bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of GP110, a neutrophil surface protein
Authors: Tripathi S C , Van Dyke T E
Published: January 2020

Localized juvenile periodontosis is associated with a defect of neutrophil chemotaxis that is characterized by selective depletion of a surface protein with a molecular mass of 110 kDa (GP110). Data on partial characterization of GP110 suggest that it is a glycoprotein which is enriched in Lys, Glu, His, Leu, and Ala residues.
A 66-kilodalton heat shock protein of Salmonella typhimurium is responsible for binding of the bacterium to intestinal mucus
Authors: Ensgraber M , Loos M
Published: January 2020

Salmonella typhimurium infections have increased during the last few years. However, the interplay of virulence factors in S. typhimurium pathogenesis is still poorly understood, particularly with regard to the mechanisms and components of the bacterium which are involved in its interaction with the intestinal mucus. We have observed that S. typhimurium is aggregated by incubation with colonic mucus (guinea pig model). To quantify this phenomenon, an aggregation assay was established. By using this assay, it was found that the aggregation profile of S. typhimurium strains freshly isolated from patients (age 9 and older) with salmonellosis correlated with the severity of the disease. An isolate with high aggregation behavior was chosen for characterization of the bacterial component involved in binding to colonic mucus material. The component of S. typhimurium responsible for aggregation was purified and characterized as a 66-kDa protein which was able to completely inhibit mucus-mediated bacterial aggregation. This protein was recognized by monoclonal antibodies against the 65-kDa heat shock protein (HSP) of Mycobacterium leprae. The 66-kDa protein of S. typhimurium was inducible by incubating the bacteria at 50 degrees C and was secreted into the supernatant, from which it could be isolated in both dimeric and polymeric forms. The monoclonal anti-HSP 65, as well as a polyclonal antibody against the 66-kDa protein of S. typhimurium, caused dose-dependent inhibition of the aggregation of S. typhimurium by crude mucus preparations. This is the first report showing that a bacterial HSP is involved in mucus-mediated interaction of pathogens with the host.
Modulation of tumor necrosis factor production by macrophages in Entamoeba histolytica infection
Authors: Wang W , Keller K , Chadee K
Published: January 2020

The macrophage-derived mediator tumor necrosis factor alpha (TNF) is a cytokine with pleiotropic effects. TNF exhibits potent immunologic and inflammatory properties in parasitic diseases. The present study examined the production of TNF by macrophages isolated from gerbils infected with Entamoeba histolytica and by naive macrophages in response to amoebae in vitro. Amoebic liver abscess-derived macrophages produced low constitutive basal levels of TNF; in response to lipopolysaccharide (LPS) stimulation, TNF production was enhanced by 14-, 11-, and 6-fold at 10, 20, and 30 days postinfection, respectively. Amoebic liver abscess-derived macrophages pretreated with either recombinant gamma interferon (IFN-gamma) or the cyclooxygenase inhibitor indomethacin augmented TNF production in response to soluble amoebic proteins and LPS. Kupffer cells and peritoneal and spleen macrophages from infected animals did not release TNF constitutively in vitro. However, TNF production in response to LPS stimulation was significantly higher at 10 and 20 days postinfection. Macrophages from infected and naive animals pretreated with recombinant IFN-gamma or indomethacin produced increased amounts of TNF in response to LPS but not in response to soluble amoebic protein stimulation. Pretreatment of naive macrophages with amoebic proteins inhibited LPS-induced TNF production by 69 to 79%; the effect of the amoebic proteins was partially reversed by indomethacin pretreatment. In contrast, IFN-gamma- and LPS-activated naive macrophages produced enhanced levels of TNF in response to live amoebae and soluble amoebic proteins. Our results demonstrate that TNF production by macrophages is altered during E. histolytica infection and in response to amoebae and suggest a role for IFN-gamma and prostaglandin E2 in regulating TNF production during the infection.
Cloning, expression, and sequencing of a protease gene (tpr) from Porphyromonas gingivalis W83 in Escherichia coli
Authors: Bourgeau G , Lapointe H , Péloquin P , Mayrand D
Published: January 2020

Porphyromonas gingivalis is a highly proteolytic organism which metabolizes small peptides and amino acids. Indirect evidence suggests that the proteases produced by this microorganism constitute an important virulence factor. In this study, a gene bank of P. gingivalis W83 DNA was constructed by cloning 0.5- to 20-kb HindIII-cut DNA fragments into Escherichia coli DH5 alpha by using the plasmid vector pUC19. A clone expressing a protease from P. gingivalis was isolated on LB agar containing 1% skim milk. The clone contained a 3.0-kb insert that coded for a protease with an apparent molecular mass of 64 kDa. Sequencing part of the 3.0-kb DNA fragment revealed an open reading frame encoding a protein of 482 amino acids with a molecular mass of 62.5 kDa. Putative promoter and termination elements flanking the open reading frame were identified. The activity expressed in E. coli was extensively characterized by using various substrates and protease inhibitors, and the results suggest that it is possibly a thiol protease.
Synthesis, characterization, and some immunological properties of conjugates composed of the detoxified lipopolysaccharide of Vibrio cholerae O1 serotype Inaba bound to cholera toxin
Authors: Gupta R K , Szu S C , Finkelstein R A , Robbins J B
Published: January 2020

Protection against cholera has been correlated with the level of serum vibriocidal antibodies. The specificity of these vibriocidal antibodies was mostly to the lipopolysaccharide (LPS). We synthesized conjugates of detoxified LPS with cholera toxin (CT) and other proteins in order to elicit serum LPS antibodies with vibriocidal activity. Treatment with hydrazine (deacylated LPS) reduced the endotoxic properties of the LPS to clinically acceptable levels and resulted in a molecule larger and more antigenic than the saccharide produced by acid hydrolysis. More immunogenic conjugates resulted from multipoint compared with single-point attachment of the deacylated LPS to the protein. The conjugates containing CT had low levels of pyrogen and no toxic activity upon Chinese hamster ovary cells and elicited booster responses of vibriocidal and CT antibodies when injected subcutaneously as saline solutions into mice; the vibriocidal titers were similar to those elicited by comparable doses of cellular vaccines. We suggest how serum vibriocidal antibodies might prevent cholera.
Membrane translocation of diphtheria toxin carrying passenger protein domains
Authors: Madshus I H , Olsnes S , Stenmark H
Published: January 2020

For diphtheria toxin to be cytotoxic, the enzymatically active part (fragment A) must be translocated to the cytosol. We here demonstrate that additional proteins linked as N-terminal extensions can be translocated along with fragment A across the plasma membrane of toxin-sensitive cells. Thus, an extra fragment A of diphtheria toxin and some of apolipoprotein AI were translocated as passenger proteins along with mutant diphtheria toxin fragment A. Translocation was monitored by the cytotoxic effect of the additional fragment A as well as by the translocation of [35S]methionine-labelled protein to a compartment protected from externally added pronase. Cytotoxicity experiments indicated that double A fragments can also be translocated across the membrane of intracellular vesicles. The results demonstrate that the translocation apparatus used for toxin translocation is not limited to a single A fragment but can accommodate additional proteins as well. The fact that proteins as large as 20 kDa can be brought into cells by way of diphtheria toxin under both in vitro and in vivo conditions opens up the possibility of using diphtheria toxin mutants for introducing molecules with biological activity into cells.
Adoptively transferred anti-idiotype pulsed B cells mediate autoimmune myocarditis
Authors: Paque R E , Miller R
Published: January 2020

Syngeneic mice receiving adoptively transferred enriched B cells or splenocytes pulsed in vitro with polyclonal anti-idiotypic antibodies (anti-Ids) against anti-CVB3 virus antibodies developed coxsackievirus B3 antigen-binding and virus-neutralizing antibodies during a 30-day period, in addition to overt expression of autoimmune myocarditis. Adoptive transfer of anti-Id pulsed T cells resulted in delayed appearance and transient expression of antiviral antibodies, and antiviral antibodies were marginal to absent in syngeneic animals receiving anti-Id pulsed macrophage populations. Proliferation analysis of recipient splenocytes indicated lack of proliferative capacity in response to monoclonal or polyclonal anti-Ids and only marginal proliferative capacity in response to coxsackievirus B3 virus antigen(s). In vitro assessment of delayed hypersensitivity in recipient animals demonstrated some specific immunity to anti-Ids in recipients receiving splenocytes or T cells. Anti-Ids expressing mimicry for heart-associated and/or viral antigen(s) interacting with B cells or other accessory cells suggest an autoantibody or anti-Id triggering of B-cell-mediated mechanisms involved in the development of myocarditis.
Antibodies to lipooligosaccharide of a Brazilian purpuric fever isolate of Haemophilus influenzae biogroup aegyptius lack bactericidal and protective activity
Authors: Peters V B , Rubin L G
Published: January 2020

The immunological basis for protection against Brazilian purpuric fever (BPF), a fulminant infection of young children associated with bacteremia with Haemophilus influenzae biogroup aegyptius, is unknown. Candidate antigens to which protective antibodies may be directed include cell surface proteins and lipooligosaccharide (LOS). We studied the activity of antisera to LOS purified from a BPF H. influenzae biogroup aegyptius isolate. Anti-LOS antisera contained anti-LOS antibody by enzyme immunoassay and immunoblot and no detectable anti-outer membrane protein antibodies by immunoblot. Anti-LOS antisera had minimal bactericidal activity and were not protective against the homologous strain in an infant rat model of bacteremia. Antiserum to whole bacterial cells had a titer of anti-LOS antibody similar to that of anti-LOS antisera and was bactericidal and protective. Removal of anti-LOS antibodies from anti-whole cell antiserum by affinity chromatography did not result in a loss of bactericidal activity. Serum from a normal adult contained anti-LOS antibodies and had bactericidal activity. However, anti-LOS antibodies purified from this serum did not have detectable bactericidal activity. These studies suggest that anti-LOS antibodies produced in rats are not bactericidal and do not contribute to protection against experimental bacteremia with BPF strains of H. influenzae biogroup aegyptius.
Serotype diversity and antigen variation among invasive isolates of Ureaplasma urealyticum from neonates
Authors: Zheng X , Watson H L , Waites K B , Cassell G H
Published: January 2020

Ureaplasma urealyticum has previously been isolated from the cultured cerebrospinal fluid of 13 of 418 newborn infants; additional bloodstream isolates were obtained from the same population. Ten of the 13 cerebrospinal fluid and 3 bloodstream isolates were available for serotyping in the present study. By the use of serotype-specific reagents, including monoclonal antibodies, 70% of the cerebrospinal fluid isolates were identifiable as serotype 1, 3, 6, 8, or 10; i.e., they represented 5 of the 14 established serotypes or both presently defined genomic clusters. One of the bloodstream isolates was identified as serotype 3. Our data support the hypothesis that the property of invasiveness for unreaplasmas is likely not limited to one or a few particular serotypes among the 14 established serovars. Additionally, our study has shown that even in isolates of the same serotype, there can be size variation in the antigens expressed. Therefore, it would appear that many serotypes are invasive and that perhaps antigen variability and host factors may be more important determinants for ureaplasma infections than different serotypes per se.
Comparison of the abilities of Mycobacterium avium and Mycobacterium intracellulare to infect and multiply in cultured human macrophages from normal and human immunodeficiency virus-infected subjects
Authors: Crowle A J , Ross E R , Cohn D L , Gilden J , May M H
Published: January 2020

Patients with AIDS commonly develop disseminated infections with Mycobacterium avium (MA) but not its close relative, M. intracellulare (MI). In non-AIDS patients who have these infections, the two species are about equally distributed. The higher incidence of infection with MA than with MI in AIDS patients might be due to the selective susceptibility of these patients to MA. This possibility was tested by comparing the abilities of MA and MI to infect and replicate in cultured macrophages from normal subjects and from patients with AIDS-related complex or AIDS. The macrophages were cultured in medium supplemented with 1 or 5% normal or patient sera or with 1% defined serum substitute. Replication of MA (serovar 4) or MI (serovars 16 and 17) in the macrophages was measured by CFU counts made from lysed samples of the macrophages taken at 0,4, and 7 days after macrophage infection. MA and MI in infected normal macrophages which were cultured in normal serum replicated in these macrophages at similar rates. MA but not MI multiplied abnormally rapidly in patient macrophages cultured in either normal serum or patient serum. The accelerated growth of MA in patient macrophages was macrophage dependent, because patient sera did not change the rate of MA replication in culture medium lacking macrophages. However, patient sera did increase the permissiveness of normal macrophages to MA but not MI. These results suggest that a selective increased susceptibility to MA compared with a retained normal resistance to MI in human immunodeficiency virus-infected patients as they progress from AIDS-related complex to AIDS accounts for the higher prevalence of MA than MI infection in AIDS patients. The results also indicate that the mechanisms of native resistance in human macrophages to MA and MI are different.
Isolation and characterization of the Porphyromonas gingivalis prtT gene, coding for protease activity
Authors: Otogoto J , Kuramitsu H K
Published: January 2020

The prtT gene, coding for trypsinlike proteolytic activity, has been isolated from Porphyromonas gingivalis ATCC 53977. This gene is present immediately downstream from the sod gene on a 5.9-kb DNA fragment from the organism isolated in Escherichia coli. The complete nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the enzyme corresponds to a 53.9-kDa protein with an estimated pI of 11.85. Gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography also indicated a similar molecular size for the protease. The enzyme was purified to near homogeneity following anion-exchange and gel-filtration chromatography. The purified enzyme also exhibited a single protein species with a size of approximately 53 kDa. Enzyme activity was strongly dependent upon the presence of reducing agents (dithiothreitol, cysteine, and 2-mercaptoethanol) and was also stimulated in the presence of calcium ions. A comparison of the properties of the prtT gene product with comparable parameters of proteases previously purified from different strains of P. gingivalis suggested that the cloned protease represents a previously uncharacterized enzyme.
Listeria ivanovii is capable of cell-to-cell spread involving actin polymerization
Authors: Karunasagar I , Krohne G , Goebel W
Published: January 2020

Listeria ivanovii has been considered to be pathogenic to animals but has rarely been found associated with human infections. It has been claimed that L. ivanovii lacks the actA gene, which in L. monocytogenes encodes a protein required for interaction with host cell actin. Using fluorescence microscopy and electron microscopy, we demonstrate that L. ivanovii can invade mammalian cells, lyse the phagosomal membrane, polymerize host cell actin, reorganize actin to form tails, and spread from cell to cell. However, no DNA homologous to the actA gene could be detected by polymerase chain reaction. Further, L. ivanovii lacks the 90-kDa surface protein which in L. monocytogenes is encoded by actA. Despite the ability to spread from cell to cell, L. ivanovii differed significantly from L. monocytogenes in being unable to form plaques on monolayers of 3T3 fibroblast cells.
Rosetting Plasmodium falciparum-infected erythrocytes express unique strain-specific antigens on their surface
Authors: Helmby H , Cavelier L , Pettersson U , Wahlgren M
Published: January 2020

Spontaneous binding of uninfected erythrocytes to Plasmodium falciparum-infected erythrocytes (rosetting) has been suggested to have a critical role in the induction of cerebral malaria. We report here that rosetting can be mediated by several molecular mechanisms involving parasite polypeptides with M(r)s of 22,000 or 28,000, termed rosettins. Antibodies to either polypeptide disrupt rosettes in a strain-specific fashion. Rosettes of five of the seven isolates examined thus far are more easily disrpted by anti-22,000-M(r) rosettin antibodies than by anti-28,000-M(r) rosettin antibodies. Polyclonal anti-22,000-M(r) rosettin antibodies raised in mice or rabbits strongly and strain specifically stain the surface of nonfixed erythrocytes infected with late asexual stages of rosetting P. falciparum. Simultaneous antibody staining and rosetting are seen when the anti-22,000-M(r) rosettin antiserum is diluted so that only partial disruption of rosettes is obtained, confirming that the fluorescence-labelled infected erythrocytes are involved in rosetting. The 22,000-M(r) rosettin is accessible for surface iodination on erythrocytes infected with strains of rosetting parasites sensitive to anti-22,000-M(r) rosettin antibodies, whereas no labelling occurred on either normal erythrocytes or nonrosetting-P. falciparum-infected erythrocytes. Purified anti-22,000-M(r) rosettin serum immunoglobulin G immunoprecipitated three parasite-derived polypeptides with M(r)s of 22,000, 45,000 (doublet), and 50,000 from lysates of [35S]methionine-labelled, parasite-infected erythrocytes. Our results suggest that rosetting is mediated by strain-specific, antigenically distinct, P. falciparum-derived polypeptides.
Role of attached lipid in immunogenicity of Borrelia burgdorferi OspA
Authors: Erdile L F , Brandt M A , Warakomski D J , Westrack G J , Sadziene A , Barbour A G , Mays J P
Published: January 2020

OspA is a protective antigen of the Lyme disease spirochete Borrelia burgdorferi. Expression of the full-length B. burgdorferi B31 OspA gene in Escherichia coli produces a protein that is processed posttranslationally by signal peptidase II and contains an attached lipid moiety. The recombinant OspA lipoprotein has been purified by detergent extraction and ion-exchange chromatography. Priming and boosting with OspA lipoprotein, either with no adjuvant or adsorbed to alum, elicited a strong, dose-dependent immunoglobulin G response. Serum from vaccinated mice inhibited spirochetal growth in vitro. Mice immunized twice with as little as 0.4 micrograms of OspA lipoprotein were protected against an intradermal challenge with 10(4) infectious spirochetes. The ability of the purified recombinant lipoprotein to induce a strong protective response in the absence of toxic adjuvants makes it an excellent candidate antigen for a human vaccine against Lyme disease. By contrast, no serum immunoglobulin G or growth inhibitory response to OspA nonlipoprotein was seen at any dose. The difference in immunogenicities of the lipoprotein and nonlipoprotein forms of OspA is not due to any difference in the antigenicities of the two proteins. These results suggest that posttranslational lipid attachment is a critical determinant of the immunogenicity of the OspA protein.
Interaction of insulin with Pseudomonas pseudomallei
Authors: Woods D E , Jones A L , Hill P J
Published: January 2020

Pseudomonas pseudomallei is the causative agent of melioidosis, a disease being increasingly recognized as an important cause of morbidity and mortality in many regions of the world. An intriguing observation regarding melioidosis is that a significant percentage of patients who develop the disease have preexisting diabetes mellitus. In this regard, we have tested the hypothesis that insulin may modulate the growth of P. pseudomallei. We have demonstrated that insulin markedly inhibits the growth of P. pseudomallei in vitro and in vivo. The growth rate of P. pseudomallei in minimal medium containing human recombinant insulin was significantly lower than that of control cultures containing no insulin. P. pseudomallei grew at an increased rate in serum samples obtained from diabetic rats compared with that in serum samples obtained from control animals. When the insulin level was restored by the addition of human recombinant insulin, the growth rate was reduced to a level similar to that seen in control serum. P. pseudomallei also grew significantly better in insulin-depleted human serum than control human serum. 125I-insulin binding studies demonstrated that P. pseudomallei possesses a specific, high-affinity binding site for human insulin. In in vivo studies, rats made diabetic by streptozotocin injection (80 mg/kg of body weight, intraperitoneally) were significantly more susceptible to P. pseudomallei septicemia than control rats. Thus, it appears that serum insulin levels may play a significant role in modulating the pathogenesis of P. pseudomallei septicemic infections.
Characterization of an immunoreactive 17.5-kilodalton outer membrane protein of Haemophilus somnus by using a monoclonal antibody
Authors: Tagawa Y , Haritani M , Yuasa N
Published: January 2020

A single outer membrane protein (OMP) of Haemophilus somnus, with an apparent molecular mass of 17.5 kDa, was identified in the sodium dodecyl sulfate (SDS)-insoluble fraction after extraction with 1% SDS-0.5 M NaCl-0.1% beta-mercaptoethanol. A hybridoma derived from mice immunized with H. somnus OMP fractions produced a monoclonal antibody (MAb), designated 20-3-5, that bound to the 17.5-kDa OMP of H. somnus. The MAb 20-3-5 epitope was present on 45 of 45 strains of H. somnus tested. MAb 20-3-5 cross-reacted with Haemophilus agni, Histophilus ovis, and Haemophilus haemoglobinophilus but not with 13 other species and subspecies of gram-negative bacteria. Immunoelectron-microscopic and antibody absorption studies revealed that the MAb 20-3-5 epitope is exposed on the surface of bacteria. In an immunoblot analysis, convalescent-phase sera obtained from calves with experimental H. somnus pneumonia contained antibodies to the 17.5-kDa OMP of H. somnus. Future studies will be directed toward examining the role of the 17.5-kDa OMP in immunity to H. somnus infections.
Use of transformation to construct antigenic hybrids of the class 1 outer membrane protein in Neisseria meningitidis
Authors: van der Ley P , van der Biezen J , Hohenstein P , Peeters C , Poolman J T
Published: January 2020

The class 1 protein of Neisseria meningitidis is an important component of candidate outer membrane vaccines against meningococcal meningitis. This porin protein contains two variable regions which determine subtype specificity and provide binding sites for bactericidal monoclonal antibodies. To determine the contribution of each of these variable regions in the induction of bactericidal antibodies, a set of isogenic strains differing only in their class 1 epitopes was constructed. This was done by transformation of meningococcal strain H44/76 with cloned class 1 genes and selection of the desired epitope combinations in a colony blot with subtype-specific monoclonal antibodies. When used for the immunization of mice, outer membrane complexes induced bactericidal antibodies only against meningococcal strains sharing at least one of their class 1 epitopes. The results demonstrate that the P1.2 and P1.16 epitopes, normally located in the fourth exposed loop of the protein, efficiently induce bactericidal antibodies independently of the particular sequence in the first variable region. The P1.5 and P1.7 epitopes, normally located in the first exposed loop, were found to induce lower bactericidal titers. Hybrid class 1 outer membrane proteins were constructed by inserting oligonucleotides encoding the P1.7 and P1.16 epitopes into the porA gene. In this way, we obtained a set of strains which carry the P1.5 epitope in loop 1, P1.2 in loop 4, and P1.7 and P1.16 (separately or in combination) in either loop 5 or loop 6. The additional epitopes were found to be exposed at the cell surface. Outer membrane complexes from several of these strains were found to induce a bactericidal response in mice against the inserted epitopes. These results demonstrate that it is feasible to construct meningococcal strains carrying multivalent class 1 proteins in which multiple subtype-specific epitopes are present in different cell surface-exposed loops.
Role of gamma interferon in late stages of murine salmonellosis
Authors: Muotiala A , Mäkelä P H
Published: January 2020

Gamma interferon (IFN-gamma) is an important mediator of natural resistance of mice to Salmonella species during the first week of infection, when it restricts the rate of intracellular growth of the bacteria but does not lead to their killing (A. Muotiala and P. H. Mäkelä, Microb. Pathog. 8:135-141, 1990). We used the experimental mouse salmonellosis model to investigate the role of IFN-gamma in the later stages of a sublethal infection and the ensuing specific immunity. When anti-IFN-gamma was administered starting 6 days after challenge, it did not prevent the cessation of intracellular bacterial growth and the formation of the plateau stage in the second week of infection. In addition, anti-IFN-gamma given 14 and 16 days after challenge did not alter the elimination of the bacteria in the clearance stage in the third week of infection. When mice immunized 2 months previously with live vaccine were infected with virulent salmonellae, depletion of IFN-gamma enhanced the early growth of the bacteria in the same manner as that seen in naive mice. However, when the immunized mice were infected with attenuated aroA bacteria, their clearance started immediately and was unaffected by IFN-gamma depletion, demonstrating that IFN-gamma is not required for the clearance. We conclude that IFN-gamma restricts the rate of intracellular bacterial multiplication in the first week of Salmonella infection in both naive and immune mice but is not a mediator of bacterial clearance in either naive or immunized mice.
Glycolytic enzymes of Candida albicans are nonubiquitous immunogens during candidiasis
Authors: Swoboda R K , Bertram G , Hollander H , Greenspan D , Greenspan J S , Gow N A , Gooday G W , Brown A J
Published: January 2020

A cDNA library was made with mRNA from Candida albicans grown under conditions favoring the hyphal form. The library was screened for sequences that encode immunogenic proteins by using pooled sera from five patients with oral candidiasis and five uninfected patients. Most of these patients were human immunodeficiency virus positive. From 40,000 cDNA clones screened, 83 positive clones were identified. Of these, 10 clones were chosen at random for further analysis. None of these 10 cDNAs were derived from a multigene family. The 5' and 3' ends of all 10 clones were analyzed by DNA sequencing. Two cDNAs were separate isolates of a sequence with strong homology to pyruvate kinase genes from other fungi (59 to 73%) and humans (60%). A third cDNA had strong sequence homology to the Saccharomyces cerevisiae and Kluyveromyces lactis alcohol dehydrogenase genes (68 to 73%). A fourth cDNA was homologous (81%) to an S. cerevisiae protein of unknown function. The functions of the remaining six C. albicans cDNAs are not known. A more detailed analysis of the clones encoding glycolytic enzymes revealed that sera from few patients recognized them as antigens. Therefore, although glycolytic enzymes constitute a group of C. albicans proteins that are immunogenic during oral and esophageal infections, their detection cannot be exploited as an accurate marker of infection.
Tumor necrosis factor alpha, interleukin-1 alpha, interleukin-6, and prostaglandin E2 production in murine peritoneal macrophages infected with Ehrlichia risticii
Authors: van Heeckeren A M , Rikihisa Y , Park J , Fertel R
Published: January 2020

Ehrlichia risticii is a gram-negative obligate intracellular bacterium which primarily infects macrophages and crypt epithelial cells in the intestinal wall and is the etiologic agent of Potomac horse fever. To understand the pathogenesis of the disease, we tested whether E. risticii induces inflammation-associated products in thioglycolate-induced mouse peritoneal macrophages. Mouse peritoneal macrophages produced larger amounts of interleukin-1 alpha (IL-1 alpha) but lower levels of tumor necrosis factor alpha (TNF-alpha), IL-6, and prostaglandin E2 (PGE2) when exposed to live or killed E. risticii than when exposed to Escherichia coli lipopolysaccharide (LPS). Preincubation of macrophages with live or killed E. risticii suppressed TNF-alpha, IL-6, and PGE2 generation but not IL-1 alpha production in response to LPS. Murine gamma interferon treatment of macrophages did not influence TNF-alpha, IL-1 alpha, IL-6, or PGE2 production regardless of exposure to E. risticii. Intracellular cyclic AMP was significantly greater in E. risticii-infected macrophages than in uninfected macrophages. These results suggest that increased levels of IL-1 alpha but not TNF-alpha or PGE2 production by macrophages may be primarily involved in the pathogenesis of the disease caused by E. risticii. Increased intracellular concentration of cyclic AMP in infected macrophages may be chiefly responsible for the high level of IL-1 alpha and inhibition of TNF-alpha production in response to LPS.
PRB3 null mutations result in absence of the proline-rich glycoprotein Gl and abolish Fusobacterium nucleatum interactions with saliva in vitro
Authors: Azen E , Prakobphol A , Fisher S J
Published: January 2020

The glycosylated proline-rich glycoprotein (Gl or PRG), a product of the PRB3 gene, is a major constituent of human parotid saliva. Important functions proposed for Gl include acting as a bacterial receptor. The Gl proteins of several subjects were typed by two polyacrylamide gel electrophoresis (PAGE) systems: acid-lactate PAGE followed by staining with the periodic acid-Schiff reagent and sodium dodecyl sulfate-PAGE followed by electrophoretic transfer and staining with amido black or concanavalin A. The results showed one subject who apparently lacked Gl. The four exons, including splice junctions, for both PRB3 alleles of this subject were completely sequenced. Unexpressed (null) mutations were detected with an identical C nucleotide insertion in the same coding region of exon 3 of both alleles. This C nucleotide insertion leads to a frameshift with a premature termination codon that probably results in markedly reduced or absent PRB3 gene expression. We then used a nitrocellulose blot overlay assay to assay the bacterial receptor activity of parotid saliva from the PRB3null subject. No interactions with Fusobacterium nucleatum, shown previously to interact selectively with Gl, were detected. Together, these results suggest that this subject does not express the PRB3 gene and that one of the consequences is an altered ability to interact with a bacterium known to colonize the oral cavity.
Variant responses of mice to Borrelia burgdorferi depending on the site of intradermal inoculation
Authors: de Souza M S , Smith A L , Beck D S , Kim L J , Hansen G M , Barthold S W
Published: January 2020

C3H/He mice inoculated intradermally at one of two sites with Borrelia burgdorferi responded differently to infection. Shoulder-inoculated mice developed spirochetemia, B. burgdorferi-specific antibody, and arthritis earlier than foot-inoculated mice. Lymphocyte populations derived from spleen tissue were elevated in the shoulder- but not the foot-inoculated mice, and those from lymph nodes were increased in both groups. Lymphocytes derived from blood and spleen tissue showed impaired proliferative responses to all mitogens for shoulder-inoculated mice only, whereas proliferation of lymph node cells was not affected, regardless of route. These results demonstrate that the site of initial B. burgdorferi inoculation is an important determinant in the pathogenesis of B. burgdorferi infection.
A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans
Authors: Munro G H , Evans P , Todryk S , Buckett P , Kelly C G , Lehner T
Published: January 2020

Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species.
Analysis of mannoproteins from blastoconidia and hyphae of Candida albicans with a common epitope recognized by anti-complement receptor type 2 antibodies
Authors: Wadsworth E , Prasad S C , Calderone R
Published: January 2020

Mannoproteins of approximately 50 kDa from blastoconidia and 60 kDa from hyphae of Candida albicans reacted in Western blots (immunoblots) with either a polyclonal rabbit antiserum (CA-7) or a monoclonal antibody (CA-A) to the C. albicans C3d-binding protein (complement receptor type 2). The glycosylated nature of these proteins was demonstrated by their reactivity with concanavalin A and by selective labeling with the biotin-hydrazide reagent following periodate oxidation. Differences in the oligosaccharides of these proteins were observed in regard to their reactivity with lectin-peroxidase reagents and sensitivity to glycosidases such as N-glycanase or endoglycosidase F (but not endoglycosidase H). The 60-kDa mannoprotein reacted with wheat germ agglutinin, while the 50-kDa mannoprotein did not. Treatment of the 60-kDa mannoprotein with the glycosidases mentioned above resulted in its conversion into a species of 40 to 45 kDa. Enzyme treatment had no obvious effect on the electrophoretic mobility of the 50-kDa species from blastoconidia. Both the 50- and 60-kDa glycoproteins remained immunoreactive after treatment with the glycosidases. Reactivities of the two mannoproteins to neuraminidase also differed. Finally, the 50-kDa (blastoconidia) and the 60-kDa (hyphae) mannoproteins were purified by using ion-exchange chromatography and electroelution. The purified proteins differed in net charge, the 60-kDa species having a more acidic pI. Functional activity of the purified mannoproteins was demonstrated, as each inhibited the rosetting of antibody-sensitized sheep erythrocytes conjugated with iC3b or C3d by hyphae. Thus, an epitope(s) common to both a mycelial and blastoconidial mannoprotein is associated with a structurally different oligosaccharide for each growth form.
The 39-kilodalton outer membrane protein of Proteus mirabilis is an OmpA protein and mitogen for murine B lymphocytes
Authors: Korn A , Kroll H P , Berger H P , Kahler A , Hessler R , Brauburger J , Müller K P , Nixdorff K
Published: January 2020

Partial amino acid sequence analysis of a major outer membrane protein of Proteus mirabilis (39-kDa protein) indicates that it is an OmpA protein. The mitogenic activities of the 39-kDa protein for murine lymphocytes were also investigated with T lymphocytes isolated by passing spleen cells over columns of nylon wool fiber and B lymphocytes obtained by treating spleen cells with monoclonal antibodies to Thy1 plus complement. The 39-kDa protein showed little activity in stimulating T cells to proliferate but was strongly mitogenic for B cells.
Alpha-2-macroglobulin functions as an inhibitor of fibrinolytic, clotting, and neutrophilic proteinases in sepsis: studies using a baboon model
Authors: de Boer J P , Creasey A A , Chang A , Abbink J J , Roem D , Eerenberg A J , Hack C E , Taylor F B
Published: January 2020

Alpha-2-macroglobulin (alpha 2M) may function as a proteinase inhibitor in vivo. Levels of this protein are decreased in sepsis, but the reason these levels are low is unknown. Therefore, we analyzed the behavior of alpha 2M in a baboon model for sepsis. Upon challenge with a lethal (4 baboons) or a sublethal (10 baboons) dose of Escherichia coli, levels of inactivated alpha 2M (i alpha 2M) steadily increased, the changes being more pronounced in the animals that received the lethal dose. The rise in i alpha 2M significantly correlated with the increase of thrombin-antithrombin III, plasmin-alpha 2-antiplasmin, and, to a lesser extent, with that of elastase-alpha 1-antitrypsin complexes, raising the question of involvement of fibrinolytic, clotting, and neutrophilic proteinases in the inactivation of alpha 2M. Experiments with chromogenic substrates confirmed that thrombin, plasmin, elastase, and cathepsin G indeed had formed complexes with alpha 2M. Changes in alpha 2M similar to those observed in the animals that received E. coli occurred in baboons challenged with Staphylococcus aureus, indicating that alpha 2M formed complexes with the proteinases just mentioned in gram-positive sepsis as well. We conclude that alpha 2M in this baboon model for sepsis is inactivated by formation of complexes with proteinases, derived from activated neutrophils and from fibrinolytic and coagulation cascades. We suggest that similar mechanisms may account for the decreased alpha 2M levels in clinical sepsis.
Cloning, expression, and mutagenesis of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: a potential staphylococcal virulence factor
Authors: Daugherty S , Low M G
Published: January 2020

Staphylococcus aureus secretes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) which is able to hydrolyze the membrane lipid PI and membrane protein anchors containing glycosyl-PI. The gene for PI-PLC (plc) was cloned from S. aureus into Escherichia coli. Oligonucleotide probes based on partial protein sequence and polyclonal antibodies raised against the purified protein were used to identify positive clones. E. coli transformed with a plasmid containing the plc gene expressed PI-PLC enzyme activity which was abolished by mutagenesis with a tetracycline resistance gene. The plc gene was present in all 15 S. aureus strains examined but not in any of 6 coagulase-negative staphylococcal species. The plc gene contained 984 bp and coded for a mature protein with a calculated molecular mass of 34,107 Da. Amino acid sequence comparisons indicated that the staphylococcal plc gene was similar (51 to 56%) to the PI-PLCs from Bacillus cereus, Bacillus thuringiensis, and Listeria monocytogenes. The recombinant PI-PLC expressed in E. coli was purified and exhibited biochemical properties identical to those of the native PI-PLC from S. aureus. PI-PLC production was decreased in agr mutant strains of S. aureus. However, PI-PLC production by both agr+ and agr mutant strains exhibited a similar dependence on the type of medium used. These data suggested that PI-PLC production was regulated by both agr-dependent and agr-independent mechanisms.
Differential susceptibilities of mice genomically deleted of CD4 and CD8 to infections with Trypanosoma cruzi or Trypanosoma brucei
Authors: Rottenberg M E , Bakhiet M , Olsson T , Kristensson K , Mak T , Wigzell H , Orn A
Published: January 2020

The role of CD4+ and CD8+ T cells in the surveillance of Trypanosoma cruzi or Trypanosoma brucei brucei was studied in mice which lacked CD4 or CD8 molecules and which were generated by embryonic stem cell technology. Whereas wild-type mice infected with T. cruzi (Tulahuén strain) displayed low levels of parasitemia and no mortality, striking increases in parasite growth and mortality occurred in both CD8- and CD4- mice. On the contrary, CD8- and, to a lesser degree, CD4- mice showed enhanced resistance to T. b. brucei. T-cell-dependent immunoglobulin G-specific responses were produced in CD8- but not CD4- mice. Normal T-cell proliferative responses were measured in both CD4- and CD8- mice. Interleukin-4 production after concanavalin A or anti-CD3 monoclonal antibody stimulation was strikingly enhanced in CD8- but not CD4- spleen cells, whereas gamma interferon production was normal in both CD4- and CD8- spleen cells. Spleen and lymph node cells from CD8- (but not CD4-) mice at 20 days postinfection with T. cruzi had higher levels of interleukin-4 mRNA than the wild-type controls, as shown in a competitive polymerase chain reaction assay. On the other hand, CD4- (but not CD8-) mice at 20 days postinfection with T. cruzi had lower levels of gamma interferon mRNA than the wild-type mice.
Activation of the human complement alternative pathway by Listeria monocytogenes: evidence for direct binding and proteolysis of the C3 component on bacteria
Authors: Croize J , Arvieux J , Berche P , Colomb M G
Published: January 2020

The capacity of the intracellular pathogen Listeria monocytogenes to activate the alternative pathway of human complement was examined. Incubation of L. monocytogenes with human serum in optimal conditions (20% Mg2+EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]-chelated serum) consumed (31.3 +/- 3.9)% of C3 hemolytic activity and led to similar amounts of C3 deposition among the 27 strains tested, except for a rough mutant and the penicillin-induced L forms of strain EGD, which bound reduced amounts of C3. The same results were obtained with strains belonging to related species (L. innocua, L. seeligeri, L. welshimeri, and L. ivanovii). Direct evidence is provided that L. monocytogenes induces the deposition of C3b and its cleavage products iC3b and C3d through ester and amide linkages, as demonstrated by the analysis of the released products of radiolabelled purified C3 after treatment with hydroxylamine. Our results clearly demonstrate that L. monocytogenes activates the alternative pathway of human complement, suggesting that bacteria in the blood or in tissues of infected patients are opsonized and targeted to C3 receptor-bearing cells such as macrophages.
Prevention of renal scarring from pyelonephritis in nonhuman primates by vaccination with a synthetic Escherichia coli serotype O8 oligosaccharide-protein conjugate
Authors: Roberts J A , Kaack M B , Baskin G , Svenson S B
Published: January 2020

Rhesus monkeys were vaccinated with a synthetic Escherichia coli serotype O8 oligosaccharide-protein conjugate. Using our experimental pyelonephritis monkey model, we tested whether such immunization was protective against the renal damage from inflammation following experimental infection with a P-fimbriated O-antigenically homologous E. coli strain. The vaccination did not significantly alter the duration of bacteriuria or interfere with the infection. However, the vaccine was efficient in renal protection, as vaccinated animals showed significantly less intratubular infiltration of neutrophils (P < 0.02) and the degree of renal scarring was also significantly less in these animals (P > 0.005) than in the control animals. Total kidney involvement in the vaccinated animals was 16.9%, compared with 32.5% in the control animals (P = 0.07).
Cytokine induction by extracellular products of oral viridans group streptococci
Authors: Takada H , Kawabata Y , Tamura M , Matsushita K , Igarashi H , Ohkuni H , Todome Y , Uchiyama T , Kotani S
Published: January 2020

During an etiological study of Kawasaki disease (mucocutaneous lymph node syndrome [MCLS]), we found that dominant viridans streptococcal strains on tooth surfaces and in the throat of both MCLS patients and non-MCLS control children formed erythrogenic and biologically active, extracellular products. In this study, we demonstrated that erythrogenic culture supernatant concentrates of representative strains (two Streptococcus mitis and two Streptococcus oralis), when injected intravenously, induced serum tumor necrosis factor alpha, interleukin-6 (IL-6), and gamma interferon in muramyldipeptide- or Propionibacterium acnes-primed C3H/HeN mice. The concentrates also induced tumor necrosis factor alpha, IL-6, and thymocyte-activating factor (essentially IL-1) in murine peritoneal macrophage, human monocyte, and human whole-blood cultures. An erythrogenic, heat-labile extracellular protein fraction (F-1) that was concentrated from the culture supernatants of a representative S. mitis strain exhibited the above-mentioned cytokine-inducing activity. This partially purified F-1 fraction also induced thymocyte-activating factor and IL-6 in human umbilical vascular endothelial cell and gingival fibroblast cultures.
Monoclonal immunoglobulin A antibodies directed against cholera toxin prevent the toxin-induced chloride secretory response and block toxin binding to intestinal epithelial cells in vitro
Authors: Apter F M , Lencer W I , Finkelstein R A , Mekalanos J J , Neutra M R
Published: January 2020

Secretory immunoglobulin A (IgA) antibodies directed against cholera toxin (CT) are thought to be important in resistance to oral challenge with virulent Vibrio cholerae, although alternative mechanisms for protection of intestinal epithelia against CT-induced fluid secretion have been proposed. The ability of anti-CT IgA to block the effects of CT on human enterocytes has not been directly tested because of the lack of a well-defined in vitro intestinal epithelial cell system to directly measure toxin action and the limited availability of purified anti-CT IgA antibodies. We have generated hybridomas that produce monoclonal IgA and IgG antibodies directed against CT by fusion of Peyer's patch cells with mouse myeloma cells after oral-systemic immunization of mice with CT and CT B-subunit protein. All of the anti-CT antibodies recognized the B subunit. Three clones (designated anti-CTB IgA-1, IgA-2, and IgA-3) which produced IgA antibodies in dimeric and polymeric forms were selected. Checkerboard immunoblotting demonstrated that IgA-1 recognized an epitope distinct from that recognized by IgA-2 and IgA-3 and that none of the antibodies were directed against the binding site of GM1, the intestinal cell membrane toxin receptor. The protective capacity of these IgAs was tested in vitro with human T84 colon carcinoma cells grown on permeable supports as confluent monolayers of polarized enterocytes. When each anti-CTB IgA was mixed with 10 nM CT and applied to the apical surfaces of T84 cell monolayers, all three IgAs blocked CT-induced Cl- secretion in a dose-dependent manner and completely inhibited binding of rhodamine-labelled CT to apical cell membranes. Thus, monoclonal anti-CTB IgA antibodies are sufficient to protect human enterocytes in vitro against CT binding and action.