bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of colloidal gold-labeled salivary proline-rich proteins to Actinomyces viscosus type 1 fimbriae
Authors: Leung K P , Nesbitt W E , Fischlschweiger W , Hay D I , Clark W B
Published: January 2020

Salivary proline-rich proteins (PRPs), which were purified from parotid saliva, were adsorbed onto 15-nm-diameter gold particles to visualize specific binding of the salivary molecules to Actinomyces viscosus type 1 fimbriae. Negatively stained preparations incubated with PRP-gold conjugates but not bovine serum albumin-gold complexes bound specifically to bacteria possessing type 1 fimbriae, A. viscosus T14V-J1 and 5519. Binding of the PRP-gold probes to strains deficient in type 1 fimbriae, i.e., strains 5951 (type 2 fimbriae only) and 147 (no fimbriae), was negligible.
Mannan composition of the hyphal form of two relatively avirulent mutants of Candida albicans
Authors: Saxena A , McElhaney-Feser G E , Cihlar R L
Published: January 2020

We have previously reported the characteristics of mannans isolated from the yeast forms of two relatively avirulent Candida albicans strains, designated 4918-2 and 4918-10. Investigations have been expanded to include an analysis of mannans from the hyphal form of these strains as well as from the hyphal form of the parental strain, 4918. After extraction, mannans were further purified by high-pressure liquid chromatography on a Bio-gel TSK DEAE-5-PW column. Subsequent to either mild acid hydrolysis, alkali hydrolysis, or acetylation followed by acetolysis, the resulting products were fractionated by high-pressure liquid chromatography on an Aminex HPX-42A column. The results of acid hydrolysis showed only minor quantitative differences in the products released from each strain, with mannose constituting the vast majority of product liberated. The profiles of mannooligosaccharides obtained from either alkali hydrolysis or acetolysis for strain 4918-2 showed distinct quantitative differences compared with profiles of the other two strains. Finally, a general characteristic noted is a decrease in the average chain length of mannooligosaccharides in hyphal mannans compared with the yeast counterpart.
Genetic evidence that the yopA gene-encoded Yersinia outer membrane protein Yop1 mediates inhibition of the anti-invasive effect of interferon
Authors: Bukholm G , Kapperud G , Skurnik M
Published: January 2020

HEp-2 cell monolayers were challenged with genetic variants of Yersinia pseudotuberculosis YPIII(pIB1) and Yersinia enterocolitica W22708(pYL4). Both strains were represented by (i) variants harboring the 70-kilobase virulence plasmid, (ii) their isogenic plasmid-cured derivatives, and (iii) two transposon mutants constructed by insertional inactivation of the plasmid genes encoding outer membrane protein Yop1 and Ca2+ dependency in strains YPIII(pIB1) and W22708(pYL4). When the HEp-2 cells were pretreated with recombinant alpha interferon subtype A, all invasive variants of Y. enterocolitica and Y. pseudotuberculosis, except those variants which expressed Yop1, showed a significantly reduced ability to localize intracellularly. The anti-invasive effect of interferon was abolished when the gene was expressed or when a sterile filtered sonic extract of a Yop1-producing strain was added to the cell cultures. To obtain further evidence of a potential role of Yop1, a DNA fragment encoding Yop1 cloned into the vector pBR322 was used. After introduction of the resultant recombinant plasmid pYMS2 into the plasmid-cured variant YPIII and the Yop1-negative transposon mutant YPIII(pIB102) of Y. pseudotuberculosis, both transformants regained the ability to produce Yop1 and showed complete inhibition of the interferon effect. Moreover, the sterile sonic extract of an Escherichia coli strain, which carried pYMS2, neutralized the anti-invasive effect of interferon. The results provide direct genetic evidence that Yop1 mediates inhibition of the anti-invasive effect of interferon in HEp-2 cell cultures. The results also demonstrated that Yop1 itself reduces the ability of Yersinia spp. to localize intracellularly in HEp-2 cells.
Moderate immunodeficiency does not increase susceptibility to Salmonella typhimurium aroA live vaccines in mice
Authors: Izhar M , DeSilva L , Joysey H S , Hormaeche C E
Published: January 2020

Salmonellae carrying appropriate mutations in genes of the aromatic biosynthesis pathway are effective as live vaccines in animals, and they are candidate typhoid vaccines for human use. They are also very effective as carriers of recombinant antigens from other pathogens to the immune system, eliciting circulatory, secretory, and cell-mediated immunity to foreign antigens. Their attenuation is believed to be due to their requirement for the metabolites p-aminobenzoic acid and 2,3-dihydroxybenzoate, which are not available in mammalian tissues. Immunosuppression (e.g., acquired immunodeficiency syndrome) is a major contraindication to the use of live vaccines. If the avirulence of Aro mutants is largely due to their auxotrophy, they should not be markedly more invasive in immunosuppressed animals. We report that wild-type Salmonella typhimurium M525 of intermediate virulence was much more invasive in sublethally irradiated BALB/c mice than in normal BALB/c mice, whereas sublethal irradiation had little if any effect on the invasiveness of an S. typhimurium aorA vaccine strain apart from a delay in its clearance from the reticuloendothelial system. xid mutant CBA/N mice carry an X-linked B-cell functional defect which results in immunoglobulin G3 agammaglobulinemia, and they are known to be more susceptible to salmonellae in late stages of the infection. We found that whereas male (CBA/N x BALB/c)F1 mice (immunodefective) were more susceptible to wild-type S. typhimurium C5 than female littermates (immunocompetent), there was no difference in the response to the S. typhimurium aroA vaccine strain. The results indicate that moderate immunosuppression does not markedly enhance susceptibility to S. typhimurium aroA live vaccines.
Accessory function of Kupffer cells in the antigen-specific blastogenic response of an L3T4+ T-lymphocyte clone to Listeria monocytogenes
Authors: Gregory S H , Wing E J
Published: January 2020

The function of Kupffer cells in the development of protective immunity to infection by Listeria monocytogenes is controversial. To determine their role in antilisterial host defenses, Kupffer cells were separated from other nonparenchymal cells of the liver by centrifugation on a metrizamide gradient followed by adherence to glass or plastic. The resultant highly enriched Kupffer cell population supported the antigen-specific blastogenic response [( 3H]thymidine incorporation) of cloned L3T4+ T lymphocytes to L. monocytogenes in vitro. Blastogenesis was dependent upon the duration of the incubation period, the concentration of the antigen, and the number of Kupffer cells in culture. Maximum reactivity was greater than that observed when the same T-cell population was incubated with adherent peritoneal exudate cells and antigen under optimal conditions. The addition of antibodies specific for murine interleukin-1 beta to cocultures of Kupffer cells and T lymphocytes eliminated the antigen-stimulated incorporation of [3H]thymidine, indicating a requirement for interleukin-1. Analysis of the culture supernatants demonstrated that, in addition to interleukin-1, granulocyte-macrophage colony-stimulating factor, interleukin-6, and gamma interferon were elaborated in cocultures containing cloned T lymphocytes, Kupffer cells, and antigen. These results suggest that Kupffer cells may serve a critical role in the development of immunity to infection by L. monocytogenes in vivo.
Human polymorphonuclear leukocyte response to Pseudomonas aeruginosa grown in biofilms
Authors: Jensen E T , Kharazmi A , Lam K , Costerton J W , Høiby N
Published: January 2020

The interaction of human neutrophils with Pseudomonas aeruginosa biofilms was examined by using a chemiluminescence assay. The biofilms induced an oxidative burst response by polymorphonuclear leukocytes which was slow and only 25% of the response to planktonic bacteria. The reduced response to P. aeruginosa biofilms could play a role in the persistence of bacteria in chronic infections.
Monoclonal antibody against a serotype antigen of Porphyromonas (Bacteroides) endodontalis and characteristics of the antigen
Authors: Hanazawa S , Sagiya T , Amano S , Nishikawa H , Kitano S
Published: January 2020

Recent studies have demonstrated the presence of three serotypes (O1K1, O1K2, and O1K-) of Porphyromonas (Bacteroides) endodontalis. In the present study, a hybridoma cell line producing monoclonal antibody (BEE11) specific for serotype O1K1 of P. endodontalis was established. The specificity of the antibody was evaluated by enzyme-linked immunosorbent assay and immunoslot blot analysis. BEE11 antibody reacted with strains ATCC 35406, HG 400, and HG 421 of the bacterium. However, it did not react with HG 422 or HG 948. Also, the antibody did not react with any of the black-pigmented Bacteroides strains tested. Although the antibody reacted with total cell envelope and capsule materials, it did not do so with lipopolysaccharide. The antibody reacted with antigen material having a molecular mass of 110 kilodaltons (kDa), as judged from fractionation by Superose 12 prep gel chromatography. When the peak fraction from the Superose 12 column was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, the reactivity was detected as a single band at an apparent molecular mass of about 52 kDa. The antigen material purified partially by high-performance liquid chromatography was sensitive to trypsin, V8 protease, and heating to 80 degrees C but not to neuraminidase. Therefore, the present study shows that BEE11 antibody recognizes a serotype antigen of P. endodontalis which may be a dimer consisting of monomers having molecular masses of approximately 52 kDa and sensitivity to proteases and heat.
Identification and mapping of an immunogenic region of Mycoplasma hyopneumoniae p65 surface lipoprotein expressed in Escherichia coli from a cloned genomic fragment
Authors: Kim M F , Heidari M B , Stull S J , McIntosh M A , Wise K S
Published: January 2020

A previously characterized lipid-modified amphiphilic surface protein of Mycoplasma hyopneumoniae, p65, has been defined by its reaction with a surface-binding monoclonal antibody (MAb) and by its exclusive partitioning into the detergent phase during Triton X-114 phase fractionation (K. S. Wise and M. F. Kim, J. Bacteriol. 169:5546-5555, 1987). In the current study, polyclonal mouse antibody (PAb) to gel-purified p65 was used to identify recombinant phage plaques expressing p65-related epitopes. Several characteristic partial tryptic fragments of p65 were recognized by both PAb and p65 and MAb to p65, but the PAb population specifically eluted from recombinant phage plaques bound only epitopes restricted to the largest of these fragments. Graded carboxypeptidase-Y digestion of intact M. hyopneumoniae generated C terminally truncated peptides that were recognized by PAb to p65 and MAb to p65, indicating that the C terminus and much of the adjoining region of p65 were present and accessible on the external face of the membrane. However, antibody eluted from recombinant phage plaques bound only to the largest truncated polypeptide, suggesting that a recombinant product corresponding to the C-terminal region of p65 was expressed in Escherichia coli. A 19-kilodalton recombinant protein (p19), which was recognized by PAb to p65 but not by MAb to p65, was detected in recombinant phage lysates. Serum antibodies from swine taken after, but not before, experimentally induced M. hyopneumoniae pneumonia preferentially recognized the native, amphiphilic p65 lipoprotein and also bound specifically to the p19 recombinant product. This confirmed that the p65 lipoprotein is a major immunogen of M. hyopneumoniae recognized during disease and identified its C-terminal region as an immunogenic domain.
Leukotriene and hydroxyeicosatetraenoic acid generation elicited by low doses of Escherichia coli hemolysin in rabbit lungs
Authors: Grimminger F , Walmrath D , Birkemeyer R G , Bhakdi S , Seeger W
Published: January 2020

Low doses of Escherichia coli hemolysin cause thromboxane-mediated hypertension and vascular leakage in blood-free perfused rabbit lungs (W. Seeger, H. Walter, N. Suttorp, M. Muhly, and S. Bhakdi, J. Clin. Invest. 84:220-227, 1989). The recirculating buffer medium and bronchoalveolar lavage fluid from lungs exposed to hemolysin (2.5 hemolytic units per ml) in the presence of cyclooxygenase inhibitor were analyzed for leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) by reverse-phase and straight-phase high-pressure liquid chromatographic techniques combined with UV spectrum analysis and post-high-pressure liquid chromatography radioimmunoassay. A rapid release of large amounts of cysteinyl-LTs and leukotriene B4 (LTB4) into the intravascular space was noted (total sum, approximately 4 to 5 micrograms). Similar quantities have hitherto been elicited only by high concentrations of the artificial calcium ionophore A 23187. Moreover, a marked liberation of 5-HETE and 12-hydroxyheptadecatrienoic acid into the buffer medium occurred, whereas LTB4 represented the predominant compound in the lavage fluid. The hemolysin-induced burst of LT and HETE generation preceded the onset of vascular leakage. The outstanding capacity of E. coli hemolysin to produce the liberation of potent lipid mediators is probably relevant to the pathways of vascular injury and amplification of inflammatory events during severe infection with hemolytic E. coli strains.
Infection of mice with Neospora caninum (Protozoa: Apicomplexa) does not protect against challenge with Toxoplasma gondii
Authors: Lindsay D S , Blagburn B L , Dubey J P
Published: January 2020

Neospora caninum and Toxoplasma gondii are structurally related protozoal parasites of mammals that may cause abortion and neonatal morbidity and mortality. Groups of mice were subcutaneously inoculated with 10(5) live zoites of the NC-1 or NC-3 isolates of N. caninum and reinoculated with an identical number of live zoites 2 weeks later. Groups of mice which were injected subcutaneously with Hanks balanced salt solution served as controls. Three weeks after the final N. caninum inoculation, one-half of the mice were inoculated subcutaneously with 2.5 x 10(4) zoites of the RH isolate of T. gondii and the other half were inoculated subcutaneously with 2.5 x 10(4) zoites of the GT-1 isolate of T. gondii. Serum samples taken from mice on the day of T. gondii inoculation were negative for specific antibodies to T. gondii, but mice inoculated with N. caninum had reciprocal titers of greater than or equal to 800 to this protozoan. All of the mice died after challenge with T. gondii, and no significant differences (P greater than 0.05) between the survival times of mice inoculated with either isolate of N. caninum and those of control mice were seen. This study indicates that N. caninum and T. gondii are distinct biologic entities and not closely related isolates.
A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences
Authors: Anderson B E , McDonald G A , Jones D C , Regnery R L
Published: January 2020

The nucleotide sequence of a Rickettsia rickettsii gene that encodes a high-molecular-mass surface antigen (190 kilodaltons), which elicits protective immunity, was determined. The 6,747-nucleotide gene coded for a 2,249-amino-acid protein with a calculated molecular weight of 224,321. A 3.8-kilobase PstI fragment proximal to the 5' end of the gene was found to consist of 13 highly related tandem repeats which constituted over 40% of the coding region. The repeated sequences could be divided into either a 225-nucleotide, 75-amino-acid unit (type I) or a 216-nucleotide, 72-amino-acid unit (type II), with extensive homology between the two types of repeating units. The deduced amino acid sequence for these repeat units, overall, was slightly hydrophobic with short hydrophilic domains. The carboxy-terminal (nonrepetitive) portion of the deduced protein sequence was hydrophilic, with potential surface-exposed epitopes. The full-length reading frame was reconstructed in Escherichia coli, and transient expression of the 190-kilodalton antigen was demonstrated; however, the protein appeared to be severely degraded by proteases and was apparently toxic to E. coli. The conservation of this unique repetitive gene structure, coupled with results from previous reports showing the protective properties of the 190-kilodalton antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to Rocky Mountain spotted fever.
Up-regulation of human neutrophil receptors for Neisseria gonorrhoeae expressing PII outer membrane proteins
Authors: Farrell C F , Rest R F
Published: January 2020

In the absence of serum, nonpiliated gonococci expressing PII outer membrane proteins (PIIs) adhere to human neutrophils whereas non-PII-expressing (PII-) gonococci do not. After an observation that neutrophils in monolayers bound more gonococci than neutrophils in suspension, we treated neutrophil suspensions with known stimulants of degranulation and measured subsequent gonococcal adherence to suspended neutrophils. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fmlp), the potent secretagogue phorbol myristate acetate, and the calcium ionophore A23187 all caused increased adherence of PII+ gonococci, but not PII- gonococci, to neutrophils in a dose-responsive manner. Increased adherence of gonococci to neutrophils was paralleled by increased degranulation of neutrophil myeloperoxidase, lysozyme, and lactoferrin. Inhibition of fmlp-induced neutrophil degranulation by pertussis toxin, the calmodulin inhibitors trifluoperazine and N-5-chloronaphthalene sulfonamide, or the intracellular calcium-binding agent trimethoxybenzoic acid also inhibited fmlp-induced gonococcal adherence to neutrophils. Neither undifferentiated nor myelocytically differentiated HL-60 cells, which possess primary but defective or nonexistent secondary granules, bound PII+ or PII- gonococci. Gonococci did not adhere to human monocytes, monocyte-derived macrophages, lymphocytes, platelets, or erythrocytes, indicating that several receptors, such as the complement receptors CR1, CR3 (CD11b/CD18), and CR4 (CD11c/CD18) or the adherence complex LFA-1 (CD11a/CD18), were probably not involved in gonococcal adherence to human neutrophils.
Altered B-lymphocyte membrane architecture indicated by ganglioside accessibility in C3H/HeJ mice
Authors: Yohe H C , Berenson C S , Cuny C L , Ryan J L
Published: January 2020

We have analyzed both the total ganglioside composition and the surface accessibility of C3H/HeN B lymphocytes and C3H/HeJ B lymphocytes. Seventeen individual resorcinol-positive moieties were visualized by two-dimensional thin-layer chromatography of the purified gangliosides from both strains. Complete homology between strains was seen in the patterns of total gangliosides purified from the endotoxin-responsive and -hyporesponsive strains, with only minor differences in the relative concentrations of four gangliosides. In comparison, only 12 individual gangliosides were accessible to surface labeling following galactose oxidase treatment in these same strains, suggesting that some gangliosides are masked at the cell surface in both strains. However, labeling of the more polar components was greatly reduced in the endotoxin-hyporesponsive (C3H/HeJ) strain, suggesting that these gangliosides have decreased accessibility to galactose oxidase at the cell surface. Therefore, while the total ganglioside compositions of the two strains were nearly equivalent, there were dramatic differences in ganglioside surface accessibility. These findings indicate that an alteration in membrane structure that is associated with the endotoxin hyporesponsiveness observed in C3H/HeJ B lymphocytes exists.
Identification of mip-like genes in the genus Legionella
Authors: Cianciotto N P , Bangsborg J M , Eisenstein B I , Engleberg N C
Published: January 2020

The mip gene of Legionella pneumophila serogroup 1 strain AA100 encodes a 24-kilodalton surface protein (Mip) and enhances the abilities of L. pneumophila to parasitize human macrophages and to cause pneumonia in experimental animals. To determine whether this virulence factor is conserved in the genus Legionella, a large panel of Legionella strains was examined by Southern hybridization and immunoblot analyses for the presence and expression of mip-related sequences. Strains representing all 14 serogroups of L. pneumophila contained a mip gene and expressed a 24-kilodalton Mip protein. Although the isolates of the 29 other Legionella species did not hybridize with mip DNA probes under high-stringency conditions, they did so at reduced stringency. In support of the notion that these strains possess mip-like genes, these species each expressed a protein (24 to 31 kilodaltons in size) that reacted with specific Mip antisera. Moreover, the cloned mip analog from Legionella micdadei encoded the cross-reactive protein. Thus, mip is conserved and specific to L. pneumophila, but mip-like genes are present throughout the genus, perhaps potentiating the intracellular infectivity of all Legionella species.
Binding of Mycobacterium avium-Mycobacterium intracellulare to human leukocytes
Authors: Catanzaro A , Wright S D
Published: January 2020

We examined nonopsonic binding of Mycobacterium avium-Mycobacterium intracellulare (MAI) by human leukocytes. Macrophages (M phi) avidly bound fluorescently labeled MAI in the absence of serum proteins. Binding appeared to be mediated by a lineage-specific, proteinaceous receptor on M phi, since (i) binding of labeled bacteria could be competitively inhibited by unlabeled MAI, (ii) treatment of M phi with trypsin ablated the ability of M phi to bind MAI, and (iii) the capacity to bind MAI was observed on monocytes, M phi, and stimulated polymorphonuclear cells but not on lymphocytes or unstimulated polymorphonuclear cells. The receptor for MAI appeared mobile in the plane of the membrane, since spreading of M phi on a carpet of immobilized, unlabeled MAI down modulated binding of labeled MAI added in suspension. The receptor required neither calcium nor magnesium for activity and appeared different from other known receptors for intracellular pathogens.
Nucleotide sequence of yst, the Yersinia enterocolitica gene encoding the heat-stable enterotoxin, and prevalence of the gene among pathogenic and nonpathogenic yersiniae
Authors: Delor I , Kaeckenbeeck A , Wauters G , Cornelis G R
Published: January 2020

The gene encoding the heat-stable enterotoxin (yst) was cloned from the chromosome of Yersinia enterocolitica W1024 (serotype O:9), and the nucleotide sequence was determined. The yst gene encodes a 71-amino-acid polypeptide. The C-terminal 30 amino acids of the predicted protein exactly correspond to the amino acid sequence of the toxin extracted from culture supernatants (T. Takao, N. Tominaga, and Y. Shimonishi, Biochem. Biophys. Res. Commun. 125:845-851, 1984). The N-terminal 18 amino acids have the properties of a signal sequence. The central 22 residues are removed during or after the secretion process. This organization in three domains (Pre, Pro, and mature Yst) resembles that of the enterotoxin STa of Escherichia coli. The degree of conservation between the E. coli and Y. enterocolitica toxins is much lower in the Pre and the Pro domains than in the mature proteins. The mature toxin of Y. enterocolitica is much larger than that of E. coli, but the active domain appears to be highly conserved. The yst gene of Y. enterocolitica introduced in E. coli K-12 directed the secretion of an active toxin. The cloned yst gene was used as an epidemiological probe among a collection of 174 strains representative of all Yersinia species except Yersinia pestis and numerous Y. enterocolitica subgroups. In Y. enterocolitica, there was a clear-cut difference between pathogenic and nonpathogenic strains: 89 of 89 pathogenic and none of 51 nonpathogenic strains contained yst-homologous DNA, suggesting that Yst is involved in pathogenesis. Among the other Yersinia species, only four strains of Yersinia kristensenii had DNA homologous to yst.
Characterization of Streptococcus gordonii (S. sanguis) PK488 adhesin-mediated coaggregation with Actinomyces naeslundii PK606
Authors: Kolenbrander P E , Andersen R N
Published: January 2020

Intergeneric coaggregation of Streptococcus gordonii (S. sanguis) PK488 and Actinomyces naeslundii PK606 was studied by using coaggregation-defective (Cog-) mutants of both strains. A streptococcal protein of 38 kilodaltons was identified with anti-S. gordonii serum absorbed with Cog- cells of the streptococcus. Absorbed immunoglobulin G specifically blocked coaggregation of the streptococcus-actinomyces pair but did not affect the coaggregation of the streptococcus with other coaggregation partners. The 38-kilodalton protein was found in the supernatant of mild sonicated cell suspensions and was extracted from whole cells with sodium barbital or with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). An immunoreactive protein of the same size was found in sonicated cell supernatants of several other oral streptococci that also coaggregated with A. naeslundii PK606. Inhibition of S. gordonii PK488-A. naeslundii PK606 coaggregation was not observed with any of 16 different sugars tested. We propose that a functionally similar adhesin that mediates coaggregations with A. naeslundii PK606 is expressed by several species of the genus Streptococcus.
Adherence of Porphyromonas (Bacteroides) gingivalis to Streptococcus sanguis in vitro
Authors: Stinson M W , Safulko K , Levine M J
Published: January 2020

Intergeneric bacterial adherence is responsible for the complexity of the microbiota in human dental plaque and is believed to enable some extraneous bacteria to initially colonize the human oral cavity. Some current evidence indicates that Streptococcus sanguis, an early colonizer of teeth, enhances subsequent colonization by Porphyromonas (Bacteroides) gingivalis, a bacterium associated with advanced adult periodontitis. In this study, selected strains of P. gingivalis and S. sanguis were tested for their adherence activities in vitro. A differential filtration assay was devised in which one member of the test pair was radiolabeled. Heterogeneous aggregates that formed in mixed suspensions were collected on polycarbonate filters (8-microns pore size) and were washed free of individual bacteria and small homologous clumps. P. gingivalis 381, W50, JKG7, and 33277 adhered to S. sanguis G9B, M5, Challis 6, and 38. P. gingivalis A7A1-28 did not adhere well to S. sanguis under these conditions. More precise measurements of intergeneric adherence were obtained with an alternative assay with radiolabeled P. gingivalis and an artificial dental plaque composed of S. sanguis coupled to cyanogen bromide-activated agarose beads. CNBr-agarose was selected as the supporting matrix for the plaque because it was uniformly and permanently coated with S. sanguis and because P. gingivalis had negligible adherence activity for streptococcus-free beads. P. gingivalis W50 grown to the early stationary phase adhered to S. sanguis-coated beads in higher numbers than either midlogarithmic- or late-stationary-phase cells. Intergeneric adherence was not inhibited or reversed by the presence of lactose or other monosaccharides or disaccharides. Pretreatment of either bacterium with trypsin or proteinase K reduced subsequent adherence by 86 to 100%. Neuraminidase treatment of P. gingivalis caused 98% reduction of adherence, whereas similar treatment of S. sanguis caused only a 2% loss. Preincubation of P. gingivalis at 60 degrees C for 30 min decreased subsequent adherence to S. sanguis-coated beads by 94%. Adherence was reduced by 96% when bacteria were assayed while suspended in human whole saliva or when pretreated with saliva and subsequently assayed in buffer. The concentration of whole human saliva required to inhibit 50% adherence in this assay was 23 micrograms per ml (1:200 dilution). Suspension of the bacteria in normal rabbit serum resulted in 94% inhibition of adherence. These data indicate that saliva and serum may be important host defense factors for controlling Porphyromonas-Streptococcus adherence.
Diphosphoryl lipid A derived from lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides inhibits activation of 70Z/3 cells by LPS
Authors: Kirkland T N , Qureshi N , Takayama K
Published: January 2020

Diphosphoryl lipid A derived from nontoxic lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides ATCC 17023 did not stimulate the murine pre-B cell line 70Z/3 to synthesize surface immunoglobulin or kappa mRNA. However, it effectively blocked Escherichia coli LPS-induced activation of 70Z/3 cells in a concentration-dependent manner. This inhibition was specific only to cells activated by LPS, since it did not inhibit activation of 70Z/3 cells by gamma interferon. Maximal inhibitory effect occurred when the antagonist was added within 2 h before adding the LPS. These results strongly suggested that R. sphaeroides diphosphoryl lipid A is competing with E. coli LPS for physiological lipid A receptors on the 70Z/3 cells.
A monoclonal antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis
Authors: Lu Y S , Lai W C , Pakes S P , Nie L C
Published: January 2020

Monoclonal antibodies (MAbs) directed against the 37.5-kDa outer membrane protein were produced by fusing myeloma cells with spleen cells obtained from mice immunized with a pathogenic strain of Pasteurella multocida isolated from a rabbit. Desirable MAbs were selected by enzyme-linked immunosorbent assay, whole-cell radioimmunoprecipitation (WC-RIP), and Western blot (immunoblot) analysis. WC-RIP and Western blot analyses, using MAb 1608 adsorbed with intact P. multocida cells and the eluted MAb, demonstrated that the antigen recognized by this MAb is exposed on the cell surface, is antibody accessible, and has an estimated molecular mass of 37.5 kDa. Treatment of outer membrane vesicles of P. multocida with proteinase K totally abrogated the MAb 1608 activity, indicating that this MAb binds to a protein antigenic determinant. Furthermore, MAb 1608 was nonreactive to purified lipopolysaccharide in Western blot analysis. Passive transfer studies showed that nine rabbits inoculated intranasally with MAb 1608 and homologously challenged intranasally had significantly reduced mortality, severity of pneumonia, prevalence of P. multocida colonization in nonrespiratory organs, and numbers of P. multocida in nasal cavities compared with the controls. Furthermore, the number of P. multocida in lungs was reduced 84,750-fold. Similarly, passive transfer experiments indicated that MAb 1608 protected mice against homologous and heterologous challenges with P. multocida strains bearing the antigenic determinant recognized by MAb 1608. However, no protection was afforded by MAb 1608 when mice were challenged with a P. multocida strain lacking the antigenic determinant recognized by MAb 1608. This study establishes that the 37.5-kDa outer membrane protein is the target for a protective MAb.
Asexual development of Cryptosporidium parvum within a differentiated human enterocyte cell line
Authors: Flanigan T P , Aji T , Marshall R , Soave R , Aikawa M , Kaetzel C
Published: January 2020

Unremitting diarrhea with malabsorption is associated with Cryptosporidium parvum infection of the small intestine in patients with AIDS. The lack of a well-defined in vitro model of C. parvum infection has severely hampered research into the biology of cryptosporidial invasion of the host epithelial cell and development of new pharmacologic and immunologic therapies. The adherent human intestinal epithelial cell line HT29 when grown in glucose-free medium develops morphologic and functional characteristics of the small intestine enterocyte and was used to develop an in vitro model of infection. Cryptosporidium oocysts obtained from AIDS patients were applied to a monolayer of cloned, differentiated HT29.74 cells. Cells were fixed and stained to estimate the degree of parasite infection. Schizonts were easily distinguished from the host cell by light microscopy. Twenty-four hours after 10(5) oocysts were added to approximately 10(6) HT29.74 cells, Cryptosporidium infection rates varied from 50 to 120 schizonts per 1,000 cells. Among 14 different experiments, the mean infection rate was 91 (+/- 18) schizonts per 1,000 cells. Electron microscopy at 6 and 24 h confirmed intracellular localization and development of schizonts. The morphologic features of the cryptosporidial schizonts within HT29.74 cells, which included the presence of a dense band and feeder layer, were identical to those described during cryptosporidial infection of human enterocytes in patients with AIDS. Fewer schizonts were observed at 5 days and beyond. Infection of differentiated HT29.74 cells (62 and 65 schizonts per 1,000 cells at 24 and 72 h, respectively) was over five times more efficient than infection of undifferentiated HT29.74 cells (9 and 5 schizonts per 1,000 cells at 24 and 72 h, respectively). In vitro infection of differentiated HT29.74 cells will allow a better understanding of the mechanisms by which C. parvum infects the small intestinal epithelium and will allow a systematic evaluation of new therapeutic agents.
Interactions between Yersinia enterocolitica and rabbit ileal mucus: growth, adhesion, penetration, and subsequent changes in surface hydrophobicity and ability to adhere to ileal brush border membrane vesicles
Authors: Paerregaard A , Espersen F , Jensen O M , Skurnik M
Published: January 2020

Interactions between Yersinia enterocolitica and rabbit ileal mucus were examined. Strains carrying the Yersinia virulence plasmid, pYV, adhered to crude mucus but not to intestinal luminal contents that had been immobilized on polystyrene. Using an Y. enterocolitica O:9 mutant in which the yadA gene (formerly called yopA), encoding the high-molecular-weight outer membrane protein YadA (formerly called protein P1 or Yop1), had been inactivated and an Escherichia coli strain carrying the cloned yadA gene, we demonstrated that the ability to adhere to mucus correlated closely to expression of YadA. Thereafter, we evaluated possible consequences of binding between pYV-carrying Y. enterocolitica O:3 strains and constituents in the mucus layer. pYV-carrying strains were able to multiply at a high rate in mucus but not in luminal contents, and the ability to adhere to mucus could therefore facilitate bacterial colonization of the mucosa. However, we also showed in vitro that mucus acted as a barrier for a mucus-adherent, pYV-carrying Y. enterocolitica strain. Furthermore, penetration through, or preincubation with, mucus reduced subsequent adhesion of the pYV-carrying strain to brush border membrane vesicles without simultaneously causing bacterial aggregation. Preincubation with mucus also changed the bacterial surface of the same strain from hydrophobic to hydrophilic. Immunoglobulins present in mucus did not seem to be of importance for our observations. Interaction of Y. enterocolitica with intestinal mucus may thus reflect a host defense mechanism that reduces the pYV-mediated adhesion to the epithelial cell membrane, possibly by rendering the bacteria less hydrophobic.
Molecular structure of the Dr adhesin: nucleotide sequence and mapping of receptor-binding domain by use of fusion constructs
Authors: Swanson T N , Bilge S S , Nowicki B , Moseley S L
Published: January 2020

The Dr hemagglutinin of uropathogenic Escherichia coli mediates adherence to the upper urinary tract. E. coli strains which express this adhesin bind to the Dr blood group antigen and mediate mannose-resistant hemagglutination (MRHA). Chloramphenicol inhibits MRHA produced by the Dr hemagglutinin and may act as an analog for the tissue receptor at the adhesin-binding site. The nucleotide sequence of the Dr hemagglutinin fimbrial subunit was determined and found to have significant homology with that of F1845, a fimbrial adhesin associated with diarrhea, and with the afimbrial adhesin AFA-I of uropathogenic E. coli. Chimeric adhesin determinants consisting of the Dr structural subunit and F1845 accessory genes or of the F1845 structural subunit and Dr accessory genes were constructed. The Dr and F1845 determinants were shown to have a close structural relationship, with functional differences concentrated in the fimbrial subunit. Oligonucleotide-directed site-specific mutagenesis was used to facilitate construction of a hybrid adhesin subunit gene containing the amino terminus of F1845 fused to the carboxy terminus of the Dr structural gene. The resulting construct confers chloramphenicol-resistant hemagglutination when introduced into an E. coli strain expressing the cloned Dr hemagglutinin. The chloramphenicol sensitivity or resistant phenotype of MRHA produced by this family of adhesins is determined solely by the fimbrial subunit gene. Domains responsible for the chloramphenicol sensitivity of Dr-mediated MRHA reside within the amino-terminal portion of the fimbrial subunit.
Effects of Cryptococcus neoformans-specific suppressor T cells on the amplified anticryptococcal delayed-type hypersensitivity response
Authors: Buchanan K L , Fidel P L , Murphy J W
Published: January 2020

Cell-mediated immunity is an important host resistance mechanism against Cryptococcus neoformans, the etiological agent of cryptococcosis. Previous studies from our laboratory have shown that the anticryptococcal cell-mediated immune response as measured by delayed-type hypersensitivity (DTH) is down-regulated by a cascade of antigen-specific T suppressor (Ts) cells. Recently, we have identified a population of CD4 T cells that up-regulate the anticryptococcal DTH response (Tamp cells). The Tamp cells are found in the spleens of donor mice at 6 days after immunization with cryptococcal antigen, and they amplify the anticryptococcal DTH response when transferred to syngeneic recipients at the time of immunization of the recipients. In this study, we determined the effects of C. neoformans-specific Ts cells on the induction of the Tamp cells in the Tamp cell-donor mice and on the induction and expression of the amplified anticryptococcal DTH response in the Tamp cell-recipient mice. When cryptococcal-specific Ts1 cells were given at the time of immunization of the Tamp cell-donor mice, induction of Tamp cells was inhibited. In contrast, when Ts1 cells were given at the time of adoptive transfer of Tamp cells, the recipients displayed amplified DTH responses, indicating that Ts1 cells do not affect the Tamp cells' function once the Tamp cells have been produced. C. neoformans-specific Ts2 cells given at the time of either immunization or footpad challenge of the Tamp cell-recipient mice did not alter, to any measurable extent, the amplified DTH response. These results indicate that in addition to amplifying the anticryptococcal DTH response, Tamp cells may protect the anticryptococcal TDH cells from suppression by C. neoformans-specific Ts cells, much like contrasuppressor cells do in other systems. However, further characterization of the Tamp cells revealed that they are not adherent to Viscia villosa lectin, indicating that the anticryptococcal Tamp cells do not have this characteristic in common with contrasuppressor cells of other antigen systems.
Production and characterization of monoclonal antibodies to a 58-kilodalton antigen of Aspergillus fumigatus
Authors: Fratamico P M , Long W K , Buckley H R
Published: January 2020

Eight monoclonal antibodies that recognize a serodiagnostically important 58-kDa antigen of Aspergillus fumigatus were produced and partially characterized. 2-7, 2-12, and 2-14 are of the immunoglobulin M class, and 2-2-1, 2-2-4, 2-2-6, 2-2-9, and 2-2-13 are all immunoglobulin G1(kappa) antibodies. Immunoblot analysis with A. fumigatus mycelial extract demonstrated that all of the monoclonal antibodies recognize a major 58-kDa antigen. The antigen was also detected by immunoblot analysis of 4- and 7-day culture filtrate preparations. 2-2-1, 2-2-4, and 2-2-6 cross-reacted with an antigen of approximately 55 kDa from an extract of Candida albicans. 2-7, 2-12, 2-14, and 2-2-4 formed a precipitin band with immunoaffinity-purified 58-kDa antigen by immunodiffusion. Results from indirect immunofluorescence assays with 2-7 and 2-2-9 showed fluorescent staining mainly on the surfaces of conidia and hyphae, indicating that the 58-kDa antigen may be cell wall associated. 2-2-9 and 2-2-13 and antibodies in patient and immune rabbit sera precipitated the [35S]methionine-labeled 58-kDa antigen. The 58-kDa antigen immunoprecipitated by each of the antibodies was enzymatically cleaved by Staphylococcus aureus V8 protease; one cleavage product, a 35-kDa fragment, was generated, indicating that the precipitated antigens share primary structure. Immunoblot analysis with an immunoaffinity-purified 58-kDa antigen showed that sera from patients with invasive aspergillosis reacted with the same antigen as that recognized by the monoclonal antibodies.
Antigenicity and immunogenicity of an extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells
Authors: Gómez A M , Rhodes J C , Deepe G S
Published: January 2020

In order to identify T-cell antigens from Histoplasma capsulatum yeast cells, we prepared a detergent extract of the cell wall and cell membrane of yeast-phase H. capsulatum G217B and analyzed its antigenicity and immunogenicity. Mice injected with viable H. capsulatum yeast cells or with 500 or 1,000 micrograms of the extract mounted a delayed-type hypersensitivity response to solubilized cell wall and cell membrane. Vaccination with this antigenic preparation conferred a protective immune response in mice that were challenged intravenously with H. capsulatum yeast cells. The extract induced in vitro proliferation by splenocytes from mice injected with either viable yeast cells or the soluble cell wall and cell membrane preparation. We also examined the profile of in vitro responses by a murine T-cell line and by cloned T cells to soluble cell wall and cell membrane by employing the technique of T-cell immunoblotting. Two prominent regions that stimulated the T-cell line and cloned T cells were identified. Fractions encompassing an area between 53 and 64 kDa caused proliferation by a T-cell line and five of six clones. Antigens recognized by the T-cell line and by three of six clones were contained in another area that extended from 69 to 82 kDa. The data demonstrate that this soluble extract from cell wall and cell membrane contains antigens recognized by T cells and mediates protective immunity. Moreover, T-cell immunoblotting provides a useful technique for mapping immunoreactive molecules from H. capsulatum yeast cells.
Polymorphism in the aerobactin-cloacin DF13 receptor genes from an enteroinvasive strain of Escherichia coli and pColV-K30 is associated only with a decrease in cloacin susceptibility
Authors: Marolda C L , Valvano M A , Crosa J H
Published: January 2020

We have cloned chromosomal genes mediating the aerobactin iron transport system from the enteroinvasive strain Escherichia coli 978-77. The physical map of the region spanning the siderophore biosynthesis genes and the upstream portion of the receptor gene in strain 978-77-derived clones was identical to the corresponding regions in pColV-K30, while the downstream portion was different. Recombinant plasmids derived from strain 978-77 encoded a 76-kDa outer membrane protein, in contrast to the 74-kDa polypeptide encoded by similar clones derived from pColV-K30. No differences were found in the uptake of ferric aerobactin mediated by either the 76-kDa- or the 74-kDa-encoding plasmids. In contrast, cells containing the 76-kDa-encoding plasmids showed a 16-fold decrease in susceptibility to cloacin compared with cells harboring the 74-kDa-encoding plasmids. Two classes of chimeric aerobactin receptor genes were constructed by exchanging sequences corresponding to the downstream portion from the aerobactin receptor gene of both systems. The pColV-K30-978-77 chimeric gene encoded a 76-kDa outer membrane protein which mediated a low level of cloacin susceptibility, whereas the 978-77-pColV-K30 type encoded a protein of 74 kDa determining a level of cloacin susceptibility identical to that mediated by pColV-K30.
Tandem insertion sequence-like elements define the expression site for variable antigen genes of Borrelia hermsii
Authors: Barbour A G , Carter C J , Burman N , Freitag C S , Garon C F , Bergström S
Published: January 2020

The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches.
Diphosphoryl lipid A obtained from the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides is an endotoxin antagonist in mice
Authors: Qureshi N , Takayama K , Kurtz R
Published: January 2020

Diphosphoryl lipid A (DPLA) obtained from the nontoxic lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides ATCC 17023 did not induce interleukin-1 release by murine peritoneal macrophages. However, it blocked this induction by toxic deep-rough chemotype LPS (ReLPS) from Escherichia coli D31m4. Previously, we obtained similar results on the induction of tumor necrosis factor (TNF) by macrophages. These results showed that DPLA is able to block in vitro the induction of two important mediators of gram-negative bacterial sepsis. We then wanted to determine whether DPLA could also block the induction of TNF by LPS in animals. Mice were treated with 100 micrograms of R. sphaeroides DPLA and challenged 60 min later with 1.0 micrograms of ReLPS from E. coli. The serum TNF level was measured after 60 min. Treatment of mice with this DPLA blocked the rapid and transient rise of TNF caused by ReLPS. This result suggested that R. sphaeroides DPLA might be able to protect animals against endotoxin shock caused by gram-negative bacterial infection.
Differential expression of Trypanosoma cruzi neuraminidase in intra- and extracellular trypomastigotes
Authors: Rosenberg I A , Prioli R P , Mejia J S , Pereira M E
Published: January 2020

The developmentally regulated expression of Trypanosoma cruzi neuraminidase in culture cells was monitored by immunofluorescence with a monoclonal antibody (TCN-2) against the enzyme. The results showed that TCN-2 reacted with all intracellular trypomastigote forms (NA+) but not with amastigotes. Immunoprecipitation of radiolabeled neuraminidase confirmed TCN-2 reactivity with trypomastigotes and the specificity of the antibody binding. During exiting from the host cells, all trypomastigotes were still NA+. However, when free in the extracellular environment, the relative proportion of NA+ parasites declined from 100% to about 20%, thereby establishing a subpopulation of trypomastigotes which did not express enzyme (NA-). The expression of neuraminidase in all intracellular trypomastigotes and in only a subpopulation of the extracellular counterpart suggests that the enzyme may play a role in parasite exiting from infected cells.