bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and expression of the gene encoding the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase of Streptococcus mutans
Authors: Hillman J D , Duncan M J , Stashenko K P
Published: January 2020

The fructose-1,6-diphosphate-dependent lactate dehydrogenase from Streptococcus mutans JH1000 was purified by a modification of published methods. The sequence of 27 amino-terminal amino acids was determined, which allowed us to construct a 17-base DNA probe that had 32-fold degeneracy. The probe was used to screen a genomic library in pBR322. Of 18 reactive clones, 1 was found that expressed lactate dehydrogenase (LDH) activity identical to that of S. mutans with regard to dependence on fructose-1,6-diphosphate, thermal inactivation profile, and inhibition by sodium oxamate. Extracts of this clone possessed a protein band that comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with purified LDH from JH1000. Compared with controls, the clone was shown to produce elevated amounts of L-(+)-lactic acid during growth in the presence of glucose, thereby indicating that the activity was expressed in vivo. This result was substantiated by demonstrating that the activity could complement a mutation in the fermentative D-(-)-LDH of Escherichia coli. Subcloning showed that the S. mutans LDH subunit is encoded by a 1.2-kilobase gene. Our ability to clone this gene is expected to have great practical significance in the construction of an effector strain for use in the replacement therapy of dental caries.
Characterization of Pseudomonas aeruginosa adherence to mouse corneas in organ culture
Authors: Singh A , Hazlett L D , Berk R S
Published: January 2020

The present study was designed to obtain further information on the nature of the corneal macromolecule(s) to which Pseudomonas aeruginosa adheres and how adherence might be prevented. Scarified adult mouse corneas in organ culture were treated with trypsin or lipase to determine whether the receptor molecule(s) was protein or lipid in nature. Trypsin (20 micrograms/ml) treatment of the cornea for 5 min had no significant effect on bacterial adherence, and longer periods of enzyme exposure resulted in extensive surface cell lysis. In contrast, lipase treatment (50,000 U/ml) for 1 h caused little visible cell lysis and significantly reduced bacterial adherence. To test further the lipid nature of the receptor, a highly purified monosialoganglioside (GM1) preparation (500 micrograms/ml) was used to preincubate (1 h) the cornea prior to bacterial application, and this also inhibited bacterial adherence. Similar corneal treatment with gangliotetraosylceramide (asialo GM1) (500 micrograms/ml) had little effect on ocular bacterial binding. Premixing of the bacterial inoculum with GM1 prior to corneal application had no significant effect on inhibiting bacterial binding, but similarly premixing the bacterial inoculum with asialo GM1 transiently decreased adherence. Lastly, premixing of the bacterial inoculum or preincubation of corneas with fibronectin (500 micrograms/ml for 1 h) both decreased bacterial adherence. These findings provide evidence that the receptor-adhesin interactions of P. aeruginosa at the ocular surface in organ culture are complex, involve a glycolipid moiety, and may be blocked by a ganglioside containing at least one sialosyl residue or by fibronectin, which may bind to membrane-associated gangliosides.
Morphological evidence for penetration of anti-O antibody through the capsule of Klebsiella pneumoniae
Authors: Meno Y , Amako K
Published: January 2020

The role of the capsule in the reaction with anti-O or anti-K serum was examined morphologically by the techniques of freeze-substitution and immunoelectron microscopy in two strains of Klebsiella pneumoniae differing in virulence for mice. Strain Chedid (O1:K2), an encapsulated virulent strain, has a thicker capsule (150 nm) than the encapsulated avirulent strain 277 (O1:K2) (60 nm). Two morphologically recognizable domains in the capsule created by the arrangement of the capsular filaments were clearly seen in strain Chedid but were less evident in strain 277. Anti-O antibody could penetrate through the capsular layer of both strains. The capsule has no function as a barrier for the penetration of the antibody. Anti-K antibody reacted only on the surface of the capsule and induced swelling of the capsule.
Production, characterization, and species specificity of monoclonal antibodies to Mycobacterium avium complex protein antigens
Authors: Rouse D A , Morris S L , Karpas A B , Probst P G , Chaparas S D
Published: January 2020

The incidence of Mycobacterium avium-Mycobacterium intracellulare complex infections has increased in recent years primarily because a significant proportion of acquired immunodeficiency syndrome patients develop disseminated M. avium complex disease. In an effort to develop new tools to study these infections, we have produced eight monoclonal antibodies directed against M. avium. Western blot (immunoblot) specificity analysis and protease sensitivity assays indicate that four of these antibodies recognize M. avium-specific protein epitopes and two react with M. avium complex-specific peptide determinants. These monoclonal antibodies may be useful clinically in the diagnosis of M. avium complex disease and in the laboratory for isolation and characterization of native and recombinant M. avium complex antigens.
Inhibition of Trichomonas vaginalis motility by monoclonal antibodies is associated with reduced adherence to HeLa cell monolayers
Authors: Krieger J N , Torian B E , Hom J , Tam M R
Published: January 2020

Adherence of trichomonads to host epithelial cells appears to be a critical step in the pathogenesis of trichomoniasis. We evaluated the effect of a panel of 10 monoclonal antibodies on attachment of [35S]methionine-radiolabeled Trichomonas vaginalis strains to HeLa cell monolayers. Of 10 monoclonal antibodies, 3 totally eliminated motility of PHS2J strain trichomonads and reduced their adherence to 48 to 60% of control values (P less than 0.001). However, none of the monoclonal antibodies affected motility or adherence of STD13 strain trichomonads. Although the antibodies all reacted with PHS2J trichomonads by immunofluorescence, there was no correlation between inhibition of adherence and findings on either immunofluorescence or radioimmunoprecipitation. Direct microscopic observations showed that incubation with the monoclonal antibodies did not cause cytolysis of T. vaginalis. In quantitative cultures there was no difference in the number of colonies produced by parasites that had been incubated with antibodies that inhibited or had no effect on adherence. We conclude that our monoclonal antibodies reduced adherence not by cytotoxic effects or by competing for specific sites mediating adherence of the protozoa, but by inhibiting motility of T. vaginalis.
Characterization of the pilin gene of Moraxella bovis Dalton 2d and expression of pili from M. bovis in Pseudomonas aeruginosa
Authors: Elleman T C , Hoyne P A , Lepper A W
Published: January 2020

The pilin gene of Moraxella bovis Dalton 2d was isolated by cloning in Pseudomonas aeruginosa. The nucleotide sequence of this gene encodes a prepilin of 156 amino acid residues. When high levels of pilin were expressed from the gene in P. aeruginosa, by using the pL promoter of bacteriophage lambda inserted upstream of the coding sequence, pili which were indistinguishable from pili of M. bovis were produced.
Immune and nonimmune regulation of the population of Trypanosoma musculi in infected host mice
Authors: Albright J W , Pierantoni M , Albright J F
Published: January 2020

A clear understanding of the population dynamics of trypanosome infections is lacking. In the case of murine Trypanosoma musculi infections, there are no answers to questions concerning (i) the nature of the prolonged plateau phase during which the number of parasites present in the host remains nearly constant (is it a static or dynamic steady state?); (ii) the origin of new parasites, if the plateau is a dynamic steady state, given the relatively early disappearance of generative forms from the bloodstream; and (iii) the role, if any, of a putative ablastin (reproduction-restricting antibody) in regulating the population dynamics of T. musculi infections. We describe here the results of studies of the number and distribution of mature and reproductive forms (RF) in the blood and peritoneal space of both immunocompetent and cyclophosphamide-treated mice throughout the course of infection. While the RF disappeared from the blood within a few days after parasite inoculation, a high fraction (20 to 30%) of the parasites in the peritoneal space were RF throughout the course of infection and, thus, represented a source of new parasites. If an ablastin is responsible for inhibiting RF in the blood, it appeared to have no effect on RF in the peritoneal space. The results of this investigation support the conclusion that the control of the dynamics of T. musculi infections is largely nonimmunological (until cure of the infection) and probably is exercised by the supply of nutrients and reproduction-inhibiting (nonimmunological) and maturation-promoting factors that affect the generative fraction of the population.
Pathogenic and nonpathogenic strains of Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin
Authors: Petri W A , Jackson T F , Gathiram V , Kress K , Saffer L D , Snodgrass T L , Chapman M D , Keren Z , Mirelman D
Published: January 2020

Entamoeba histolytica infection results in either asymptomatic colonization or invasive colitis and liver abscess. E. histolytica isolates from patients with invasive disease have characteristic isoenzyme profiles (pathogenic zymodemes), suggesting a role for parasite factors in determining the severity of infection. A galactose-specific cell surface lectin from a pathogenic zymodeme was shown to mediate in vitro adherence to human colonic mucins and contact-dependent killing of target cells. Six nonoverlapping antigenic determinants were identified on the 170-kilodalton heavy subunit of the pathogenic lectin. Anti-lectin monoclonal antibodies (MAb) directed against epitopes 1 and 2 enhanced adherence whereas MAb to epitopes 3 through 6 either inhibited or had no effect on adherence. We tested 50 pathogenic and nonpathogenic strains for reactivity to these anti-lectin MAb by radioimmunoassay. MAb to epitopes 1 through 6 reacted in the radioimmunoassay with all 16 pathogenic zymodeme strains tested. In contrast, only MAb to epitopes 1 and 2 bound to the lectin from nonpathogenic strains. Western immunoblots with anti-lectin antibodies showed that the 170-kilodalton heavy subunit was present in the nonpathogenic amebae. Adherence of the nonpathogenic SAW 760 strain to human erythrocytes was enhanced by MAb to epitope 1 and blocked by galactose, confirming the presence of a functionally active lectin. A lectin radioimmunoassay based on MAb to epitopes 1 and 3 proved to be a simple and rapid method to distinguish pathogenic from nonpathogenic amebae in culture. Further exploration of the functional consequences of the antigenic differences demonstrated for the lectin may lead to a better understanding of its role in pathogenesis.
Heterogeneity of intestinal receptors for Escherichia coli heat-stable enterotoxin
Authors: Ivens K , Gazzano H , O'Hanley P , Waldman S A
Published: January 2020

The structure of rat intestinal cell receptors for Escherichia coli heat-stable enterotoxin (ST) was investigated by affinity cross-linking to 125I-ST and analysis by denaturing gel electrophoresis. Cross-linking of labeled toxin to intestinal membranes and analysis by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed five specifically labeled proteins with molecular masses of 160, 136, 78, 71, and 56 (kilodaltons) kDa. Exhaustive reduction of these samples resulted in a similar pattern of labeling. Affinity-labeled proteins were further analyzed by nonreducing SDS-PAGE, reduction of the resulting separated proteins, and further separation by SDS-PAGE in the presence of beta-mercaptoethanol. Thus, the 160-kDa band on nonreducing gels consisted of two different receptors: a 160-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 78-kDa subunit. Similarly, the 136-kDa band on nonreducing gels consisted of a 136-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 71-kDa subunit. The 78-, 71-, and 56-kDa subunits were not further reducible. These data suggest heterogeneity of the ST receptor subunit structure and organization in rat intestinal epithelia.
Identification of lambda gt11 clones encoding the major antigenic determinants expressed by Theileria parva sporozoites
Authors: Iams K P , Hall R , Webster P , Musoke A J
Published: January 2020

An antiserum, C16, was raised in cattle against freeze-thawed extracts of sporozoites of Theileria parva (Muguga). This antiserum, which neutralizes sporozoite infectivity in vitro, identified theileria-specific antigens having approximate molecular masses of 105, 90, 85, 69, 67, 52, 47, and 43 kilodaltons (kDa) on Western blots (immunoblots) of infected tick salivary gland extracts. The antiserum was used to screen an expression library of T. parva (Muguga) genomic DNA fragments. Three recombinant bacteriophage clones carrying different theileria DNA inserts were studied. The expressed gene product from each clone was used to affinity purify antibodies from C16 antiserum for use in probing Western blots of uninfected and infected tick salivary gland extracts. The population of antibodies selected by each clone specifically recognized a subset of the antigens identified by C16 antiserum. The antigens fell into three distinct groups as defined by their reactivity with each set of selected antibodies. One group included antigens of 105, 90, 85, and 35 kDa, a second group included antigens of 69, 67, 52, 47, and 43 kDa, and the third group included an apparently distinct pair of antigens of 47 and 43 kDa. Thus, antibodies that reacted with determinants encoded by the three recombinant phage clones recognized all of the major antigens seen on Western blots probed with whole C16 antiserum. These results suggest that there may be only three immunodominant antigens expressed in T. parva (Muguga) sporozoites. Additionally, monoclonal antibodies have been raised which neutralize sporozoite infectivity in vitro. These antibodies react with epitopes of the antigens with Mrs of 69,000, 67,000, 52,000, 47,000, and 43,000 which are encoded in clone pgT-42 and have been used to localize these epitopes on the sporozoite surface.
Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide
Authors: Schlageter A M , Kozel T R
Published: January 2020

A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgG1) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgG1, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a greater than IgG1 much greater than IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a greater than IgG1 greater than IgG2b.
Liposomes containing anti-idiotypic antibodies: an oral vaccine to induce protective secretory immune responses specific for pathogens of mucosal surfaces
Authors: Jackson S , Mestecky J , Childers N K , Michalek S M
Published: January 2020

By using a gnotobiotic rat model system to study the induction of protective immune responses by anti-idiotype (anti-id) vaccines specific for antibodies directed at the cariogenic microorganism Streptococcus mutans, it was shown that administration of such an anti-id vaccine provided partial protection against dental caries after challenge with virulent microorganisms. Protective effects were first demonstrated by direct parenteral administration of the anti-id vaccine into salivary gland regions, as determined by reductions in microbial colonization and caries scores. Subsequently, the anti-id was incorporated into liposomes and administered by gastric intubation. Immunization by this regimen also resulted in a significant reduction in caries as well as S. mutans colonization of the oral cavity, with concomitant increases in salivary immunoglobulin A anti-S. mutans antibodies. These data provide evidence that anti-id vaccines specifically targeted at the secretory immune system can induce protective immune responses to pathogens of mucosal surfaces.
The gene coding for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria monocytogenes
Authors: Köhler S , Leimeister-Wächter M , Chakraborty T , Lottspeich F , Goebel W
Published: January 2020

The gene of Listeria monocytogenes that encodes a major extracellular protein (p60) was cloned in Escherichia coli. The gene was designated iap, as p60 was previously shown to represent an invasion-associated protein (M. Kuhn and W. Goebel, Infect. Immun. 57:55-61, 1989). The recombinant E. coli clone expressed p60, as shown by immunoblotting. The complete nucleotide sequence of iap was determined. The deduced amino acid sequence of p60 (484 amino acids) contains a putative N-terminal signal sequence of 27 amino acids and an extended repeat region consisting of 19 threonine-asparagine units. Hybridization with the entire iap gene revealed the presence of homologous sequences in most other Listeria species. In contrast, a 400-base-pair internal iap probe which contained the whole repeat region hybridized only with genomic DNA from L. monocytogenes. Four oligonucleotides previously described as specific probes for the detection of L. monocytogenes (A. R. Datta, B. A. Wentz, D. Shook, and M. W. Trucksess, Appl. Environ. Microbiol. 54:2933-2937, 1988) were shown to be part of the iap gene.
Effects of anti-inflammatory agents on mucosal inflammation induced by infection with gram-negative bacteria
Authors: Linder H , Engberg I , van Kooten C , de Man P , Svanborg-Edén C
Published: January 2020

Gram-negative bacterial infections of the urinary tract elicit a mucosal inflammatory response. Interleukin-6 is secreted into the urine, and polymorphonuclear leukocytes (PMNL) are recruited. In the present study we examined the effect of anti-inflammatory agents on these parameters and on bacterial clearance from the kidneys. Dexamethasone reduced interleukin-6 secretion, the PMNL response, and bacterial clearance. Diclofenac abolished the urinary interleukin-6 response but reduced the PMNL response and bacterial clearance only at the highest concentrations. Indomethacin drastically decreased bacterial clearance without the corresponding effect on interleukin-6 production or the PMNL response. The results demonstrate that the inhibition of inflammation impairs bacterial clearance from the kidneys. This is, however, not a direct function of inhibited interleukin-6 production or PMNL recruitment.
Functional analysis of the sialic acid-binding adhesin SfaS of pathogenic Escherichia coli by site-specific mutagenesis
Authors: Morschhäuser J , Hoschützky H , Jann K , Hacker J
Published: January 2020

The gene coding for the sialic acid-specific adhesin SfaS produced by the S fimbrial adhesin (sfa) determinant of Escherichia coli has been modified by oligonucleotide-directed, site-specific mutagenesis. Lysine 116, arginine 118, and lysine 122 were replaced by threonine, serine, and threonine, respectively. The mutagenized gene clusters were able to produce S fimbrial adhesin complexes consisting of the S-specific subunit proteins including the adhesin SfaS. The mutant clones were further characterized by hemagglutination and by enzyme-linked immunoassay tests with antifimbria- and anti-adhesin-specific monoclonal antibodies, one of which is able to block S-specific binding (Moch et al., Proc. Natl. Acad. Sci. USA 84:3462-3466, 1987). The lysine-122 mutant clone was indistinguishable from the wild-type clone in these assays. Replacement of lysine 116 and arginine 118, however, abolished hemagglutination and resulted in clones which showed a weak (lysine 116) or a negative (arginine 118) reaction with the antiadhesin-specific antibody A1. We therefore suggest that lysine 116 and arginine 118 have an influence on binding of SfaS to the sialic acid residue of the receptor molecule. Substitution of arginine 118 by serine also had a negative effect on the amount of SfaS adhesin proteins isolated from the S fimbrial adhesin complex.
Measurement of the human immune response to meningococcal lipooligosaccharide antigens by using serum to inhibit monoclonal antibody binding to purified lipooligosaccharide
Authors: Estabrook M M , Mandrell R E , Apicella M A , Griffiss J M
Published: January 2020

We developed a human inhibition monoclonal enzyme-linked immunosorbent assay (HIMELISA) to investigate the human immune response to the lipooligosaccharides (LOS) of Neisseria meningitidis. Monoclonal antibodies (MAb) were used to define seven epitopes on four LOS molecules of a meningococcal strain (126E) previously shown to express immunogenic LOS epitopes. The assay could distinguish epitope-specific antibody within whole sera. Neither the specificity nor the amount of the antibody measured by HIMELISA in sera of vaccinates changed during the immune response to meningococcal capsular polysaccharides, a chemically unrelated antigen. By using the HIMELISA, it was determined that sera from adults convalescing from meningococcal disease strongly inhibited MAb binding to two of the seven defined epitopes. The 3.6-kilodalton LOS of strain 126E expressed both of these epitopes. In addition, one of the inhibited epitopes was also expressed on the 4.0-kilodalton LOS of strain 126E. The convalescent-phase sera inhibited MAb binding to these two epitopes when they were expressed on LOS of diverse meningococcal strains. An acute-phase serum blocked MAb to the two epitopes to a lesser degree than did a convalescent-phase serum from the same patient. Immunoblotting the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated LOS with convalescent-phase sera confirmed the specificity of the human anti-LOS antibody identified by HIMELISA.
Partial purification and characterization of the enterotoxin produced by Campylobacter jejuni
Authors: Daikoku T , Kawaguchi M , Takama K , Suzuki S
Published: January 2020

Campylobacter jejuni enterotoxin was partially purified from culture supernatant. The purified fraction after gel filtration indicated three bands at 68, 54, and 43 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This fraction enhanced the adenylate cyclase activity of HeLa cell membranes by 1.5-fold over that of the control. The study with anti-cholera toxin immunoglobulin G (IgG) and ganglioside affinity column chromatographies revealed that the eluent from the anti-cholera toxin IgG column chromatography exhibited a single band (68 kDa) on SDS-PAGE and native PAGE, whereas the eluent from ganglioside column chromatography exhibited two bands (68 and 54 kDa) on SDS-PAGE. These suggest that the 68-kDa polypeptide should have an immunological relationship with cholera toxin, and the 68- and 54-kDa polypeptides might be responsible for the recognition of ganglioside.
The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing
Authors: Szeto L , Shuman H A
Published: January 2020

The Legionella pneumophila major secretory protein (Msp) is a Zn2+ metalloprotease whose function in pathogenesis is unknown. The structural gene for the Msp protease, mspA, was isolated from an L. pneumophila genomic library. In Escherichia coli which contain plasmids with the mspA gene, Msp protein and activity are found in the periplasmic space and the cytoplasm. Transposon mutagenesis with Tn9 of an mspA-containing plasmid in E. coli yielded mutants which no longer expressed protease activity and others with increased protease activity. These results suggested that mspA expression might be regulated. Msp was shown to be produced at a much higher level in L. pneumophila grown in rich compared to semidefined media. A Tn9 insertion which abolishes Msp expression was introduced into the L. pneumophila genome. This mspA::Tn9 L. pneumophila strain showed no detectable production of Msp by immunoblot analysis, and it had less than 0.1% of the protease activity found in the wild-type strain. This mutant was fully capable of growing within and killing human macrophages derived from the HL-60 cell line.
Immunization against chlamydial genital infection in guinea pigs with UV-inactivated and viable chlamydiae administered by different routes
Authors: Rank R G , Batteiger B E , Soderberg L S
Published: January 2020

Female guinea pigs were immunized with viable or UV light-inactivated chlamydiae (agent of guinea pig inclusion conjunctivitis), belonging to the species Chlamydia psittaci, by intravenous, subcutaneous, oral, or ocular routes. All animals were then inoculated vaginally with viable chlamydiae to determine the extent of protection against challenge infection induced by the various regimens. The course of genital infection was significantly reduced in intensity in all groups of animals except the unimmunized controls and those animals immunized orally with inactivated antigen. Guinea pigs immunized with viable antigen were more likely to develop resistance to challenge infection and, in general, had a significantly greater degree of protection than animals immunized with inactivated antigen. No one route seemed superior in producing a protective response. Animals in all groups demonstrating protection developed serum and secretion immunoglobulin G antibody responses to chlamydiae. Lymphocyte proliferative reactions to chlamydial antigen were variable among groups. Immunoblot analysis of serum and secretions indicated a wide range of antibody specificities, but most protected animals produced antibodies to the major outer membrane protein, lipopolysaccharide, and the 61-kilodalton protein. No definitive associations could be made between the increased ability of immunization with viable organisms to produce resistance to challenge infection and a particular immune parameter. These data indicate that viable chlamydiae given by various routes are able to induce a strong immune response which can provide resistance against reinfection in some cases or at least reduce the degree of infection to a greater degree than inactivated antigen. However, complete resistance to genital tract infection may be difficult to obtain and alternate immunizations strategies may have to be developed.
Synthetic peptide segments from the Escherichia coli porin OmpF constitute leukocyte activators
Authors: Vordermeier H M , Hoffmann P , Gombert F O , Jung G , Bessler W G
Published: January 2020

After characterization of the porin OmpF and selection of molecular structures responsible for leukocyte activation by using computer-assisted epitope analysis, the analogs OmpF (153-174) (containing amino acids 153 to 174), OmpF (157-174), and OmpF (275-285) were synthesized and tested. Like the native protein, the segments were mitogenic for BALB/c splenocytes and induced B lymphocyte differentiation into antibody-producing plasma cells and tumor cytotoxicity of macrophages against the fibroblast cell line L929. We thus demonstrated that defined peptide segments are responsible for the leukocyte-activating properties of a major bacterial surface protein.
Production of immunoglobulin A protease by Streptococcus pneumoniae from animals
Authors: Proctor M , Manning P J
Published: January 2020

Human isolates of Streptococcus pneumoniae tested by traditional immunochemical methods produce a protease that cleaves human immunoglobulin A1 (IgA1) into Fab and Fc fragments. The protease may be an important virulence factor, but studies of its pathogenetic significance have been hampered by lack of a suitable animal model. Since S. pneumoniae is a respiratory pathogen for several species of animals, we sought to determine whether isolates of this organism from animals with pneumococcal infection, including fatal diplococcal pneumonia, produced an IgA protease. Isolates from six animal species including the mouse, rat, dog, guinea pig, rhesus monkey, and chimpanzee were tested for protease activity against IgA preparations from the mouse, rat, dog, guinea pig, rabbit, rhesus and cynomolgus monkeys, gorilla, and human. Cleavage of IgA was demonstrated by the appearance of Fc fragments in Western blots (immunoblots) treated with specific antisera. All these isolates except that from the guinea pig produced a protease that cleaved IgA of human, rhesus monkey, and gorilla origin. Cleavage was inhibited by 5 mM EDTA. IgA cleavage from the other species could not be demonstrated. Although S. pneumoniae can colonize the respiratory tracts of several animal species, it is a significant pathogen principally of humans and some other primates. Our data suggest that some species of nonhuman primates including the rhesus monkey could be suitable for experimental studies on the significance of IgA protease in the pathogenesis of pneumococcal disease.
Two soluble antigens of Plasmodium falciparum induce tumor necrosis factor release from macrophages
Authors: Taverne J , Bate C A , Kwiatkowski D , Jakobsen P H , Playfair J H
Published: January 2020

The production of cytokines such as tumor necrosis factor (TNF) may contribute to the pathology of malaria. We showed previously that crude preparations of heat-stable exoantigens from parasite cultures induce the release of TNF in vitro and in vivo. When separated from the culture medium by affinity chromatography, in which immune immunoglobulin G was used as ligand, the mixture of exoantigens of Plasmodium falciparum retained the capacity to induce the secretion of TNF, both by human monocytes from Gambian children and by mouse macrophages. Two individual antigens, Ag1 and Ag7, further purified by affinity chromatography and identified by crossed immunoelectrophoresis, also stimulated TNF production by both types of cell but differed in other functional properties. Thus, the activity of Ag7, but not that of Ag1, was inhibited by polymyxin B, and antisera made against boiled exoantigens of the rodent parasite Plasmodium yoelii which blocked the ability of these antigens to induce the production of TNF also inhibited the activity of Ag7 without affecting Ag1. Since the prevalence of antibody against Ag7 in sera from children in endemic areas appears to correlate with the development of immunity against the manifestations of the disease, this antigen may be one cause of pathology, perhaps through its ability to induce the production of TNF. Its serological relationship with rodent exoantigens suggests that it might be a candidate for an anti-disease vaccine which has the advantage that its active moiety is not subject to significant antigen polymorphism.
Virulence of a Legionella anisa strain associated with Pontiac fever: an evaluation using protozoan, cell culture, and guinea pig models
Authors: Fields B S , Barbaree J M , Sanden G N , Morrill W E
Published: January 2020

Legionella anisa and the amoeba Hartmannella vermiformis were isolated from an indoor fountain implicated as the infectious reservoir in an outbreak of Pontiac fever. We evaluated the ability of this strain of L. anisa to multiply in cultures of an amoeba (H. vermiformis), a ciliated protozoan (Tetrahymena pyriformis), and human mononuclear cells and to infect guinea pigs. These bacteria multiplied in the culture of H. vermiformis but failed to infect guinea pigs or the cultures of T. pyriformis and human mononuclear cells. These findings suggest that some Legionella spp. may multiply only in specific protozoan hosts. The inability of this strain of L. anisa to multiply in human phagocytic cells may be related to the development of Pontiac fever rather than pneumonic legionellosis in exposed individuals. Further studies are necessary to determine whether the ability of legionellae to infect certain host cells can be correlated to differences in human disease.
Cooperative complement- and bacterial lectin-initiated bactericidal activity of polymorphonuclear leukocytes
Authors: Kurashima C , Sandberg A L , Cisar J O , Mudrick L L
Published: January 2020

The recognition of glycoconjugate receptors on sialidase-treated polymorphonuclear leukocytes (PMNs) by the Gal/GalNAc-reactive fimbrial lectin of Actinomyces viscosus T14V has previously been shown to initiate lactose-inhibitable phagocytosis and subsequent killing of the bacteria. Although a mutant lacking fimbriae, A. viscosus 147, was not destroyed by this mechanism, the present studies demonstrate that the deposition of C3 fragments on this bacterium by anti-A. viscosus 147 immunoglobulin M (IgM) prior to incubation with either untreated or sialidase-treated PMNs correlated with a reduction in viability of approximately 2 log10. This bactericidal activity was unaffected by lactose. A similar decrease in viability was observed following the addition of untreated PMNs to A. viscosus T14V preincubated with anti-A. viscosus 147 IgM and complement, conditions favorable for C3- but not lectin-mediated bactericidal activity. Neither IgM nor complement alone was opsonic for either strain, and individually they did not alter killing of A. viscosus T14V by sialidase-treated PMNs or inhibition of this bactericidal activity by lactose. The number of viable A. viscosus T14V cells was decreased by approximately 3.5 log10 when the bacteria were incubated with IgM and complement prior to the addition of sialidase-treated PMNs, and lactose only partially inhibited this response. Thus, the PMN-dependent bactericidal activity initiated by the participation of both the actinomyces lectin and complement was significantly greater than that achieved by either ligand alone.
Properties of a rat monoclonal antibody reactive with both the mannan of Candida species and the O-antigen 6,7 polysaccharide of serogroup C1 salmonellae
Authors: Nnalue N A , Weintraub A , Lindberg A A
Published: January 2020

Monoclonal antibody MASC1-MR9 of isotype immunoglobulin M was generated in LOU/C rats by immunization with heat-killed Salmonella thompson (serogroup C1, O-antigen 6,7 lipopolysaccharide) bacteria which had been further enriched for O-antigen by coating with homologous lipopolysaccharide. Eight monoclonal antibodies were selected by screening against the lipopolysaccharide of S. thompson. In a subsequent test against Candida mannan, only one antibody, MASC1-MR9, was reactive. Immunofluorescence microscopy of various yeasts and bacteria with MASC1-MR9 showed that this antibody bound to the surfaces of 142 of 148 Candida strains of 10 different species tested but failed to bind to the surface of Saccharomyces cerevisiae. The Candida strains which failed to bind MASC1-MR9 were all five strains of Candida krusei and the single strain of Candida utilis tested. Several (11 of 33) Salmonella strains belonging to serogroup C1 reacted with this antibody, as expected; however, 1 of 11 strains of Morganella morganii and 4 of 64 strains of Escherichia coli were also reactive. Serum-free supernatants of MASC1-MR9 agglutinated S. thompson, one strain of Salmonella choleraesuis, and 109 of 110 Candida strains tested. The immunochemical properties of MASC1-MR9 as studied by enzyme-linked immunosorbent inhibition assays show that it recognizes an epitope present in the mannan of Candida species as well as in the O-6,7 antigen of Salmonella species.
Thrombospondin binds to Staphylococcus aureus and promotes staphylococcal adherence to surfaces
Authors: Herrmann M , Suchard S J , Boxer L A , Waldvogel F A , Lew P D
Published: January 2020

Bacterial adherence to surfaces is the determining first step in staphylococcal infections. Activated platelets mediate adherence of staphylococci to tissues during inflammation or infection; however, the molecular mechanisms of this interaction are not clearly understood. Thrombospondin, a large multifunctional glycoprotein, is the principal platelet-stored glycoprotein. It is secreted upon platelet activation and either bound to receptors on the platelet surface or released and incorporated into blood clots and extracellular matrices. To characterize thrombospondin binding to staphylococci, we incubated [125I]thrombospondin with Staphylococcus aureus Cowan 1 in the presence of albumin and separated bound and free thrombospondin by centrifugation. We found that binding was (i) specific, since it was up to 76% inhibitable and up to 60% reversible in the presence of a 100-fold excess of unlabeled thrombospondin, (ii) saturable, with an apparent dissociation constant (Kd) of 5.6 x 10(-9) M and a maximal number of 2,600 binding sites per microorganism, and (iii) Ca2+ dependent, since omission of this ion from the medium decreased significantly the binding capacity. The binding reaction was insensitive to previous trypsin treatment of bacteria, but it was strongly inhibited in the presence of heparin. Protein A-negative and -positive strains had similar binding characteristics. To determine the promotion of staphylococcal adherence to surfaces by solid-phase thrombospondin, we incubated 3H-labeled S. aureus Cowan 1 and 26 pathogenic staphylococcal isolates with thrombospondin-coated polymethylmethacrylate disks and found that adherence was significantly promoted as a function of adsorbed thrombospondin. These results indicate a role for thrombospondin as an important mediator of staphylococcal adherence to activated platelets, to blood clots, or to extracellular matrices in pyogenic infections.
Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene
Authors: Domann E , Leimeister-Wächter M , Goebel W , Chakraborty T
Published: January 2020

The entire nucleotide sequence of an open reading frame located immediately downstream of the listeriolysin gene from a virulent Listeria monocytogenes serotype 1/2a strain was determined. The product of the open reading frame was 510 amino acids with a predicted molecular weight of 57,400. The deduced amino acid sequence of this open reading frame is highly similar to that of a family of secreted metalloproteases produced by various members of the genus Bacillus, of which thermolysin is the prototype. Immunoblots performed with specific antisera raised against thermolysin from Bacillus stearothermophilus allowed the detection of a 60-kDa polypeptide, corresponding to the pro-form of the protease, in culture supernatants of L. monocytogenes strains. In maxicell experiments, Escherichia coli recombinants harboring this open reading frame also specifically directed production of a 60-kDa protein. Protease activity was low to undetectable in both Listeria strains and E. coli recombinants. This is due to lack of processing of the inactive pro-form of the protease to its mature active form in both species. We have designated this gene mpl for metalloprotease of L. monocytogenes. The gene was present only in pathogenic L. monocytogenes strains, in which it was physically linked to the listeriolysin gene.
Inhibition of Ehrlichia risticii infection in murine peritoneal macrophages by gamma interferon, a calcium ionophore, and concanavalin A
Authors: Park J , Rikihisa Y
Published: January 2020

Ehrlichia risticii incubated with mouse peritoneal macrophages elicited with thioglycolate broth survived and replicated, thereby allowing examination of the effects of several immunopotentiating agents. Treatment of the macrophages with recombinant murine gamma interferon (rMuIFN-gamma) in vitro at 1 day before or 3 h after infection made the macrophages resistant to infection with E. risticii, and macrophages treated with rMuIFN-gamma at 1 to 3 days after infection developed the capacity to eradicate intracellular E. risticii. Similar effects were seen with macrophages treated with the Ca2+ ionophore A23187 before or after E. risticii infection in vitro. Concanavalin A treatment before or 3 h after infection caused the macrophages to become resistant to infection with E. risticii but could confer neither ehrlichiacidal nor ehrlichiastic activity to them once infection had been established for more than 1 day. Bacterial products such as lipopolysaccharide and muramyl dipeptide were less or not at all effective, respectively, in conferring antiehrlichial activity to macrophages. Finally, protein kinase C activator, phorbol myristate acetate, and recombinant tumor necrosis factor did not induce any antiehrlichial activity in macrophages when the macrophages were treated either before or after infection.
Antimalarial antibodies of the immunoglobulin G2a isotype modulate parasitemias in mice infected with Plasmodium yoelii
Authors: White W I , Evans C B , Taylor D W
Published: January 2020

Previous studies have demonstrated the importance of antibodies in mediating immunity to malaria, but the relative contribution of the different immunoglobulin isotypes has not been assessed. In this study, hyperimmune plasma was generated against Plasmodium yoelii and separated by protein A-Sepharose chromatography into fractions containing immunoglobulin G1 (IgG1), IgG2a, IgG2b, or IgG3 antibodies and the remaining nonbinding plasma proteins, including IgM. Following concentration, the antimalarial titer of each isotypic fraction was approximately equivalent to the corresponding isotype in hyperimmune plasma. The isotypic fractions were passively transferred to BALB/c and outbred ICR mice prior to challenge with virulent P. yoelii 17XL and to CBA/CaJ mice challenged with avirulent P. yoelii 17XNL. Only mice receiving IgG2a antibodies experienced an altered course of infection. Immunoprecipitation studies showed that all four IgG isotypes appear to recognize a similar set of antigens. These results suggest that antimalarial antibodies of the IgG2a isotype play a dominant role in modulating P. yoelii parasitemias.
Characteristics of adherence of enteroaggregative Escherichia coli to human and animal mucosa
Authors: Yamamoto T , Endo S , Yokota T , Echeverria P
Published: January 2020

An Escherichia coli strain (serotype O127a:H2) that had been isolated from a child with diarrhea in Thailand and that was negative for the virulence factors of the four categories of diarrheagenic E. coli (enterotoxigenic, enteropathogenic, enteroinvasive, and enterohemorrhagic) and that showed an aggregative pattern of adherence to HeLa cells was investigated for adherence to native or Formalin-fixed human and animal mucosa. The hemagglutinating activity and adherence ability of the bacteria were resistant to D-mannose and were strictly regulated by environmental conditions. Genetic data supported the close relation between the hemagglutinating activity and adherence ability. In accordance with the adherence pattern on tissue-cultured cells, the bacteria adhered to human and animal mucosa, as evidenced by a direct gold-labeling analysis. In human intestines, Formalin-fixed mucous coatings, epithelial cells of colonic mucosa, epithelial cells of ileal single lymphoid follicles and Peyer's patches, and the absorptive cells of jejunal or ileal villi provided adherence targets. Adherence to M cells in the Peyer's patch-associated epithelium was also confirmed. The adherence levels to native jejunal or ileal human villi were low, as was the case with the corresponding Formalin-fixed villi. In human urinary tract, the superficial epithelial cells of both native and Formalin-fixed ureter provided striking adherence targets. In animal (porcine and rabbit) small intestines, the bacteria adhered to the native villi to a lesser extent than to the Formalin-fixed villi. The adherence levels were compared with those of enterotoxigenic E. coli with colonization factor antigen (CFA)/I pili or CFA/II pili. The data suggested unique mucosa adherence characteristics of the enteroaggregative E. coli strain. The possibility of the adherence ability as a virulence factor was discussed.
Characterization of bradyzoite-specific antigens of Toxoplasma gondii
Authors: Tomavo S , Fortier B , Soete M , Ansel C , Camus D , Dubremetz J F
Published: January 2020

Monoclonal antibodies that react specifically with bradyzoite antigens of Toxoplasma gondii were selected by differential immunofluorescence among hybridomas produced against these organisms. These antigens were further characterized by immunofluorescence on living bradyzoites and Western immunoblotting. Four pellicular antigens (36, 34, 21, and 18 kDa) were identified; three of these are exposed on the surface of the organism and accessible to either antibodies, trypsin cleavage, or both of these surface-probing procedures. These antigens were found on recent human isolates of T. gondii, as well as on laboratory strain bradyzoites obtained from either brain cysts or in vitro-grown parasites.
Direct expression of Bordetella pertussis filamentous hemagglutinin in Escherichia coli and Salmonella typhimurium aroA
Authors: Guzmán C A , Walker M J , Rohde M , Timmis K N
Published: January 2020

Nonfused (i.e., nonhybrid) filamentous hemagglutinin (FHA) of Bordetella pertussis was efficiently expressed in Escherichia coli K-12 and Salmonella typhimurium aroA at levels higher than those found in wild-type B. pertussis when the upstream signals of the gene were replaced and the translation initiation region was engineered to optimize translational efficiency. Inclusion of part of the C-terminal FHA open reading frame, whose translation product does not appear to be part of the major secreted species of FHA, was shown to be important in achieving protein expression in both E. coli and S. typhimurium aroA; removal of the downstream gene sequence abolished recombinant FHA production. The levels of expression observed varied widely according to the construct and host bacterium used.