bluuebunny/crossref_metadata_2025 huggingface dataset:
A recombinant non-fatty acylated form of the Hi-PAL (P6) protein of Haemophilus influenzae elicits biologically active antibody against both nontypeable and type b H. influenzae
Authors: Green B A , Metcalf B J , Quinn-Dey T , Kirkley D H , Quataert S A , Deich R A
Published: January 2020

An approximately 15,000-dalton outer membrane lipoprotein of Haemophilus influenzae, the Hi-PAL (P6) protein, has been shown to elicit bactericidal and protective antibodies against both type b and nontypeable H. influenzae strains and is a vaccine candidate for these organisms. To determine whether the lipid modification of this protein is required for immunogenicity or the elicitation of biologically active antibodies, a genetic fusion was constructed that contains the sequence of mature Hi-PAL fused to the polylinker region of pUC19. The protein expressed by this clone does not contain detectable lipid and was purified to homogeneity. This recombinant fusion protein, rPAL, elicited a strong immune response when injected into rabbits, and the antiserum reacted well with native Hi-PAL. The antiserum was bactericidal against a number of clinical nontypeable strains, duplicating the activity of anti-Hi-PAL. The anti-rPAL antiserum was also protective against type b bacteremia in the infant rat model. These results demonstrate that purified rPAL elicits antibodies with biological activities that are similar to those of anti-Hi-PAL antibodies. Thus, the lipid component of Hi-PAL is not required for either immunogenicity or elicitation of biologically active antibodies.
Antigenic conservation of the 15,000-dalton outer membrane lipoprotein PCP of Haemophilus influenzae and biologic activity of anti-PCP antisera
Authors: Deich R A , Anilionis A , Fulginiti J , Metcalf B J , Quataert S , Quinn-Dey T , Zlotnick G W , Green B A
Published: January 2020

A gene from Haemophilus influenzae encoding an outer membrane lipoprotein of about 15,000 daltons and which comigrates with the peptidoglycan-associated lipoprotein (PAL) of H. influenzae on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been previously reported and designated pcp gene, and its product has been designated PCP. in order to obtain specific immunologic probes for the analysis of PCP expression, cellular location, and antigenic conservation in H. influenzae, pcp was fused to the lac polylinker region of plasmid pUC19 and the hybrid gene was expressed in Escherichia coli. PCP purified from these cells was used to generate rabbit and mouse polyclonal antisera and mouse monoclonal antibody against PCP. Western immunoblot analysis with anti-PCP monoclonal antibody demonstrated that PCP is present and antigenically conserved in 30 tested strains of H. influenzae, including 27 clinical nontypeable strains. Polyclonal antiserum against PCP killed 9 of 11 clinical H. influenzae strains in a complement-mediated bactericidal assay, and bactericidal activity was additive with bactericidal activity of antisera against PAL. These results indicate that PCP is a potentially valuable component for a subunit vaccine against nontypeable H. influenzae disease, especially in combination with PAL or other components.
Immunoaccessible peptide sequences of the major outer membrane protein from Chlamydia trachomatis serovar C
Authors: Zhong G M , Brunham R C
Published: January 2020

The antigenicity of the major outer membrane protein of Chlamydia trachomatis serovar C was assessed by using overlapping hexapeptide homologs of serovar C major outer membrane protein and rabbit antisera in a peptide enzyme-linked immunosorbent assay. Five immunogenic sites were found distributed within variable sequences of the protein: four were immunodominant and three were surface exposed on native elementary bodies of serovar C. None was surface exposed on serovars H, I, and J.
Reduced lymphocyte responses to mitogens in natural and experimental trichomoniasis
Authors: Mason P R , Gwanzura L
Published: January 2020

Proliferative responses to mitogens were determined by using peripheral blood mononuclear cells from women with active trichomoniasis, with serological evidence of past infection with Trichomonas vaginalis, and with no evidence of current or past infection. Even after the human immunodeficiency virus antibody status of the patients was taken into account, cells from women with active trichomoniasis showed reduced responses to phytohemagglutinin, concanavalin A, pokeweed mitogen, and bacterial lipopolysaccharide. Similar findings were obtained by using spleen cells from mice inoculated subcutaneously with live trichomonads. Reduction in proliferative responses by these cells could be detected 3 days after inoculation. There was some evidence to suggest that more-pathogenic strains of the parasite induced a greater degree of immunosuppression. The responses of spleen cells from mice inoculated with trichomonad-free culture supernatants were within normal limits, indicating that live trichomonads were needed to induce suppression. Support for this was gained from studies with cells from women who were treated successfully. Cells from these women rapidly regained normal lymphoproliferative function. Interleukin-2 (IL-2) production by spleen cells from infected mice was determined from measurements of mitochondrial activity in an IL-2-dependent T-cell line following incubation with stimulated spleen cell culture supernatants. These tests demonstrated lower IL-2 activity in supernatants from cell cultures from infected mice than in those from uninfected mice. The reduction in IL-2 activity did not, however, appear to correlate with the degree of reduction of mitogen-induced lymphoproliferation. Suppression of T-cell-mediated immunity may be one of the mechanisms by which T. vaginalis is able to evade host responses to infection.
High-frequency spontaneous mutation of classical Vibrio cholerae to a nonmotile phenotype
Authors: Mostow P , Richardson K
Published: January 2020

The species Vibrio cholerae contains within it two biotypes, classical and El Tor, both of which are motile. Phenotypic expression of motility was unaffected by type of growth medium, salt concentration, pH, or temperature of incubation. However, seven strains of classical V. cholerae produced spontaneous nonmotile mutants at an unusually high frequency (ca. 10(-4)), while no mutants were detected for all three El Tor strains examined. No revertants of these nonmotile mutants were detected. Four independent mutants of classical strain 395 were isolated to characterize this phenomenon. By transmission electron microscopy, one of the nonmotile mutants was found to be flagellated, while the other three were found to be aflagellate. Chromosomal DNA from the mutants and parental wild-type strain 395 was examined by Southern blot analysis with, as probes, V. cholerae mutagenic prophages VcA-1 and VcA-2 and six cloned motility gene regions isolated from transposon insertion motility mutants of strains 395 and N16961 (El Tor, Inaba). The parental wild-type strain and all of the mutants exhibited the same pattern of bands when probed with VcA-1 and VcA-2 DNAs. Four of the cloned motility gene regions hybridized to the same fragments of DNA in both the wild-type and mutant isolates. However, two other probes detected a new fragment for a single aflagellate mutant. The observations that spontaneous nonmotile mutants occurred at a high frequency and that these mutants did not revert at a detectable frequency suggested that a genetic event is involved. The phenomenon appears to be limited to classical V. cholerae and may explain why classical V. cholerae is only sporadically associated with disease in the current pandemic.
Insertional inactivation of the gene encoding a 76-kilodalton cell surface polypeptide in Streptococcus gordonii Challis has a pleiotropic effect on cell surface composition and properties
Authors: Jenkinson H F , Easingwood R A
Published: January 2020

A library of Streptococcus gordonii DL1-Challis DNA was constructed in lambda gt11. Phage plaques were screened for production of antigens that reacted with antiserum to S. gordonii cell surface proteins. A recombinant phage denoted lambda gt11-cp2 was isolated that carried 1.85 kb of S. gordonii DNA and that expressed an antigen with a molecular mass of 29 kDa in Escherichia coli. Antibodies that reacted with the expression product were affinity purified and were shown to react with a single polypeptide antigen with a molecular mass of 76 kDa in S. gordonii DL1-Challis. A segment (0.85 kb) of the cloned DNA within the transcription unit was ligated into a nonreplicative plasmid carrying an erythromycin resistance determinant and transformed into S. gordonii DL1-Challis. The plasmid integrated onto the chromosome, and expression of the 76-kDa polypeptide antigen was abolished. The gene inactivation had no obvious effect on bacterial growth or on a number of phenotypic properties, including hydrophobicity and adherence. However, it abolished serum-induced cell aggregation, mutant cells had reduced aggregation titers in saliva and in colostrum immunoglobulin A, and it also reduced coaggregation with some Actinomyces species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of cell envelope proteins from wild-type and mutant strains showed that as well as lacking the surface-exposed 76-kDa polypeptide, mutant cell envelopes were deficient in several other polypeptides, including those that bound to immunoglobulin A. Expression of the gene encoding the 76-kDa polypeptide in S. gordonii appeared to be critical for functional conformation of the cell surface.
Enterotoxin and cytotoxin production by enteroinvasive Escherichia coli
Authors: Fasano A , Kay B A , Russell R G , Maneval D R , Levin M M
Published: January 2020

It has long been suspected that besides their ability to invade enterocytes, enteroinvasive Escherichia coli (EIEC) strains have the ability to elaborate an enterotoxin. We tested 35 EIEC strains for cytotoxins and 9 (1 per serogroup) for enterotoxins. All 35 strains exhibited low levels of Vero cell cytotoxins that are immunologically and genetically distinct from Shiga-like toxin I or II of enterohemorrhagic E. coli. Sterile supernatants and cell lysates of two EIEC strains were tested in rabbit ileal loops, and both stimulated moderate fluid accumulation (circa 0.5 ml/cm) without tissue damage; secretory activity was confirmed in Ussing chambers, where these two strains and the seven others tested significantly increased short circuit current without altering tissue conductance. Curing the 140-MDa invasiveness plasmid from an EIEC strain did not diminish enterotoxin production. Culture in minimal Fe2+ medium is necessary to detect expression of the enterotoxin which is circa 68 to 80 kDa in size and is distinct from the EIEC cytotoxin.
Streptococcus pyogenes clinical isolates and lipoteichoic acid
Authors: Leon O , Panos C
Published: January 2020

Minimally subcultured clinical isolates of virulent nephritogenic and nonnephritogenic Streptococcus pyogenes of the same serotype showed major differences in lipoteichoic acid (LTA) production, secretion, and structure. These were related to changes in coccal adherence to and destruction of growing human skin cell monolayers in vitro. A possible relationship between cellular LTA content and group A streptococcal surface hydrophobicity was also investigated. Nephritogenic S. pyogenes M18 produced twice as much total (i.e., cellular and secretory) LTA as did the virulent, serologically identical, but nonnephritogenic isolate. Also, the LTAs from these organisms differed markedly. The polyglycerol phosphate chain of LTA from the nephritogenic isolate was longer (1.6 times) than was that from the nonnephritogenic isolate. Likewise, both LTAs indicated the presence of alanine and the absence of glucose. Amino sugars were found in LTA from only nephritogenic S. pyogenes. Teichoic acid, as a cellular component or secretory product, was not detected. The adherence of two different nephritogenic group A streptococcal serotypes (M18 and M2) exceeded that of the serologically identical but nonnephritogenic isolates (by about five times), indicating a correlation between virulent strains causing acute glomerulonephritis and adherence to human skin cell monolayers. Likewise, LTA from nephritogenic S. pyogenes M18 was more cytotoxic (1.5 times) than was that from the nonnephritogenic isolate for human skin cells, as determined by protein release. This difference was not perceptible by the more sensitive dye exclusion method (i.e., requiring less LTA), which emphasizes changes in host cell morphology and death. Also, the secretion of LTA by only virulent nephritogenic S. pyogenes M18 was exacerbated by penicillin (a maximum of four times). Finally, while the adherence of nephritogenic S. pyogenes M18 decreased markedly after continued subculturing in vitro, the surface hydrophobicity did not.
Schistosoma mansoni larvicidal activity of murine bronchoalveolar lavage cells
Authors: Lewis F A , White-Ziegler C A , Ball J E , Niemann G M
Published: January 2020

We have investigated the ability of cells obtained from both normal and immune mice by bronchoalveolar lavage (BACs) to kill Schistosoma mansoni larvae in vitro. In cultures with mechanically derived schistosomules, high levels of larvicidal activity were displayed by BACs from both normal and irradiated cercaria-immunized C57BL/6 mice. Based on effector-to-target-cell ratios, BAC-mediated killing was two- to threefold more efficient than killing mediated by macrophage-rich cell populations obtained from the peritoneal cavity. BACs from normal A/J mice were essentially as larvicidal as normal C57BL/6 cells. However, BACs from a strain of mouse (P/J) with a known macrophage defect possessed negligible larvicidal activity. Macrophages made up 85 to 95% of BACs from all three strains tested. In contrast to cells of the IC-21 macrophage cell line, B6 BACs did not show enhanced killing activity when preincubated with lymphokine-containing supernatants. Lung schistosomules harvested 10 days after cercarial penetration were refractory to BAC-mediated killing.
Suppression of T-lymphocyte responses to Entamoeba histolytica antigen by immune sera
Authors: Salata R A , Martinez-Palomo A , Canales L , Murray H W , Trevino N , Ravdin J I
Published: January 2020

Lymphocytes from patients cured of amebic liver abscesses proliferate and produce gamma interferon upon incubation with soluble Entamoeba histolytica antigen: however, amebic liver abscesses exhibit a relentless progression without treatment. To determine whether suppressive factors are present in sera, we studied T-lymphocyte responses to total soluble E. histolytica antigen by using cells from five patients treated for amebic liver abscesses in the presence of 15 different immune sera and 10 control sera. In the presence of immune sera, E. histolytica antigen-induced lymphocyte proliferation decreased by 63% and production of gamma interferon was reduced by 93.2% (P less than 0.01). Immune sera had no effect on the mitogenic responses of patient lymphocytes to phytohemagglutinin or on the proliferative responses of control lymphocytes to phytohemagglutinin or tetanus toxoid. The suppressive activity of immune sera diminished as the time between therapy for amebic liver abscesses and serum collection increased (P less than 0.05). Suppressive activity did not correlate with the titers of serum anti-amebic antibody and was not affected when serum was absorbed with viable amebic trophozoites. In conclusion, soluble factors present in the sera of amebic liver abscess patients suppressed in vitro lymphocyte responses to E. histolytica antigen and may have contributed to the lack of development of effective in vivo cell-mediated immune responses following the onset of amebic liver abscesses.
Inhibition of heat-labile toxin from Bordetella parapertussis by fatty acids
Authors: Endoh M , Nagai M , Burns D L , Manclark C R , Nakase Y
Published: January 2020

The ability of heat-labile toxin (HLT) from Bordetella parapertussis to induce skin lesions in guinea pigs was found to be inhibited by lipids isolated from skin layers of adult mice, which are refractory to the lesion-inducing activity of HLT. These lipids were identified as linoleic and oleic acids. Other long-chain unsaturated fatty acids were also found to inhibit HLT; however, fatty alcohols, neutral lipids, phospholipids, cholesterol, prostaglandin, and leukotriene had no measurable effects on HLT action. The data presented in this report indicate that the ability of HLT to induce skin lesions in animals may depend, at least in part, on the free fatty acid content of the skin layer.
Adherence of Staphylococcus epidermidis to fibrin-platelet clots in vitro mediated by lipoteichoic acid
Authors: Chugh T D , Burns G J , Shuhaiber H J , Bahr G M
Published: January 2020

The adherence of two strains of Staphylococcus epidermidis to human fibrin-platelet clots in vitro was investigated. Both strains were noncapsulated, nonhemagglutinating, and nonslime producers. Binding was not related to surface charge, carbohydrate profile, or hydrophobicity of the bacteria. Adherence was reduced four- to sixfold (P less than 0.001) on pretreatment of bacteria with lipase, while neuraminidase, trypsin, phospholipase C, and sodium periodate did not alter their binding. Pretreatment of bacteria with substances known to bind lipoteichoic acid (LTA), such as human albumin and anti-LTA antibodies, also resulted in a fourfold (P less than 0.001) reduction in adherence. Prior incubation of clots with free LTA, but not with deacylated LTA, produced a fourfold (P less than 0.001) decrease in the adherence of homologous and heterologous strains of S. epidermidis. A similar reduction was also observed when LTAs derived from Staphylococcus aureus and Streptococcus pyogenes were used. These data provide evidence that the lipid moiety of LTA has a central role in the adherence of S. epidermidis to fibrin-platelet clots in vitro.
Effects of Pseudomonas aeruginosa elastase on alveolar epithelial permeability in guinea pigs
Authors: Azghani A O , Connelly J C , Peterson B T , Gray L D , Collins M L , Johnson A R
Published: January 2020

Elastase-deficient mutants of Pseudomonas aeruginosa are less virulent than the wild type and are easily cleared from the lungs of guinea pigs. The effect of P. aeruginosa elastase on lung epithelium, however, is not yet understood. We addressed the hypothesis that breach of the epithelial barrier by elastase from P. aeruginosa allows invading organisms and toxic substances to penetrate the interstitium. We measured the clearance of aerosolized technetium-labeled albumin (molecular weight, 69,000) from the lungs of anesthetized guinea pigs with the aid of a gamma camera and a dedicated computer. Aerosols of the elastase (0.1 to 5 micrograms) increased the rate of clearance of labeled albumin from the lungs in proportion to the elastase dose. Electron microscopic studies using horseradish peroxidase as a tracer revealed that elastase interrupts intercellular tight junctions of the epithelial lining, thereby increasing the permeability to macromolecules. The amounts of elastase used in this report did not cause interstitial or alveolar edema, as determined by both postmortem extravascular lung water volume measurement and morphological examination. The data indicate that the elastase is a potentially important virulence factor in acute lung infection.
Polyclonal anti-idiotypic antibodies exhibit antigenic mimicry of limited type 1 fimbrial proteins of Escherichia coli
Authors: Paque R E , Miller R , Thomas V
Published: January 2020

Polyclonal anti-idiotypic antibodies (anti-Ids)(fim) developed against idiotypes on antibodies (Ab-1s) that specifically bind structural, organelle fimbrial proteins of Escherichia coli were able to modulate immune function in anti-Id(fim)-immunized mice. Proliferation or suppression of splenic lymphoid cell responses by polyclonal anti-Ids in tissue culture appeared to be dose dependent. Anti-Ids were able to induce a dose-dependent T-cell-mediated immunity specific for type 1 fimbrial antigen(s) in immunized animals when assessed in vitro, but they failed to elicit in vivo positive ear-swelling skin reactions. Anti-Ids were unable to induce protective immunity against an in vivo infectious challenge with E. coli in anti-Id-immunized adult animals, but they stimulated a specific, secondary antibody response in anti-Id-challenged mice. Anti-Ids stimulated the development of anti-anti-Ids (Ab-3s) specifically binding a fimbrial antigen(s) and revealed the presence of antibody idiotypes binding E. coli adhesin proteins in the 27- to 29-kilodalton range. Results suggest discrete, but subtle, immunomodulatory effects of the anti-Ids and potential vaccinoid properties capable of stimulating a specific humoral and cellular response in vivo.
Protein phosphorylation by protein kinase C in HEp-2 cells infected with enteropathogenic Escherichia coli
Authors: Baldwin T J , Brooks S F , Knutton S , Manjarrez Hernandez H A , Aitken A , Williams P H
Published: January 2020

Infection of HEp-2 monolayers with enteropathogenic Escherichia coli 2036-80 (O119) stimulated phosphorylation of several target cell proteins, the most prominent of which had apparent molecular weights of 21,000 and 29,000. Proteins of the same size were phosphorylated in response to known activators of the calcium-phospholipid-dependent protein kinase C. Screening of clinical isolates of various O serogroups revealed that all strains able to form the characteristic attaching and effacing lesion of enteropathogenic E. coli showed elevated phosphorylation of 21,000- and 29,000-dalton protein species.
Multiple mechanisms in serum factor-induced resistance of Haemophilus influenzae type b to antibody
Authors: Kuratana M , Hansen E J , Anderson P
Published: January 2020

Incubation of Haemophilus influenzae type b at less than or equal to 10(7) CFU/ml with serum ultrafiltrate induces a phenotypic conversion in which complement-mediated bactericidal activity by somatic antibodies decreases while killing by capsular antibody is unchanged. Conversion had been shown to occur in a capsule-deficient (b-) mutant of strain Eag (thus appearing independent of capsulation), to include an increase in lipopolysaccharide content, and to be inhibited by chloramphenicol or puromycin. In the present study, in several strains not previously examined, conversion was not inhibited by the drugs and the corresponding b- mutants did not convert. Incubation in ultrafiltrate was also found to increase capsulation, as detected by radioassay, only 1.6-fold in Eag but 4.5-fold in DL26, the strain with the largest increase in resistance; moreover, complement-mediated opsonization by capsular antibody was greatly decreased. Thus, multiple mechanisms, capsule dependent as well as independent, appear to contribute to the serum factor-induced resistance of H. influenzae type b to antibody.
Assay for enterotoxigenic Escherichia coli heat-stable toxin b in rats and mice
Authors: Whipp S C
Published: January 2020

The Escherichia coli heat-stable enterotoxin STb is the most prevalent toxin associated with diarrheagenic isolates of porcine origin. This report demonstrates that when endogenous protease activity was blocked with soybean trypsin inhibitor, STb evoked a dose-dependent secretory response in infant mice and jejunal loops of rats. Infant mice were much less sensitive to STb than rats were. The response of rat jejunal loops to STb was linearly related to the log of the dose of STb through 10 twofold dilutions. The anterior 25% of the jejunum was less sensitive than was the remainder of the gut. An incubation period of 2.5 h provided a maximal response. The linear response to STb in rats could be used as a bioassay.
New mouse models for chronic Cryptosporidium infection in immunodeficient hosts
Authors: Ungar B L , Burris J A , Quinn C A , Finkelman F D
Published: January 2020

Cryptosporidium sp. causes fulminant diarrhea and chronic infection in immunocompromised, particularly human immunodeficiency virus-infected, persons. The lack of in vitro cultivation and a suitable animal model has limited development of effective treatment. We describe two new mouse models of chronic symptomatic cryptosporidiosis in adult athymic mice and in T-cell subset-depleted mice. A progressive infection, fatal within 4 months, occurred in most adult athymic mice; a few developed stable infections. Symptoms included dehydration, weight loss, intermittent diarrhea, and jaundice. Pathologic abnormalities and organisms localized in the intestine in stable infections but involved the hepatobiliary tree and pancreas in others. Lymphoid cells from histocompatible, Cryptosporidium sp.-immune mice cured infected nude mice. Identical infections occurred in neonatally infected BALB/c mice treated with anti-CD4 monoclonal antibodies alone or also with anti-CD8 monoclonal antibodies; the mice were cured when the monoclonal antibody treatments were stopped. These models will be useful in definition of the immune defects that permit chronic cryptosporidiosis to develop and in assessment of treatment modalities.
Expression of the cloned gene for enterotoxin STb of Escherichia coli
Authors: Lawrence R M , Huang P T , Glick J , Oppenheim J D , Maas W K
Published: January 2020

This study involved the construction of hybrid plasmids to produce heat-stable enterotoxin type II of Escherichia coli (STb). The translation of the open reading frame for the STb gene estA was demonstrated in several ways. Studies using in vivo labeling with [35S]cysteine demonstrated a radiolabeled protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expected molecular weight of 5,000 for toxin STb. Insertion of translational or transcriptional termination signals into the BglII site of the estA gene blocked the expression of estA. The estA gene was cloned into high-expression vector pKC30 downstream from the strong pL promoter. Northern (RNA) blotting assays revealed a 10- to 20-fold increase in mRNA produced by strain C600F(pKC30STb) over other STb-producing strains, compared with little or no increase in enterotoxin activity demonstrated by bioassay. The estA gene, with its own promoter and Shine-Delgarno region and a portion of the sequence for the signal peptide deleted, was also inserted under the control of the tac promoter. Even after induction of the tac promoter by addition of isopropyl-beta-D-thiogalactopyranoside, no biologic enterotoxin activity could be identified. Neutralizing antibodies to STb were produced in rabbits by using either a purified OmpF-STb-beta-galactosidase fusion protein or a 19-amino-acid synthetic STb peptide coupled to keyhole limpet hemocyanin.
Protein L, a bacterial immunoglobulin-binding protein and possible virulence determinant
Authors: Kastern W , Holst E , Nielsen E , Sjöbring U , Björck L
Published: January 2020

Protein L, an immunoglobulin kappa light chain-binding protein, is expressed on the surfaces of certain strains of Peptostreptococcus magnus. Thirty strains of P. magnus were isolated from clinical specimens, and four of them were found to express protein L. Among the 30 strains, 7 were isolated from the vaginas of patients with bacterial vaginosis, and the 4 immunoglobulin-binding strains all belonged to this group, results demonstrating that expression of protein L is correlated to peptostreptococcal virulence (P less than 0.001 in the chi-square test) and indicating that the molecule could be a virulence determinant. Similar amounts of protein L were expressed by the four strains, and when protein L was isolated from three of them and analyzed in Western blots, the same immunoglobulin-binding patterns were obtained. The N-terminal amino acid sequences of tryptic fragments of protein L were determined, and on the basis of these sequence data, oligonucleotides were synthesized and used to screen a genomic library of peptostreptococcal DNA in the lambda ZAP vector. The nucleotide sequence was determined for one of the clones detected in this way. In dot blots and Southern blots of peptostreptococcal DNA, another synthetic oligonucleotide probe based on this sequence showed no hybridization with DNA samples from the nonexpressing strains, whereas similar Southern blot patterns were seen when DNA samples from protein L-expressing strains were analyzed. These results suggest that the protein L gene is missing rather than being down regulated in protein L-negative strains of P. magnus. Finally, the probe did not hybridize with DNA purified from immunoglobulin-binding streptococcal and staphylococcal strains or with Escherichia coli DNA, suggesting that the protein L gene is unique to protein L-expressing strains of P. magnus.
Outer membrane and porin characteristics of Serratia marcescens grown in vitro and in rat intraperitoneal diffusion chambers
Authors: Malouin F , Campbell G D , Halpenny M , Becker G W , Parr T R
Published: January 2020

The composition and antibiotic permeability barrier of the outer membrane of Serratia marcescens were assessed in cells grown in vivo and in vitro. Intraperitoneal diffusion chambers implanted in rats were used for the in vivo cultivation of bacteria. Outer membranes isolated from log-phase bacterial cells recovered from these chambers were compared with membranes isolated from cells grown in vitro. Analysis revealed that the suspected 41-kilodalton porin and the OmpA protein were recovered on sodium dodecyl sulfate-polyacrylamide gels in equal quantities. Several high-molecular-weight proteins, thought to be iron starvation induced, appeared in the diffusion chamber-grown cells. The outer membrane permeability barriers to cephaloridine were similar in in vivo- and in vitro-grown cells based on permeability coefficient calculations. The permeability coefficient of cephaloridine in S. marcescens cells (30.3 x 10(-5) to 38.9 x 10(-5) cm s-1) was greater than that obtained for an Escherichia coli strain expressing only porin OmpC but smaller than those obtained for the E. coli wild type and a strain expressing only porin OmpF. Functional characterization of the suspected porin was performed by using the planar lipid bilayer technology. The sodium dodecyl sulfate-0.4 M NaCl-soluble porin from both in vitro- and in vivo-grown cells showed an average single-channel conductance in 1 M KCl of 1.6. A partial amino acid sequence (19 residues) was obtained for the S. marcescens porin. The sequence showed a very high homology to the E. coli OmpC porin. These data identified the S. marcescens outer membrane 41-kilodalton protein as a porin by both functional and amino acid analyses. Also, the methodology used allowed for efficient growth and recovery of diffusion chamber-grown bacterial cells and permitted identification of specific in vivo-induced changes in bacterial cell membrane composition.
Mapping antigenic sites on the major outer membrane protein of Chlamydia trachomatis with synthetic peptides
Authors: Zhong G M , Reid R E , Brunham R C
Published: January 2020

The antigenicity of the major outer membrane protein (MOMP) of Chlamydia trachomatis was comprehensively evaluated by using overlapping hexapeptide homologs of serovar B MOMP and polyclonal rabbit antisera in a peptide enzyme-linked immunosorbent assay. Of 367 hexapeptides, 152 showed reactivities with at least one antiserum. Seven hexapeptides located within variable domain (VD) IV (residues 288 to 316) were found to be most reactive in terms of their binding titer and frequency, suggesting that VD IV is the immunodominant region within the MOMP as detected by this assay. Peptide-reactive antibodies could also recognize corresponding epitopes on either viable or acetone-permeabilized organisms. The antigenic specificity and immunoaccessibility of epitopes located in VD IV were resolved by absorbing antisera with chlamydial elementary bodies. Six antigenic sites were found in this region and included a B-type-specific site (S1), four subserogroup-specific sites (S2 and S4 to 6), and one species-specific site (S3), each displaying varying degrees of surface exposures on elementary bodies from different C. trachomatis serovars.
Modification of the glycolipid-binding specificity of vero cytotoxin by polymyxin B and other cyclic amphipathic peptides
Authors: Head S , Ramotar K , Lingwood C
Published: January 2020

Polymyxin B, an amphipathic cyclic decapeptide produced by Bacillus polymyxa, is routinely used in the extraction of the components from the periplasmic space of gram-negative bacteria. Vero cytotoxin 1 (VT1) is an Escherichia coli-elaborated subunit toxin which binds to the glycolipid globotriosylceramide (Gal-alpha 1-4-Gal beta 1-4-Glc-ceramide [Gb3]) and has been strongly implicated in the etiology of the hemolytic uremic syndrome and hemorrhagic colitis. We now show by in vitro glycolipid-binding assays that in the presence of low concentrations of polymyxin B, globotetraosylceramide (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide [Gb4]) is also recognized by both the VT1 B (binding) subunit and holotoxin. Melittin, a 26-amino-acid cyclic peptide of similar amphipathic nature, produced the same effect, whereas a hydrophobic blocking agent did not. Triton X-100 did not increase binding of VT1 to Gb4 but prevented glycolipid binding in toto at concentrations above 0.5%. Caution is therefore advised in the analysis of VT1 glycolipid binding in the presence of amphipathic peptides.
Immune T cells sorted by flow cytometry confer protection against infection with Treponema pallidum subsp. pertenue in hamsters
Authors: Liu H , Steiner B M , Alder J D , Baertschy D K , Schell R F
Published: January 2020

The role of cell-mediated immunity against infection with Treponema pallidum subsp. pertenue in humans or experimental animals is unclear. Hamsters injected subcutaneously in the hind paws with 4 x 10(6) unfractionated lymph node cells or enriched lymph node T cells (immunoglobulin negative, Ia negative) from T. pallidum subsp. pertenue-immune hamsters were resistant to challenge with T. pallidum subsp. pertenue. The popliteal lymph nodes of hamsters that received immune cells weighed less and had significantly fewer treponemes than did lymph nodes from hamsters infused with cells from nonimmune donors. Furthermore, recipients of immune T cells failed to develop antitreponemal antibodies 21 days after challenge. Enriched T cells were obtained by flow cytometric separation by using monoclonal anti-Ia antibody 14-4-4s, which identified hamster B cells. Flow cytometric analysis by two-color immunofluorescent staining with anti-hamster-immunoglobulin and monoclonal anti-Ia antibody 14-4-4s confirmed that monoclonal anti-Ia antibody 14-4-4s recognized B cells. In addition, lymph node cells obtained after treatment with anti-Ia monoclonal antibody 14-4-4s and complement were 97% T cells, as determined by monoclonal antibody 20, a hamster T-cell marker. These results demonstrated that highly enriched T cells (immunoglobulin negative, Ia negative) from T. pallidum subsp. pertenue-immune hamsters conferred partial protection on hamsters against infection with T. pallidum subsp. pertenue.
Cytotoxic activity of the Proteus hemolysin HpmA
Authors: Swihart K G , Welch R A
Published: January 2020

We previously showed that hpmA is the hemolysin determinant most commonly found among Proteus isolates. To assess the potential contribution of HpmA to virulence, we first characterized the toxic activities of this hemolysin. Hemolytic activity was present in total cell cultures and cell-free supernatants of Proteus clinical isolates as well as Escherichia coli containing cloned hpm genes. HpmA also possesses cytotoxic activity which was detected by a chromium release assay against a variety of target cell lines (Daudi, Raji, T24, U937, and Vero). Analysis of the dose response of bacterial cells against both T24 cells and erythrocytes showed that E. coli containing cloned hpm genes was 30-fold more cytotoxic than Proteus mirabilis BA6163. Also, 10(5)-fold more bacterial cells were needed to lyse T24 cells than to lyse erythrocytes. HpmA- mutants of two Proteus strains in which the central portion of hpmA was deleted were constructed. These HpmA- mutants, which have lost the hemolytic and cytotoxic activities exhibited by their respective parent strains, demonstrate that HpmA is needed for both of these activities. In an ascending model of murine urinary tract infection, the hpmA mutant strain WPM111 behaved no differently from its parent strain, BA6163, with respect to either the level of kidney colonization or histopathological changes in the kidney. However, WPM111 had a sixfold higher 50% lethal dose than BA6163 when injected intravenously into C3H mice.
Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling
Authors: Galán J E , Curtiss R
Published: January 2020

The ability to enter intestinal epithelial cells is an essential virulence factor of salmonellae. We have previously cloned a group of genes (invA, B, C, and D) that allow S. typhimurium to penetrate tissue culture cells (J. E. Galán and R. Curtiss III, Proc. Natl. Acad. Sci. USA 86:6383-6387, 1989). Transcriptional and translational cat and phoA fusions to invA (the proximal gene in the invABC operon) were constructed, and their expression was studied by measuring the levels of alkaline phosphatase or chloramphenicol acetyltransferase activity in mutants grown under different conditions. It was found that when strains containing the fusions were grown on media with high osmolarity, a condition known to increase DNA superhelicity, the level of invA transcription was approximately eightfold higher than that in strains grown on media with low osmolarity. The osmoinducibility of invA was independent of ompR, which controls the osmoinducibility of other genes. Strains grown in high-osmolarity media in the presence of subinhibitory concentrations of gyrase inhibitors (novobiocin or coumermycin A1), which reduce the level of DNA supercoiling, showed reduced expression of invA. Nevertheless, invA was poorly expressed in topA mutants of S. typhimurium, which have increased DNA superhelicity. In all cases, the differential expression of the invasion genes was correlated with the ability of S. typhimurium to penetrate tissue culture cells. These results taken together indicate that expression of S. typhimurium invasion genes is affected by changes in DNA supercoiling and suggest that this may represent a way in which this organism regulates the expression of these genes.
Characteristics of Candida albicans adherence to mouse tissues
Authors: Cutler J E , Brawner D L , Hazen K C , Jutila M A
Published: January 2020

An ex vivo binding assay originally described for determining lymphocyte homing receptors was adapted for studying Candida albicans-host cell interactions in unfixed tissue sections. BALB/cByJ mice were sacrificed, and various organs were removed, rapidly frozen on dry ice, and sectioned. C. albicans yeast cells were suspended to 1.5 x 10(8) cells per ml in Dulbecco modified Eagle medium supplemented with 5% newborn calf serum, and 100 microliters of the suspension was added to tissue sections for 15 min with rotation at 4 degrees C or at 22 to 24 degrees C. The sections were then fixed in glutaraldehyde, washed, and examined. Stationary-phase yeast cells adhered better than log-phase cells, and adherence characteristics were similar at 4 degrees C and 22 to 24 degrees C. Yeast cells from nine strains of C. albicans showed similar tissue specificity. Adherence to lymph node tissue was confined to subcapsular spaces and trabecular sinuses. In the spleen, yeast cells bound to the marginal zones. In both tissues, an association of yeast cells with tissue macrophages was suggested by results with macrophage-specific monoclonal antibodies and fluorescent or immunoperoxidase staining techniques. C. albicans adhered to convoluted tubules, glomeruli, and the tunica media of arterioles in the kidney. During experimentally induced fungemia in mice, C. albicans yeast cells associated with the same tissue sites as in the ex vivo assay, except that binding to renal arterioles was not seen in the in vivo test. A strain of Saccharomyces cerevisiae showed some adherence patterns in common with C. albicans, which indicates that tissue adherence is not sufficient for virulence. Mechanisms of attachment were not determined, but strains of C. albicans varied quantitatively in their ability to attach, and binding was inhibited by chelators of divalent cations.
Failure to demonstrate a major role for Kupffer cells and radiosensitive leukocytes in immunoglobulin-mediated elimination of Trypanosoma musculi
Authors: Kongshavn P A , Shaw K , Ghadirian E , Ulczak O
Published: January 2020

Previous studies have indicated that elimination of parasitemia in Trypanosoma musculi infection is brought about by immunoglobulin G2a antibodies, C3, and an effector cell. Experiments were designed to identify the putative effector cell by using several approaches. Infected C5-deficient or C5-sufficient mice treated with silica particles or given 900 rads of radiation 3 days earlier effectively eliminated trypanosomes following administration of immune plasma (IP). Silica-treated, noninfected mice given T. musculi preincubated with IP also cleared the parasites. Radiolabeling studies revealed that uptake of the cleared trypanosomes by the liver in normal mice was relatively low (24%) and fell only slightly (19%) in silica-treated mice. In contrast, uptake of radiolabeled sheep erythrocytes by the liver was normally much higher (47%) and fell drastically (7%) in silica-treated mice. Mice were then immunocompromised by 900 rads of radiation, silica particles, and anti-platelet serum combined before IP-sensitized trypanosomes were given. Leukocyte and platelet counts were both reduced by 95% and sheep erythrocyte uptake by the liver fell from 77 to 5%; however, greater than 99% of the injected trypanosomes were cleared in these mice and uptake of radiolabeled trypanosomes by the liver was similar to that of normal mice. Lastly, in anesthetized mice in which Kupffer cells were excluded surgically from the circulation, greater than 99% of the IP-sensitized trypanosomes disappeared rapidly from the blood. Only 7% of the radiolabel was found in the liver versus 60% in sham-operated mice. The results are interpreted as showing that hepatic Kupffer cells play a minor role in the immune elimination of T. musculi. Likewise, radiosensitive leukocytes and platelets are unlikely to be sole candidates for the putative effector cell that mediates a cure of murine trypanosomiasis.
Restriction fragment length polymorphism in the cytadhesin P1 gene of human clinical isolates of Mycoplasma pneumoniae
Authors: Dallo S F , Horton J R , Su C J , Baseman J B
Published: January 2020

Genomic DNA obtained from Mycoplasma pneumoniae clinical isolates spanning a 30-year period was analyzed for the presence of polymorphism in their P1 cytadhesin genes. All clinical isolates expressed a 170-kilodalton P1 protein that reacted with anti-P1 monoclonal antibodies. However, Southern blot analysis of specific M. pneumoniae isolates with subclones of the P1 structural gene revealed the presence of restriction fragment length polymorphism, permitting the classification of their P1 genes into two distinct categories.
The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope
Authors: Stover C K , Marana D P , Carter J M , Roe B A , Mardis E , Oaks E V
Published: January 2020

Lasting immunity against Rickettsia tsutsugamushi, the causative agent of scrub typhus fever, has been demonstrated to be strain specific. Two protein antigens of 110 and 56 kilodaltons (kDa) have been shown to exhibit strain-specific epitopes. The 56-kDa scrub typhus antigen (Sta56) is an abundant outer membrane protein of R. tsutsugamushi and is an antigen often recognized by humans infected with this obligate intracellular bacterium. In this study the complete gene encoding Sta56 (strain Karp) was cloned into pBR322 on a 2.3-kilobase genomic HindIII DNA fragment and the complete 56-kDa polypeptide was expressed in Escherichia coli. DNA sequence analysis of the 2.3-kilobase HindIII fragment revealed an open reading frame large enough to encode a 56-kDa polypeptide. A putative signal sequence was identified at the deduced amino terminus of the Sta56 polypeptide, and pulse-chase analysis of maxicells labeled with [35S]methionine demonstrated that a higher-molecular-weight precursor matures into the 56-kDa polypeptide. Epitope scanning analysis with synthetic peptides derived from the deduced amino acid sequence identified an octapeptide (located from amino acid residues 117 to 124) that was reactive with a Karp strain-specific monoclonal antibody (K13F88A). Other epitopes recognized by different monoclonal antibodies, including another Karp strain-specific monoclone (K1E106), were localized to different regions of the protein based on their reactivities with lambda gt11 recombinants expressing various portions of the sta56 gene.
Cryptococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipid lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), a possible adhesion receptor for yeasts
Authors: Jimenez-Lucho V , Ginsburg V , Krivan H C
Published: January 2020

The role of glycosphingolipids as adhesion receptors for yeasts was examined. Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae, as well as Histoplasma capsulatum and Sporotrichum schenckii (in their yeast phases), bound specifically to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), as measured by overlaying glycosphingolipid chromatograms with 125I-labeled organisms. An unsubstituted galactosyl residue was required for binding, because the yeasts did not bind to glucosylceramide (Glc beta 1-1Cer) derived from lactosylceramide by treatment with beta-galactosidase or to other neutral or acidic glycosphingolipids tested that contained internal lactosyl residues. Interestingly, the yeasts preferentially bound to the upper band of the lactosylceramide doublet in human lung and bovine erythrocytes, suggesting that the ceramide structure also affects binding. Active metabolism of the yeasts was required for binding to lactosylceramide, as binding was maximal in buffer containing glucose and was almost completely abolished in nutrient-deficient medium. C. neoformans also bound to human glioma brain cells grown in monolayers, and this binding was inhibited by liposomes containing lactosylceramide but not by liposomes containing glucosylceramide. Lactosylceramide is a major glycosphingolipid in these cells and the only one to which the yeasts bound. As lactosylceramide is widely distributed in epithelial tissues, this glycosphingolipid may be the receptor for yeast colonization and disseminated disease in humans.