytes (PMN) was investigated in vitro. Rat immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgA monoclonal antibodies specific for the dinitrophenyl hapten were used. Both monomeric and polymeric IgA showed chemotactic activity in a dose-dependent manner. IgG1 and IgG2b also induced a dose-dependent locomotor response of PMN, but the nature of the induced migration was chemokinetic (enhancing random migration). IgG2a was chemotactic and induced maximal migration at a relatively low concentration. IgG1- and IgG2b-immune complexes induced stronger migration than antibody alone; however, IgA- and IgG2a-immune complexes did not. IgA was shown to modify the chemotactic movement of PMN induced by N-formylmethionyl-leucyl-phenylalanine (FMLP). In the presence of both IgA and FMLP in the lower chamber, the migration towards suboptimal concentrations of FMLP was enhanced. By contrast, IgA in the upper chamber decreased migration towards the optimal or higher concentrations of FMLP. These findings suggest that IgA may work synergistically with luminal chemoattractants to mobilize PMN to the locus of infection on the mucosal surface. In addition, the intense activity of IgG2a alone and IgG1- or IgG2b-immune complexes in inducing PMN migration may play an important role in inflammatory processes. The data indicate that immunoglobulins have a direct effect on PMN mobility.
Sequence analysis and expression in Escherichia coli of the hyaluronidase gene of Streptococcus pyogenes bacteriophage H4489A
Authors: Hynes W L , Ferretti J J
Published: January 2020

The hyaluronidase gene (hylP) from Streptococcus pyogenes bacteriophage H4489A was previously cloned into Escherichia coli plasmid pUC8 as a 3.1-kilobase ThaI fragment. Southern hybridization experiments confirmed the origin of this fragment in bacteriophage H4489A before determination of the nucleotide sequence of the entire fragment. Two open reading frames (ORFs) were found, the first of which specified a 39,515-molecular-weight protein identified as the bacteriophage hyaluronidase. The second ORF encoded a 65,159-molecular-weight protein of unknown function. Putative transcription and translation control sequences for each ORF were identified by using a plasmid containing a promoterless chloramphenicol acetyltransferase gene. Controlled exclusive expression of the hylP gene via the T7 polymerase-promoter system in E. coli resulted in a 40,000-dalton protein, a result consistent with the coding capacity of the hylP gene.
Purification, location, and immunological characterization of the iron-regulated high-molecular-weight proteins of the highly pathogenic yersiniae
Authors: Carniel E , Antoine J C , Guiyoule A , Guiso N , Mollaret H H
Published: January 2020

We have previously shown that under iron limitation, different Yersinia species synthesize new polypeptides. Two of them, the high-molecular-weight proteins (HMWPs), are expressed only by the highly pathogenic strains. In the present study, the HMWPs from Y. enterocolitica serovar O:8 were purified by gel filtration, and specific antibodies were obtained. Using these antibodies, we show that the two polypeptides were synthesized de novo during iron starvation and that they were found essentially in the bacterial outer membrane fractions, although the majority of the molecules were not exposed on the cell surface. We also demonstrate that the two proteins had common epitopes and that the HMWPs of the high-virulence-phenotype species Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serovar O:8 (a strain different from the one used to purify the proteins) are antigenically related. The less pathogenic and nonpathogenic strains did not exhibit cross-reacting material, suggesting that these strains do not synthesize even an altered form of the HMWPs.
Schistosomal egg granuloma-derived fibroblast-stimulating factor is apparently distinct from interleukin-1
Authors: Prakash S , Dinarello C A , Danko K , Stadecker M J , Wyler D J
Published: January 2020

We have previously reported that egg granulomas isolated from livers of Schistosoma mansoni-infected euthymic mice in vitro elaborate a factor(s) that stimulates a variety of fibroblast responses including fibroblast proliferation and enhanced synthesis of extracellular matrix proteins. We have postulated that these factors play a role in the pathogenesis of hepatic fibrosis in schistosomiasis mansoni. Serum-free supernatants from egg granuloma cultures also stimulate thymocyte proliferation in an assay that defects interleukin-1 (IL-1). Thymocytes and fibroblasts are stimulated to proliferate by the same fractions of egg granuloma culture supernatant separated by gel filtration, isoelectric focusing, and ion-exchange chromatography. This suggested that granuloma-derived IL-1 is responsible for the observed fibroblast stimulation. Here we report that the ability of granuloma culture supernatants to stimulate the IL-1-sensitive D10.G4.1 cells but not fibroblasts is removed by treatment with immobilized anti-IL-1 antibody. We also observed that dialyzed culture supernatants from egg granulomas obtained from infected congenitally athymic (nude) mice also stimulate fibroblast proliferation. Treatment with anti-IL-1 antibody did not abrogate this response. In contrast to our experience with egg granulomas isolated from euthymic mice, IL-1- and fibroblast-stimulating activity could be separated by gel filtration and isoelectric focusing. We conclude that the fibroblast growth-stimulating activities elaborated by egg granulomas from S. mansoni-infected euthymic and athymic mice may be different but both appear to be distinct from IL-1.
A soluble Bacteroides by-product impairs phagocytic killing of Escherichia coli by neutrophils
Authors: Rotstein O D , Vittorini T , Kao J , McBurney M I , Nasmith P E , Grinstein S
Published: January 2020

The effect of Bacteroides culture filtrate on killing of Escherichia coli by neutrophils was examined as a potential mechanism for E. coli-Bacteroides microbial synergy. A low-molecular-weight heat-stable factor present in the 22-h culture filtrate of Bacteroides fragilis 9032 impaired neutrophil killing function. To determine whether short-chain fatty acids present in the filtrate could account for the inhibition, the fatty acid content of the culture filtrate was determined and sterile medium supplemented with measured concentrations of fatty acids was tested for its effect on neutrophil function. Succinic and acetic acids were measured in high concentrations, while lactic, formic, and fumaric acids were present in lower concentrations. Reconstituted media mimicked the inhibitory effect of B. fragilis filtrate on neutrophil killing capacity. In further support of the hypothesis that short-chain fatty acids were responsible for the inhibition, the filtrates of other Bacteroides strains were found to be inhibitory only after bacterial growth had entered the stationary phase, a period during which fatty acid production is maximized. Further studies investigating the mechanism of impaired neutrophil killing showed that B. fragilis 9032 culture filtrate inhibited both phagocytosis of [3H]thymidine-labeled E. coli by neutrophils and the intrinsic microbicidal functions of the neutrophil. Impairment of neutrophil superoxide production was mediated via the ability of short-chain fatty acids present in B. fragilis filtrate to reduce neutrophil cytoplasmic pH. These studies suggest that Bacteroides strains capable of reaching stationary phase in vivo may contribute to the pathogenesis of mixed infections by direct inhibition of neutrophil function.
Genetic linkage among cloned genes of Streptococcus mutans
Authors: Perry D , Kuramitsu H K
Published: January 2020

Mapping vectors containing antibiotic resistance markers inserted adjacent to or within different cloned genes from Streptococcus mutans were used to determine the relative positions of these genes on the chromosome. The gtfA, ftf, and scrB genes were inserted into streptococcal mapping vector pVA891 adjacent to an Emr gene, whereas the Emr marker was inserted directly into the gtfB gene. These chimeric plasmids were transformed into S. mutans GS-5, selecting for Emr transformants. To determine the positions of the cloned genes relative to each other, it was necessary to construct plasmids labeled with a different antibiotic resistance marker. Thus, a Tetr gene was inserted adjacent to gtfB in the appropriate mapping vector and within the ftf and scrB genes with a mini-Mu transposon (Mu dT). The chimeric plasmids were transformed into the appropriate Emr recipients, and the DNA from the resulting Emr Tetr transformants was used in linkage studies. Based on the cotransfer data, gtfB was not closely linked to gtfA, ftf, or scrB. However, gtfA cotransferred with ftf and scrB at frequencies of approximately 96 and 80%, respectively. The percent cotransfer of ftf and scrB was approximately 92. These data indicate that the three genes are clustered on the GS-5 chromosome, with ftf located between gtfA and scrB. Little, if any, linkage was observed between these genes and a variety of other random markers.
Epitopes on the S1 subunit of pertussis toxin recognized by monoclonal antibodies
Authors: Kim K J , Burnette W N , Sublett R D , Manclark C R , Kenimer J G
Published: January 2020

To identify the neutralizing epitopes on the S1 subunit (A promoter) of pertussis toxin, we characterized anti-S1 monoclonal antibodies (MAbs) X2X5, 3CX4, and 6FX1. We confirmed by immunoblot analysis that these MAbs bind to the S1 subunit and not to the B oligomer of pertussis toxin and that they recognize different epitopes by a competitive binding enzyme-linked immunosorbent assay. These MAbs had differential abilities to neutralize the lymphocytosis-promoting factor activity of pertussis toxin in mice: 3CX4 and 6FX1 had partial neutralizing abilities, while MAb X2X5 had none. With these MAbs, the epitopes on the S1 subunit were examined by using trypsinized S1 peptides, recombinant truncated S1 molecules, and synthetic peptides. The non-neutralizing MAb X2X5 bound in immunoblots to tryptic peptides of various sizes as small as 1.5 kilodaltons; the neutralizing MAbs 3CX4 and 6FX1 bound only to a 24-kilodalton tryptic peptide band. Immunoblot studies with recombinant truncated S1 molecules demonstrated that amino acid residues 7 to 14 and 15 to 26 play an important role in the binding of neutralizing MAbs and the non-neutralizing MAb, respectively. The binding of these MAbs was not dependent upon the presence of C-terminal amino acid residues 188 to 234. To further define B-cell epitopes, the binding of the MAbs we tested to synthetic peptides representing the entire S1 subunit were examined. Neutralizing MAbs 3CX4 and 6FX1 bound to none of these peptides, further suggesting that these MAbs recognize conformational epitopes. The non-neutralizing MAb X2X5 bound to peptides 11 to 26 and 16 to 30, demonstrating that the major antigenic determinant recognized by this MAb is a linear epitope located within residues 16 to 26.
Genetic diversity of penicillin G-resistant Neisseria meningitidis from Spain
Authors: Mendelman P M , Caugant D A , Kalaitzoglou G , Wedege E , Chaffin D O , Campos J , Saez-Nieto J A , Viñas M , Selander R K
Published: January 2020

Genotypic and phenotypic diversity among 16 penicillin G-resistant (Penr) isolates of Neisseria meningitidis recovered from human blood or cerebrospinal fluid in Spain was compared with that among 12 penicillin-susceptible (Pens) isolates by the use of multilocus enzyme electrophoresis, serotyping, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Thirteen distinctive multilocus enzyme genotypes (electrophoretic types [ETs] ) were identified among the 28 isolates. There was slightly less genetic diversity among the eight ETs of Penr isolates (H = 0.385) than among the eight ETs of Pens isolates (H = 0.431). Cluster analysis demonstrated two distinctive complexes of ETs and one ET that was not closely related to either complex. The possibility of a singular clonal origin of penicillin G-resistant isolates was excluded by the observations that resistance occurred in isolates of each of the two distantly related complexes of ETs, that three of the four ETs represented by multiple isolates included both susceptible and resistant strains, and that serotypes and growth requirements were not associated with the resistance phenotype. The 28 isolates showed a relatively homogeneous pattern of four PBPs, with apparently reduced penicillin G binding by PBP 3 of the Penr isolates.
Purification and chemical characterization of a cholera toxin-cross-reactive cytolytic enterotoxin produced by a human isolate of Aeromonas hydrophila
Authors: Rose J M , Houston C W , Coppenhaver D H , Dixon J D , Kurosky A
Published: January 2020

A bacterial protein toxin possessing hemolytic, enterotoxic, and cytotoxic activities as well as cross-reactivity to cholera toxin was purified from culture filtrates of a human diarrheal isolate of Aeromonas hydrophila (SSU). This cytolytic enterotoxin was purified by ammonium sulfate precipitation, hydrophobic chromatography using phenyl-Sepharose, anion-exchange chromatography on DEAE-Bio-Gel A, and size-exclusion high-performance liquid chromatography. The factor was a single polypeptide with an apparent molecular weight of 52,000 as determined by polyacrylamide gel electrophoresis. Automated amino acid sequence analysis confirmed that the toxin was a single chain and established a 25-residue N-terminal segment which was identical to that of aerolysin purified from culture supernatants of A. hydrophila isolate Ah65 originally obtained from rainbow trout as reported by Howard et al. (S. P. Howard, W. J. Garland, M. J. Green, and J. T. Buckley, J. Bacteriol. 169:2869-2871, 1987). However, the amino acid compositional analysis of the toxin produced by our human isolate (SSU) differed significantly from that of the Ah65 isolate. Taken together, these results strongly indicated that several toxic phenomena associated with A. hydrophila (SSU) culture filtrates, including hemolysis, cytotoxicity, and enterotoxicity as well as cross-reactivity to cholera toxin, all can occur on a single polypeptide. In addition, these results underline the fact that although aerolysin-related toxins isolated from culture filtrates of A. hydrophila are biologically similar, significant chemical and immunological differences may exist between toxins produced by individual isolates.
Differential clearance and host-pathogen interactions of YopE- and YopK- YopL- Yersinia pestis in BALB/c mice
Authors: Straley S C , Cibull M L
Published: January 2020

This study characterized infections in BALB/c mice by the nonpigmented Yersinia pestis KIM and its derivatives lacking the low-Ca2+-response virulence plasmid pCD1 or failing to express selected yersinial outer membrane proteins (YOPs). The parent Y. pestis showed net growth in the spleen by 2 h and in the liver after 7 h; exponential growth in both the liver and spleen culminated in death of the mice starting on day 4, with total bacterial numbers of less than 10(8) in the blood, liver, and spleen together. The histopathology progressed from microabscesses to extensive coagulative necrosis unaccompanied by further immigration of inflammatory cells. This, together with the relatively low bacterial numbers, suggests a toxigenic mechanism. YopE- or YopK- YopL- yersiniae were cleared from the spleen but grew in the liver after an initial lag. Their growth was curbed after 1 to 2 days and entered a plateau that lasted 5 to 6 days; viable numbers then decline rapidly. This suggests that these Yop- mutations distinguish, at least kinetically, between host responses in liver and spleen. Both strains caused acute inflammation in liver that evolved into structured lesions surrounded by progressively mononuclear inflammation suggestive of a granulomatous response. Accordingly, YOP E and YOPs K and L are necessary in the early days of the infection for net growth in spleen and prolonged growth in the liver; their absence is reflected morphologically by the emergence of cell-mediated immunity in the liver. The YopE- and YopK- YopL- mutants bound only slightly increased amounts of C3, suggesting that YOPs E, K, and L are protective through mechanisms other than interfering with the binding of complement.
Characterization of a 29.4-kilodalton structural protein of Giardia lamblia and localization to the ventral disk [corrected]
Authors: Aggarwal A , Adam R D , Nash T E
Published: January 2020

The amino acid sequence of a 29.4-kilodalton [corrected] structural protein located in the ventral disk and axostyle of Giardia lamblia was determined. Clone lambda M16 from a mung bean expression library in lambda gt11 expressed a fusion protein recognized by three different isolate-specific antisera and sera from G. lamblia-infected gerbils. One of the three EcoRI fragments (M16; 1.26 kilobases) encoded the recognized protein. Sequence analysis revealed a single open reading frame of 813 base pairs. Two areas showed conservation of the positions of some amino acids. The abundance of arginine, glutamic acid, and threonine was increased. Two potential alpha-helical regions were deduced in the regions of repeats. Antisera to the M16 fusion protein reacted specifically with internal components of the ventral disk and axostyle, as well as Giardia fractions enriched for ventral disk structural proteins. An identical protein was recognized in different isolates by anti-M16, and a single identical band was recognized in Southern blots using the M16 1.26-kilobase fragment as a probe. Therefore, the 29.4-kilodaltion [corrected] protein appears to be highly conserved compared with variant surface proteins.
Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids
Authors: Lee C K , Roberts A , Perrin S
Published: January 2020

Pertussis toxin is produced only by strains of Bordetella pertussis. Cloned genes encoding pertussis toxin from B. pertussis were transferred into Bordetella bronchiseptica and Bordetella parapertussis by conjugation. These transconjugants expressed pertussis toxin at levels comparable to those expressed by B. pertussis. The toxin made by these strains was biologically active in the Chinese hamster cell clumping assay, contained all five subunits, and was mostly periplasmic. Toxin expression appeared to be modulated in the same way as are the vir-regulated genes of B. pertussis. Introduction of these plasmids into B. pertussis failed to produce hypertoxigenic strains. Instead, these transconjugants underwent plasmid loss, gene deletions, or conversion to the avirulent phase.
Purification and partial characterization of a type-specific antigen of Rickettsia tsutsugamushi
Authors: Ohashi N , Tamura A , Ohta M , Hayashi K
Published: January 2020

A type-specific antigen (54- to 56-kilodalton polypeptide) in the envelope of Rickettsia tsutsugamushi was purified from each of three prototype strains (Gilliam, Karp, and Kato) by a combination of mild anionic detergent treatment, gel filtration, and reverse-phase high-performance liquid chromatography. The purified antigens from the three strains were shown to have similar amino acid compositions: primarily aspartic acid, glutamic acid, and glycine, with lesser amounts of cysteine, methionine, and tyrosine. The N-terminal amino acid sequences of the antigens were 74.3% homologous among the three strains.
Independence of neutrophil respiratory burst oxidant generation from the early cytosolic calcium response after stimulation with unopsonized Candida albicans hyphae
Authors: Wysong D R , Lyman C A , Diamond R D
Published: January 2020

We previously noted differences between neutrophil responses to unopsonized Candida albicans hyphae and responses to other particulate stimuli such as opsonized hyphae or zymosan; these differences include delayed rises in cytosolic calcium [( Ca2+]i), 1,4,5-inositol trisphosphate, and superoxide release and the total absence of early membrane depolarization. Respiratory burst stimulation is required for killing of C. albicans hyphae. Since an early rise in [Ca2+]i may act as a second messenger for burst activation by most agonists, we chelated (Ca2+)i and extracellular Ca2+ [( Ca2+)e] to compare requirements for superoxide responses to hyphae and other stimuli. Intracellular chelation, which ablated early [Ca2+]i rises, eliminated the fMet-Leu-Phe-induced respiratory burst and profoundly reduced that response to opsonized zymosan (by 96.7%), but chelation of both (Ca2+)i and (Ca2+)e only partially inhibited responses to opsonized and unopsonized hyphae (60.5 and 23.3%, respectively; the latter exceeded absolute responses evoked by opsonized zymosan, a 12-fold-more-potent stimulus for unchelated cells). Simultaneous (Ca2+)i and (Ca2+)e chelation further decreased superoxide responses to opsonized zymosan and hyphae (99.4 and 90.4%, respectively) but not to unopsonized hyphae (26.7% inhibition). Though both ingestible (zymosan) and uningestible (hyphae) opsonized particulate stimuli elicited reduced but significant respiratory bursts without early [Ca2+]i rises, the greater superoxide responses and sensitivity to chelation with opsonized zymosan suggest important differences in initiation and/or regulation of responses to these particulate stimuli. In contrast, the respiratory burst elicited by unopsonized hyphae appeared largely Ca2+ independent. If different events mediate neutrophil activation by opsonized and unopsonized hyphae, candidacidal activity in vivo may vary under divergent conditions with specific localized sites of infection.
Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli
Authors: Benz I , Schmidt M A
Published: January 2020

The adherence of enteropathogenic Escherichia coli (EPEC) to the small bowel mucosa is an important step in the pathogenesis of diarrheal diseases. It has been shown that many EPEC strains adhere to HEp-2 and especially HeLa cells in characteristic patterns termed localized adherence (LA) and diffuse adherence (DA). A plasmid-derived DNA fragment encoding a factor specific for LA hybridized only to EPEC strains expressing LA, which demonstrated that LA and DA are mediated by two genetically distinct adhesins. EPEC strain 2787 (O127:H27), isolated from a case of infantile diarrhea, exhibited three major properties: (i) it showed DA to HeLa cells, (ii) it carried two large (ca. 100-kilobase [kb]) plasmids and one small plasmid of about 3 kb, and (iii) no fimbriae could be detected by electron microscopy in organisms grown on agar plates or in liquid cultures. Whole isolated plasmid DNA was partially digested with EcoRI and cloned into the vector pBR322. One recombinant clone (pIB6) was found to exhibit the same DA pattern on HeLa cells as did the parent strain. This clone contained an 11-kb DNA fragment derived from the largest of the three plasmids, as shown by Southern hybridization. By deletion analysis, a 6.0-kb DNA fragment was shown to be sufficient for expression of the DA phenotype. This insert encoded the production of a 100,000-dalton protein mediating adhesion to HeLa cells.
Comparison of the genomes of pathogenic treponemes of human and animal origin
Authors: de Wergifosse P , Coene M M
Published: January 2020

The aim of this study was to compare the genomes of two strains of intestinal treponemes, which were isolated from patients suffering from intestinal disorders, with that of Treponema hyodysenteriae, the known etiological agent of swine dysentery (bloody scours). The guanine-plus-cytosine contents of the three DNAs were found to be 28.5 to 30.0%. DNA-DNA hybridization in liquid phase indicated a high degree of homology (56 to 95%) among the human strains and with T. hyodysenteriae. One of the human strains in particular displayed a very high homology (91 to 95%) with T. hyodysenteriae. The overall conclusion is that treponemal strains pathogenic for humans and animals are clustered within the same species (we propose T. hyodysenteriae), which suggests the possibility of exchange of pathogenic microorganisms between domestic animals and humans.
Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo
Authors: Dorman C J , Chatfield S , Higgins C F , Hayward C , Dougan G
Published: January 2020

The ompC, ompD, and ompF genes encode the three major porins of Salmonella typhimurium. ompR encodes a positive regulator required for the expression of ompC and ompF. Transposon-generated mutations in ompC, ompD, ompF, and ompR were introduced into the S. typhimurium mouse virulent strain SL1344 by P22-mediated transduction. Following preliminary characterization in vitro, the strains were used to challenge BALB/c mice by using the oral or intravenous route. Strains harboring ompC or ompF mutations were as virulent as SL1344 after oral challenge. Strains harboring ompD mutations had a slight reduction in virulence. In contrast, ompR mutants failed to kill BALB/c mice after oral challenge and the intravenous 50% lethal dose was reduced by approximately 10(5). The ompR mutants persisted in murine tissues for several weeks following oral or intravenous challenge. Furthermore, mice orally immunized with these ompR mutant strains were well protected against challenge with virulent SL1344.