Louisa B. Tabatabai

Basic fibroblast growth factor induces 3T3 fibroblasts to synthesize and secrete a cyclophilin-like protein and β2-microglobulin

Abstract When stimulated by fibroblast growth factor (FGF) BALB/c 3T3 cells synthesize and secrete elevated amounts of five proteins called the ‘superinducible proteins’, or SIPs. The expression of these proteins is greatly enhanced if the cells are treated with cycloheximide during induction. The 24 kDa protein 0SIP24) has been purified and antiserum raised against it. This protein is N-glycosylated and probably structurally constrained by one or more intramoleular disulfide bonds. The amino acid sequences of three of four peptides show significant identity with cyclophilin, an abundant cytoplasmic protein believed to mediate the immunosuppressive effects of cycloporin A. Several members of the cyclophilin family have been identified, and cDNA clones of two cyclophilin-like proteins with signal sequences have been reported. Here we show that at least one cyclophilin-like protein is secreted and that its expression is regulated by growth factors. The 12.5 kDa protein (SIP12.5) was found to be immunoprecipitated by an antiserum raised to human β2-microglobulin. This protein is strongly induced by interferon, which is a characteristic of the β2-microglobulin gene. Thus, FGF stimulates mouse embryo 3T3 cells to produce two proteins related to immune regulatory molecules. This may reflect an interaction between immune cells and nonimmune cells that occurs in vivo during processes such as wound healing when growth factors are released locally.

Identification of Five Outer Membrane-Associated Proteins among Cross-Protective Factor Proteins of Pasteurella multocida

ABSTRACT Fowl cholera is caused by Pasteurella multocida serovars A:1, A:3, and A:4. The 39-kDa cross-protective factor protein and four other membrane proteins of the membrane proteome of P. multocida were identified. We determined that the 39-kDa cross-protective protein was Pasteurella lipoprotein B, or PlpB.

Conservation of Antigenicity in a 31-kDa Brucella Protein

A 31-kilodalton (kDa) protein extracted from Brucella abortus was previously cloned into Escherichia coli and expressed at high levels. The E. coli-derived protein can be purified by a simple 2-step procedure entailing detergent extraction followed by ion-exchange chromatography. Subsequent analyses show that the E. coli-derived protein is identical to the Brucella-derived protein in molecular weight and isoelectric point. A partial amino acid sequence of the N-terminus of the protein of E. coli origin matches the predicted sequence, based on DNA sequence data. Using specific antiserum raised against the E. coli-derived protein, 34 strains of Brucella, representing all 6 recognized species, were examined for expression of the 31-kDa protein by Western blotting. This protein was detectable in all, but one Brucella species (B. ovis), including all 8 biovars of B. abortus tested. This degree of conservation supports further study of the 31-kDa protein for potential exploitation as a vaccine or diagnostic component.

Sialic acid uptake is necessary for virulence of Pasteurella multocida in turkeys.

Many pathogenic bacteria employ systems to incorporate sialic acid into their membranes as a means of protection against host defense mechanisms. In Pasteurella multocida, an opportunistic pathogen which causes diseases of economic importance in a wide range of animal species, sialic acid uptake plays a role in a mouse model of systemic pasteurellosis. To further investigate the importance of sialic acid uptake in pathogenesis, sialic acid uptake mutants of an avian strain of P. multocida P-1059 (A:3) were constructed, characterized, and an in-frame sialic acid uptake deletion mutant was assessed for virulence in turkeys. Inactivation of sialic acid uptake resulted in a high degree of attenuation when turkeys were challenged either intranasally or intravenously. Resistance of the sialic acid uptake mutant to killing by turkey serum complement was similar to that of the parent, suggesting other mechanisms are responsible for attenuation of virulence in turkeys.

Immune responses to superoxide dismutase and synthetic peptides of superoxide dismutase in cattle vaccinated with Brucella abortus strain 19 or RB51

Antibody and lymph node cell-mediated immune responses to recombinant Brucella abortus strain 19 Cu-Zn superoxide dismutase (rSOD) and to three synthetic strain 19 Cu-Zn SOD peptides were measured during 2 to 12 weeks following vaccination of cattle with B. abortus strain 19 or RB51. Cattle vaccinated with strain 19 or RB51 did not produce antibody to rSOD and to the SOD peptides. Lymph node cells from cattle vaccinated with strain 19, but not with strain RB51, proliferated when incubated with either rSOD or one of the three tested SOD peptides (GGDNYSDKPEPLGG). These results suggest that neither the strain 19 nor the strain RB51 vaccine induces antibody production to SOD and only the strain 19 vaccine induces lymph node cell-mediated immune responses to SOD.

Vaccination of cattle with chemically modified and unmodified salt-extractable proteins from Brucella abortus

Beef heifers were vaccinated on Day 0 with either salt-extractable protein (CSP) or chemically modified CSP (dCSP) from Brucella abortus Strain 19 in Freunds complete adjuvant (FCA). Six weeks later, vaccination was repeated, and heifers received either the homologous or heterologous vaccine. Another group of heifers received only FCA and saline. Vaccinations with CSP or dCSP stimulated marked antibody responses to B. abortus, as detected by standard serologic tests, an enzyme-linked immunosorbent assay, or a quantitative fluorometric immunoassay. Twelve percent of the heifers were seropositive by the CARD test 1 year after vaccination. Vaccination stimulated an increased cell-mediated immune response as measured by lymphocyte blast transformation (LBT) to B. abortus antigens. Fifty-six weeks after the initial vaccination, the heifers were challenged intraconjunctivally with 1.9 X 10(7) colony-forming units of B. abortus strain 2308. Sixty to 83% of the heifers aborted in each group and 70-83% of the heifers were culture positive. There were no significant differences (P greater than 0.05) among groups with respect to the number of abortions or the number of culture-positive heifers. Antibody responses increased rapidly within 4 weeks after challenge. Overall, antibody responses were greater for heifers that aborted than for those that did not abort. These differences were significant (P less than 0.05) only as measured by the fluorometric procedure. The LBT responses appeared to be higher for vaccinates than for the control group, but these differences were not significant (P greater than 0.20). There was a significantly lower (P less than 0.05) LBT response to heat-killed B. abortus in those heifers that aborted compared to those that did not.

Immunogenicity of Brucella abortus salt-extractable proteins

The immunogenic properties of salt-extractable proteins and chromatographic fractions thereof from Brucella abortus were evaluated in lemmings (Dicrostonyx rubricatus). The efficacy of the Brucella proteins as immunogens was determined after challenge with virulent B. abortus strain 2308 and was based on protection against clinical signs and gross lesions of brucellosis, as well as on numbers of viable Brucella in the spleen. Vaccination of lemmings with as little as 0.1 microgram of salt-extractable proteins (CSP) suppressed splenic infection, resulting in reduced numbers of viable organisms per spleen of 5-6 logs compared to non-vaccinated controls. Protein fractions separated by column chromatography were generally effective in reducing splenic infection, and contained proteins with molecular weights of 30,000, 20,000 and 12,000. Vaccines containing chemically modified dodecanoyl-CSP offered no additional advantage over unmodified CSP vaccines.

Characterization of salt-extractable protein antigens from Brucella abortus by crossed immunoelectrophoresis and isoelectricfocusing

Salt-extractable protein antigens (CSP) from Brucella abortus strains 19 and 2308 (vaccine and virulent strains, respectively) were analysed by crossed immunoelectrophoresis (CIE) using rabbit antisera to protein antigens and by isoelectricfocusing (IEF) in polyacrylamide gels. The reference immunoelectrophoretic profiles developed for proteins from strain 19 and 2308 of B. abortus contained 20 and 25 immunoprecipitates, respectively. Serum from cows experimentally infected or hyperimmunized with live organisms produced up to 5 immunoprecipitates in CIE with the protein antigens. Absorption of rabbit sera with homologous B. abortus cells reduced, but did not eliminate all of the immunoprecipitates from rabbit sera, suggesting that the majority, but not all of the protein components, are exposed on the surface of the cell. In contrast, antibody to protein antigens in agglutinin-free absorbed serum from infected cattle could still be demonstrated by CIE, even though CIE with protein extracts from whole cells radioiodinated with the cell surface labeling reagent, diazoiodusulfanilic acid, indicated that these antigens may be at or near the surface of the cell. From CIE in heterologous systems we concluded that all proteins present in strain 19 preparations were partially or completely identical to those in strain 2308. The IEF studies paralleled the CIE studies and revealed that the protein profile from strain 2308 was more complex than the profile from strain 19. Major differences between the 2 strains were found in the pH region from 3.9 to 5.0, where strain 2308 exhibited 4 additional protein bands.

Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis

BackgroundHaemophilus parasuis, the causative agent of Glässer’s disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer’s disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate.ResultsCharacterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome’s G + C content was 39.93%. Twenty protein homologs to bacteriophage proteins, including 15 structural proteins, one lysogeny-related and one lysis-related protein, and three DNA replication proteins were identified by mass spectrometry. One of the tail proteins, gp36, may be a virulence-related protein.ConclusionsBacteriophage SuMu was characterized by genomic and proteomic methods and compared to enterobacteriophage Mu.

Ornithobacterium rhinotracheale North American field isolates express a hemolysin-like protein.

Abstract Ornithobacterium rhinotracheale is a gram-negative bacterium responsible for the sporadic outbreaks of airsacculitis in poultry, accounting for millions of dollars in losses to the poultry industry annually. Although the organism was originally classified as non–β-hemolytic, recent North American field isolates of O. rhinotracheale obtained from pneumonic lungs and air sacs indicated hemolytic activity on blood agar plates upon extended incubation for 48 hr at room temperature in air after initial incubation at 37 C for 48 hr under 7.5% CO2. This report characterizes the β-hemolytic activity of O. rhinotracheale isolates by using in vitro kinetic hemolysis assays with sheep red blood cells, western blotting with leukotoxin-specific monoclonal antibodies, and isobaric tagging and relative and absolute quantitative (iTRAQ) analysis of O. rhinotracheale outer membrane protein digest preparations. The kinetic analyses of the hemolytic activity with red blood cells indicated that the protein is a pore former. iTRAQ analysis with membrane preparations revealed four peptides with homology to Mannheimia haemolytica leukotoxin and two peptides with homology to Actinobacillus actinoacetemcomitans leukotoxin. This is the first report that North American field isolates of O. rhinotracheale may express a hemolysin-like activity.