ABSTRACT
Columnaris disease (CD), caused by Flavobacterium columnare, is an emerging disease affecting rainbow trout aquaculture. Objectives of this study were to 1) estimate heritability of CD resistance in a rainbow trout line (ARS-Fp-R) previously selected 4 generations for improved bacterial cold water disease (BCWD) resistance; 2) estimate genetic correlations among CD resistance, BCWD resistance, and growth to market BW; and 3) compare CD resistance among the ARS-Fp-R, ARS-Fp-S (selected 1 generation for increased BCWD susceptibility), and ARS-Fp-C (selection control) lines. Heritability of CD resistance was estimated using data from a waterborne challenge of 44 full-sib ARS-Fp-R families produced using a paternal half-sib mating design, and genetic correlations were estimated using these data and 5 generations of BCWD resistance, 9-mo BW (approximately 0.5 kg), and 12-mo BW (approximately 1.0 kg) data from 405 ARS-Fp-R full-sib families. The CD and BCWD challenges were initiated at approximately 52 and 84 d posthatch, or approximately 650 and 1,050 degree days (°C × d), respectively. Survival of ARS-Fp-R families ranged from 0 to 48% following CD challenge and heritability estimates were similar between CD (0.17 ± 0.09) and BCWD (0.18 ± 0.03) resistance, and the genetic correlation between these 2 traits was favorable (0.35 ± 0.25). Genetic correlations were small and antagonistic (-0.15 ± 0.08 to -0.19 ± 0.24) between the 2 resistance traits and 9- and 12-mo BW. Two challenges were conducted in consecutive years to compare CD resistance among ARS-Fp-R, ARS-Fp-C, and ARS-Fp-S families. In the first challenge, ARS-Fp-R families (83% survival) had greater CD resistance than ARS-Fp-C (73.5%; P = 0.02) and ARS-Fp-S (68%; P < 0.001) families, which did not differ (P = 0.16). In the second challenge, using an approximately 2.5-fold greater challenge dose, ARS-Fp-R families exhibited greater CD resistance (56% survival) than ARS-Fp-S (38% survival; P = 0.02) families. The favorable genetic correlation between CD and BCWD resistance is supported by greater CD resistance of the ARS-Fp-R line compared to the ARS-Fp-C and ARS-Fp-S lines and suggests that both traits will be improved simultaneously when selection is practiced on only 1 trait. In summary, these data indicate the feasibility of further selective breeding of the BCWD-resistant ARS-Fp-R line for increased CD resistance to produce a double pathogen-resistant line of rainbow trout.
Subject(s)
Cold Temperature , Disease Resistance/genetics , Fish Diseases/genetics , Flavobacteriaceae Infections/veterinary , Oncorhynchus mykiss/genetics , Oncorhynchus mykiss/microbiology , Animals , Aquaculture/methods , Disease Resistance/physiology , Disease Susceptibility/physiopathology , Disease Susceptibility/veterinary , Female , Fish Diseases/physiopathology , Flavobacteriaceae Infections/genetics , Flavobacteriaceae Infections/physiopathology , Flavobacterium/physiology , Genetic Predisposition to Disease/genetics , Inbreeding , Kaplan-Meier Estimate , Male , Oncorhynchus mykiss/physiology , PhenotypeSubject(s)
Fish Diseases/microbiology , Fish Diseases/pathology , Flavobacteriaceae Infections/veterinary , Flavobacterium/isolation & purification , Oncorhynchus mykiss , Animals , Aquaculture , Bacterial Load , Flavobacterium/genetics , Gills/microbiology , Gills/pathology , RNA, Ribosomal, 16S/geneticsABSTRACT
Flavobacterium psychrophilum is the aetiological agent of bacterial coldwater disease (CWD), and this pathogen has large economic impacts on salmonid aquaculture worldwide. Previously, it was demonstrated that high levels of protection against F. psychrophilum challenge were conferred to rainbow trout, Oncorhynchus mykiss (Walbaum), by immunization with distinct molecular mass fractions of the bacterium, and specific antibodies were correlated with protection. In this study, an immunoproteomic analysis of F. psychrophilum was performed using two-dimensional polyacrylamide gel electrophoresis and Western blotting with serum from fish immunized with high- and mid-molecular mass fractions of the bacterium. Mass spectrometry was used to determine the protein identity, and 15 immunogenic proteins were positively identified following Mascot searches of the F. psychrophilum genome. Based on known function and immunogenicity of homologous proteins in other bacterial pathogens, antibodies specific for several of the identified proteins may be important for protective immunity from CWD. These include outer membrane protein OmpA (P60), trigger factor, ClpB, elongation factor G, gliding motility protein GldN and a conserved hypothetical protein. This work increases the understanding of the protective humoral immune response of rainbow trout against these distinct molecular mass fractions of F. psychrophilum and provides new potential targets for recombinant protein vaccine development.
Subject(s)
Bacterial Proteins/immunology , Fish Diseases/immunology , Flavobacteriaceae Infections/veterinary , Flavobacterium/immunology , Immunity, Humoral , Oncorhynchus mykiss/immunology , Animals , Antibodies, Bacterial/blood , Bacterial Outer Membrane Proteins/immunology , Bacterial Proteins/chemistry , Flavobacteriaceae Infections/immunology , Flavobacterium/chemistry , ImmunizationABSTRACT
Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. We previously detected genetic variation for BCWD resistance in our rainbow trout population, and a family-based selection program to improve resistance was initiated at the National Center for Cool and Cold Water Aquaculture (NCCCWA). This study investigated evidence of major trait loci affecting BCWD resistance using only phenotypic data (without using genetic markers) and Bayesian methods of segregation analysis (BMSA). A total of 10,603 juvenile fish from 101 full-sib families corresponding to 3 generations (2005, 2007, and 2009 hatch years) of the NCCCWA population were challenged by intraperitoneal injection with Flavobacterium psychrophilum, the bacterium that causes BCWD. The results from single- and multiple-QTL models of BMSA suggest that 6 to 10 QTL explaining 83 to 89% of phenotypic variance with either codominant or dominant disease-resistant alleles plus polygenic effects may underlie the genetic architecture of BCWD resistance. This study also highlights the importance of polygenic background effects in the genetic variation of BCWD resistance. The polygenic heritability on the observed scale of survival status is slightly larger than that previously reported for rainbow trout BCWD resistance. These findings provide the basis for designing informative crosses for QTL mapping and carrying out genome scans for QTL affecting BCWD resistance in rainbow trout.
Subject(s)
Fish Diseases/microbiology , Flavobacteriaceae Infections/veterinary , Genetic Predisposition to Disease , Models, Genetic , Oncorhynchus mykiss/genetics , Animals , Bayes Theorem , Breeding , Female , Fish Diseases/genetics , Flavobacteriaceae Infections/genetics , Flavobacteriaceae Infections/microbiology , Flavobacterium/classification , Flavobacterium/pathogenicity , Male , Quantitative Trait Loci , SoftwareABSTRACT
A family-based selection program was initiated at the National Center for Cool and Cold Water Aquaculture in 2005 to improve resistance to bacterial cold water disease (BCWD) in rainbow trout. The objective of this study was to estimate response to 2 generations of selection. A total of 14,841 juvenile fish (BW = 3.1 g; SD = 1.1 g) from 230 full-sib families and 3 randomly mated control lines were challenged intraperitoneally with Flavobacterium psychrophilum, the bacterium that causes BCWD, and mortalities were observed for 21 d. Selection was applied to family EBV derived from a proportional-hazards frailty (animal) model while constraining rate of inbreeding to Subject(s)
Fish Diseases/microbiology
, Flavobacteriaceae Infections/veterinary
, Flavobacterium/immunology
, Oncorhynchus mykiss
, Selection, Genetic/immunology
, Animals
, Breeding/methods
, Female
, Fish Diseases/genetics
, Fish Diseases/immunology
, Flavobacteriaceae Infections/genetics
, Flavobacteriaceae Infections/immunology
, Flavobacteriaceae Infections/microbiology
, Kaplan-Meier Estimate
, Male
, Proportional Hazards Models
, Quantitative Trait, Heritable
, Selection, Genetic/genetics
ABSTRACT
The objectives of this study were to estimate the heritabilities for and genetic correlations among resistance to bacterial cold-water disease and growth traits in a population of rainbow trout (Oncorhynchus mykiss). Bacterial cold-water disease, a chronic disease of rainbow trout, is caused by Flavobacterium psychrophilum. This bacterium also causes acute losses in young fish, known as rainbow trout fry syndrome. Selective breeding for increased disease resistance is a promising strategy that has not been widely used in aquaculture. At the same time, improving growth performance is critical for efficient production. At the National Center for Cool and Cold Water Aquaculture, reducing the negative impact of diseases on rainbow trout culture and improving growth performance are primary objectives. In 2005, when fish averaged 2.4 g, 71 full-sib families were challenged with F. psychrophilum and evaluated for 21 d. Overall survival was 29.3% and family rates of survival varied from 1.5 to 72.5%. Heritability of postchallenge survival, an indicator of disease resistance, was estimated to be 0.35 +/- 0.09. Body weights at 9 and 12 mo posthatch and growth rate from 9 to 12 mo were evaluated on siblings of the fish in the disease challenge study. Growth traits were moderately heritable, from 0.32 for growth rate to 0.61 for 12-mo BW. Genetic and phenotypic correlations between growth traits and resistance to bacterial cold-water disease were not different from zero. These results suggest that genetic improvement can be made simultaneously for growth and bacterial cold-water disease resistance in rainbow trout by using selective breeding.
Subject(s)
Fish Diseases/genetics , Flavobacteriaceae Infections/veterinary , Immunity, Innate/genetics , Oncorhynchus mykiss/growth & development , Oncorhynchus mykiss/genetics , Animals , Body Weight/physiology , Breeding , Fish Diseases/mortality , Flavobacteriaceae Infections/genetics , Flavobacteriaceae Infections/mortality , Flavobacterium/physiology , Phenotype , Survival Analysis , Time FactorsABSTRACT
Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease, but the pathogenic mechanisms of this important fish pathogen are not fully understood. Identifying bacterial genes of F. psychrophilum differentially expressed in vivo may lead to a better understanding of pathogenesis and provide targets for vaccine development. Therefore, the present study used a proteomic approach to identify and quantify proteins of F. psychrophilum following growth in vivo and under iron-limited growth conditions. As determined by 2D polyacrylamide gel electrophoresis (2D-PAGE), numerous proteins exhibited different spot intensities following culture of the bacterium in vivo, and of these, 20 were selected and identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis and Mascot searches of the F. psychrophilum genome. Eighteen proteins exhibited increased spot intensities in vivo, and these included: several chaperone and stress proteins, gliding motility protein GldN, outer membrane protein OmpH, 2 probable outer membrane proteins (OmpA family), probable aminopeptidase precursor, probable lipoprotein precursor, 3-oxoacyl-[acyl-carrier-protein]-reductase, and several proteins with unknown function. Two proteins exhibited decreased spot intensities in vivo and were identified as ferritin FtnA and outer membrane protein OmpA (P60). Culture of F. psychrophilum in iron-limited media resulted in similar protein spot intensity changes for 6 of the 20 proteins identified following growth in vivo. Results from the present study suggest a role of upregulated proteins in the pathogenesis of F. psychrophilum and these may represent potential vaccine candidate antigens.
Subject(s)
Culture Media/chemistry , Flavobacterium/drug effects , Flavobacterium/physiology , Iron/chemistry , Iron/pharmacology , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriological Techniques , Fish Diseases/microbiology , Flavobacteriaceae Infections/microbiology , Flavobacteriaceae Infections/veterinary , Gene Expression Profiling , Gene Expression Regulation, Bacterial/drug effects , Oncorhynchus mykissABSTRACT
Antiviral immunity in fish is not well understood. In mammals, Toll-like receptor (TLR) 3 is involved in double-stranded RNA recognition and host immune response activation. Here, we report the first identification of a rainbow trout TLR3 ortholog (rtTLR3), its genomic structure, and mRNA regulation. Six exons and five introns were identified from bacterial artificial chromosome (BAC) and expressed sequence tag (EST) sequencing, and this genomic organization is similar to mammalian and fish TLR3 genes. The putative 913 amino acid protein has a Toll/interleukin (IL)-1R (TIR) domain, a transmembrane domain, and leucine-rich repeats. In healthy trout, rtTLR3 is highly expressed in the liver, pyloric ceca, intestine, spleen, and anterior and trunk kidney tissues. To investigate whether rtTLR3 is involved in antiviral immunity, transcriptional regulation in vivo was examined by quantitative real-time polymerase chain reaction (PCR) after poly inosinic:cytidylic (I:C) and infectious hematopoietic necrosis virus (IHNV) treatments. TLR3 mRNA expression peaked 1 day after poly (I:C) injection of live animals, while the peak of gene expression after live IHNV challenge was observed on day 3. In vitro stimulation of rainbow trout anterior kidney leukocytes with poly (I:C) also enhanced rtTLR3 expression. Up-regulation was specific to viral challenge as there was no significant up-regulation of rtTLR3 mRNA levels in the spleen and a modest down-regulation in the anterior kidney after bath challenge with a gram-negative bacterial trout pathogen, Yersinia ruckeri. The sequence conservation of trout TLR3 and mRNA regulation after poly (I:C) or RNA virus exposures strongly suggest a role for trout TLR3 in antiviral immunity.
Subject(s)
Membrane Glycoproteins/genetics , Oncorhynchus mykiss/genetics , Oncorhynchus mykiss/immunology , Receptors, Cell Surface/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/genetics , Gene Expression Regulation/drug effects , Immunity, Innate , Infectious hematopoietic necrosis virus/pathogenicity , Membrane Glycoproteins/chemistry , Molecular Sequence Data , Poly I-C/pharmacology , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cell Surface/chemistry , Sequence Homology, Amino Acid , Species Specificity , Toll-Like Receptor 3 , Toll-Like Receptors , Yersinia ruckeri/pathogenicityABSTRACT
Engineered antibodies have come to the forefront as research reagents and clinical therapeutics. However, reduced stability or expression levels pose a major problem with many engineered antibodies. As a model for understanding functional consequences of variable region mutation, we have studied the assembly and trafficking of anti-phenylphosphocholine antibodies. Previously, we identified severe secretion defects because of mutations in the heavy chain second complementarity determining region, which is involved in antigen binding. Here we demonstrate that immunoglobulin secretion is increased up to 27-fold by incubating stably transfected PCG1-1 cells with cognate hapten p-nitrophenylphosphocholine. Secretion was unaffected by nonbinding analogs. Radiotracer and metabolic labeling experiments demonstrated specific cellular uptake of p-nitrophenylphosphocholine and increased intracellular heavy and light chain assembly. Brefeldin A inhibited hapten-mediated immunoglobulin secretion but not assembly, indicating that assembly occurs early within the biosynthetic pathway. Recovery of secretion correlated with antigen binding capacity, suggesting that the rescue mechanism involves stabilization of heavy and light chain variable domains. This model system provides the first demonstration that cognate ligands can increase intracellular assembly of functional anti-hapten antibody within mammalian cells and suggests that small molecules of appropriate specificity and affinity acting as chemical chaperones may find application for increasing or regulating immunoglobulin expression.
Subject(s)
Haptens/metabolism , Immunoglobulins/metabolism , Nitrobenzenes/metabolism , Phosphorylcholine/metabolism , Arginine/chemistry , Cell Line , Immunoglobulins/chemistry , Isoleucine/chemistry , Ligands , Phosphorylcholine/analogs & derivatives , TransfectionABSTRACT
During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51-->Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1-3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51-->Arg or Ile51-->Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51-->Arg or Ile51-->Lys mutants were partially rescued when expressed with the J558L lambda1 L chain. These results represent the first demonstration that single somatic mutations in V(H) residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.
Subject(s)
Complementarity Determining Regions/genetics , Conserved Sequence/genetics , Conserved Sequence/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Immunoglobulins/biosynthesis , Immunoglobulins/deficiency , Mutation/immunology , Amino Acid Sequence , Animals , Arginine/genetics , Binding Sites, Antibody/genetics , Complementarity Determining Regions/biosynthesis , Histones/metabolism , Humans , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/metabolism , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/metabolism , Immunoglobulins/genetics , Immunoglobulins/metabolism , Isoleucine/genetics , Lysine/genetics , Mice , Molecular Chaperones/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed/immunology , Phosphorylcholine/metabolism , Transfection , Tumor Cells, Cultured/immunology , Tumor Cells, Cultured/metabolismABSTRACT
The immune response to phosphocholine (PC)-protein is characterized by a shift in antibody repertoire as the response progresses. This change in expressed gene combinations is accompanied by a shift in fine specificity toward the carrier, resulting in high affinity to PC-protein. The somatically mutated memory hybridoma, M3C65, possesses high affinity for PC-protein and the phenyl-hapten analogue, p-nitrophenyl phosphocholine (NPPC). Affinity measurements using related PC-phenyl analogues, including peptides of varying lengths, demonstrate that carrier determinants contribute to binding affinity and that somatic mutations alter this recognition. The crystal structure of an M3C65-NPPC complex at 2.35-A resolution allows evaluation of the three light chain mutations that confer high-affinity binding to NPPC. Only one of the mutations involves a contact residue, whereas the other two have indirect effects on the shape of the combining site. Comparison of the M3C65 structure to that of T15, an antibody dominating the primary response, provides clear structural evidence for the role of carrier determinants in promoting repertoire shift. These two antibodies express unrelated variable region heavy and light chain genes and represent a classic example of the effect of repertoire shift on maturation of the immune response.
Subject(s)
Antibody Affinity , Gene Rearrangement , Immunoglobulins/genetics , Immunoglobulins/immunology , Phosphorylcholine/immunology , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Haptens/immunology , Hemocyanins/chemistry , Hemocyanins/immunology , Hybridomas , Immunoglobulins/chemistry , Immunologic Memory , Models, Molecular , Molecular Sequence Data , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/chemistry , Surface PropertiesABSTRACT
Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish. Biological activities of secreted p57 include agglutination of salmonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4C11, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a monomer that retains salmonid leucocyte agglutinating activity. The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57. When combinations of the MAbs were used in an antigen capture ELISA, the epitopes recognized by the 3 MAbs were shown to be sterically separate. However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed. These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer. Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed. Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity. A model in which p57 is released from R. salmoninarum as a biologically active monomer during infection of salmonid fish is proposed.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Fish Diseases/immunology , Gram-Positive Bacteria/immunology , Gram-Positive Bacterial Infections/veterinary , Kidney Diseases/veterinary , Oncorhynchus mykiss , Animals , Antibodies, Bacterial/analysis , Antibodies, Monoclonal , Bacterial Outer Membrane Proteins/isolation & purification , Bacterial Outer Membrane Proteins/metabolism , Blotting, Western/veterinary , Chromatography, Gel/veterinary , Chromatography, Ion Exchange/veterinary , DNA Primers/chemistry , Electrophoresis, Polyacrylamide Gel/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Epitopes/immunology , Fish Diseases/microbiology , Gram-Positive Bacterial Infections/immunology , Gram-Positive Bacterial Infections/microbiology , Hemagglutination Tests/veterinary , Kidney Diseases/immunology , Kidney Diseases/microbiology , Molecular Weight , Polymerase Chain Reaction/veterinary , Rabbits , Recombinant Proteins/chemistryABSTRACT
A panel of mutant antibodies of the phosphocholine (PC)-binding antibody, T15, was tested for binding to PC-protein, Streptococcus pneumoniae, Trichinella spiralis and Ascaris suum. Relative to wildtype T15, all the mutant antibodies showed differential recognition of the panel of PC-associated antigens. These mutant antibodies contain amino acid replacements in the CDR2 region of the heavy chain variable region, indicating the importance of CDR2 in recognition of carrier determinants. A model of T15 is shown that illustrates the strategic placement of mutations that could allow interaction with determinants associated with PC. A direct implication of this finding is that the T15 antibody combining site accommodates structures larger than phosphocholine and that recognition of associated carrier determinants could be a significant force in shaping the immune response to PC-containing pathogens.
Subject(s)
Antigens, Bacterial/immunology , Antigens, Helminth/immunology , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Variable Region/metabolism , Phosphorylcholine/immunology , Phosphorylcholine/metabolism , Amino Acid Sequence , Amino Acid Substitution/genetics , Amino Acid Substitution/immunology , Animals , Ascaris suum/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Idiotypes/genetics , Immunoglobulin Variable Region/genetics , Models, Molecular , Molecular Sequence Data , Mutation/immunology , Myeloma Proteins/genetics , Sequence Alignment , Streptococcus pneumoniae/immunology , Trichinella spiralis/immunologyABSTRACT
Renibacterium salmoninarum is a Gram-positive diplo-bacillus and the causative agent of bacterial kidney disease, a prevalent disease of salmonid fish. Virulent isolates of R. salmoninarum have a hydrophobic cell surface and express the 57-58 kDa protein (p57). Here we have investigated parameters which effect cell hydrophobicity and p57 degradation. Incubation of R. salmoninarum cells at 37 degrees C for > 4 h decreased cell surface hydrophobicity as measured by the salt aggregation assay, and decreased the amount of cell associated p57. Incubation of cells at lower temperatures (22, 17, 4 or -20 degrees C) for up to 16 h did not reduce hydrophobicity or the amount of cell associated p57. Both the loss of cell surface hydrophobicity and the degradation of p57 were inhibited by pre-incubation with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Cell surface hydrophobicity was specifically reconstituted by incubation with extracellular protein (ECP) concentrated from culture supernatant and was correlated with the reassociation of p57 onto the bacterial cell surface as determined by western blot and total protein stain analyses. The ability of p57 to reassociate suggests that the bacterial cell surface is not irreversibly modified by the 37 degrees C treatment and that p57 contributes to the hydrophobic nature of R. salmoninarum. In summary, we describe parameters effecting the removal of the p57 virulence factor and suggest the utility of this modification for generating a whole cell vaccine against bacterial kidney disease.
Subject(s)
Bacterial Outer Membrane Proteins/drug effects , Fish Diseases/microbiology , Gram-Positive Bacteria/pathogenicity , Gram-Positive Bacterial Infections/veterinary , Kidney Diseases/veterinary , Animals , Blotting, Western/veterinary , Cell Membrane/microbiology , Electrophoresis, Polyacrylamide Gel/veterinary , Gram-Positive Bacteria/drug effects , Gram-Positive Bacterial Infections/microbiology , Hot Temperature , Kidney Diseases/microbiology , Phenylmethylsulfonyl Fluoride/pharmacology , Protease Inhibitors/pharmacology , Salmon , Sodium Chloride/pharmacology , Surface Properties , Time FactorsABSTRACT
A whole cell Renibacterium salmoninarum vaccine was developed using 37 degrees C heat treated cells that were subsequently formalin fixed; this treatment reduced bacterial hydrophobicity and cell associated p57. Coho salmon Oncorhynchus kisutch were immunized with the p57- vaccine by either a combination of intraperitoneal (i.p.) and intramuscular (i.m.) injections or per os. In the first experiment, i.p./i.m. vaccination of coho salmon with p57- cells in Freund's Incomplete Adjuvant (FIA) conferred a statistically significant increase in mean time to death after the salmon were i.p. challenged with 4.1 x 10(6) colony forming units (cfu) of R. salmoninarum. There was no significant difference in response between fish immunized with R. salmoninarum cell surface extract in FIA and those immunized with extracellular protein (ECP) concentrated from culture supernatant in FIA. The i.p. challenge dose resulted in complete mortality of all fish by Day 43. In a second experiment, fish were orally vaccinated with p57- R. salmoninarum cells encased in a pH protected, enteric-coated antigen microsphere (ECAM). Fish were bath challenged with 4.2 x 10(6) cfu ml-1 on Day 0 and sampled at time points of 0 (pre-challenge), 50, 90, or 150 d immersion challenge. Vaccine efficacy was determined by monitoring the elaboration of p57 in the kidneys of vaccinated and control fish. Fish vaccinated orally demonstrated a significantly lower concentration of p57 (p < 0.01) at Day 150 post challenge compared to fish receiving ECAMs alone. Fish receiving p57 cells without ECAM coating also showed a significantly lower p57 level (p < 0.03) versus control. In contrast, fish injected intraperitoneally with the p57- cells or fish fed p57+ R. salmoninarum cells in ECAMs demonstrated no significant difference (p > 0.05) versus controls. In summary, these studies suggest the preliminary efficacy of 37 degrees C treatment of R. salmoninarum cells as an oral bacterial kidney disease vaccine.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines , Fish Diseases/immunology , Gram-Positive Bacteria/immunology , Gram-Positive Bacterial Infections/veterinary , Kidney Diseases/veterinary , Administration, Oral , Animals , Antibodies, Bacterial/chemistry , Antibodies, Monoclonal , Antigens, Bacterial/immunology , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/immunology , Blotting, Western/veterinary , Electrophoresis, Polyacrylamide Gel/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Fish Diseases/microbiology , Fish Diseases/prevention & control , Gram-Positive Bacteria/pathogenicity , Gram-Positive Bacterial Infections/immunology , Gram-Positive Bacterial Infections/prevention & control , Hot Temperature , Injections, Intramuscular/veterinary , Injections, Intraperitoneal/veterinary , Kidney Diseases/immunology , Kidney Diseases/microbiology , Kidney Diseases/prevention & control , Microspheres , Oncorhynchus kisutch , Surface PropertiesABSTRACT
Several studies on disposal of nonsecreted Ig L chains have identified the endoplasmic reticulum as the site of degradation. Here, we examine degradation of a nonsecreted Ig L chain, T15L, and an experimentally endoplasmic reticulum-retained secretion-competent L chain, D16L, in the absence of H chains. We demonstrate that 1) degradation is specifically impaired by the proteasome-specific inhibitors carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) and lactacystin, 2) L chain degradation occurs early in the biosynthetic pathway, and 3) degradation does not require vesicular transport. Our findings indicate that previous assertions of L chain disposal within the endoplasmic reticulum must be modified. To our knowledge, we provide the first direct evidence supporting a new paradigm for removal of nonsecreted Ig L chains via dislocation to cytosolic proteasomes.
Subject(s)
Cysteine Endopeptidases/physiology , Immunoglobulin Light Chains/metabolism , Multienzyme Complexes/physiology , Protein Processing, Post-Translational/immunology , Animals , Biological Transport/drug effects , Biological Transport/immunology , Cysteine Endopeptidases/immunology , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Cytoplasm/enzymology , Cytoplasm/immunology , Cytoplasm/metabolism , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/enzymology , Golgi Apparatus/immunology , Immunoglobulin Idiotypes/metabolism , Lysosomes/drug effects , Lysosomes/immunology , Lysosomes/metabolism , Mice , Multienzyme Complexes/immunology , Multienzyme Complexes/metabolism , Multiple Myeloma , Myeloma Proteins/metabolism , Proteasome Endopeptidase Complex , Protein Processing, Post-Translational/drug effects , Tumor Cells, CulturedABSTRACT
We previously described secretion defects in four mutants of the murine anti-phosphocholine Ab, T15. The mutant heavy (H) chains had amino acid replacements in the V(H) complementarity-determining region 2 (HCDR2) and were expressed at normal intracellular levels. Here, the intracellular fate of the secretion-defective mutant heavy chains was investigated. Metabolic labeling demonstrated that the T15 wild-type Ab was secreted within a 4-h chase. In contrast, the mutant H chains accumulated with intracellular t(1/2) values ranging from 10 to 24 h. The mutant H chains were associated with increased levels of the molecular chaperones BiP and GRP94, and remained endoglycosidase H sensitive, suggesting retention in the endoplasmic reticulum. Assembly of the mutant H chains with T15 light (L) chain was arrested at the H2 and H2L intermediate stages of the T15 wild-type pathway (H2 --> H2L --> H2L2). Even though some assembly with L chain occurred, it was not as a secretion-competent H2L2 Ig moiety. The T15 L chains coexpressed with mutant H chains were degraded efficiently except for a minor L chain population with a long t(1/2) that was apparently protected at the H2L stage. To our knowledge, this is the first study demonstrating that intracellular half-lives of Ig H and L chains can be influenced by somatic mutations in HCDR2.
Subject(s)
Autoantigens/metabolism , DNA-Binding Proteins/metabolism , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Variable Region/metabolism , Leucine Zippers/genetics , Animals , Autoantigens/genetics , Autoantigens/immunology , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Half-Life , Hexosaminidases/metabolism , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Immunoglobulin Light Chains/metabolism , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Kinetics , Leucine Zippers/immunology , Mutagenesis , Peptide MappingABSTRACT
The ability of somatic mutation to modify the course of an immune response is well documented. However, emphasis has been placed almost exclusively on the ability of somatic mutation to improve the functional characteristics of representative antibodies. The harmful effects of somatic mutation, its dark side, have been far less well characterized. Yet evidence suggests that the number of B cells directed to wastage pathways as a result of harmful somatic mutation probably far exceeds the number of cells whose antibodies have been improved. Here we review our recent findings in understanding the structural and functional consequences of V-region mutation.
Subject(s)
Antibody Diversity/genetics , Genes, Immunoglobulin/genetics , Mutation , Animals , Antibodies, Antiphospholipid/genetics , B-Lymphocytes/immunology , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Models, Molecular , Phosphorylcholine/immunologyABSTRACT
The extent to which somatic mutation impairs the Ig complementarity-determining region (CDR) and framework region (FRW) structure/function is not clear. Previously, we found that the VH CDR2 of the murine T15 Ab is highly sensitive to mutation; 56% (26 of 46) of Abs mutated in vitro had reduced or no Ag binding capability, and 9% were secretion impaired. Here we test whether the T15 VH CDR2 structure is unique by mutating the VH CDR2 of the anti-PC-protein murine Ab, PCG1-1. PCG1-1 VH is encoded by the M141 gene and is unrelated in sequence or structure to that of T15 VH1. The majority (54%, 20 of 37) of PCG1-1 mutants carrying one to five mutations in VH CDR2 had reduced or abolished Ag binding, while 10% were secretion impaired. Taken together, mutational analysis of the VH1 and VH M141 genes demonstrates that impaired binding and secretion may be common outcomes of CDR2 somatic mutation. We also tested the tolerance of the VH FRW2 of T15 to mutation, expecting this sequence-conserved region to be highly sensitive to alterations. However, FRW2 accommodated many nonconservative changes, and only 12% (3 of 25) of secreted mutants had impaired Ag binding. Moreover, mutations in FRW2 caused secretion defects in 24% (8 of 33), a frequency twice that of VH CDR2 mutants. A total of 16 unique secretion mutants have now been identified. These findings suggest that B cell losses from somatic mutation may be extensive and due to varied causes not all related to Ag binding.
Subject(s)
Antibodies, Monoclonal/genetics , Binding Sites, Antibody/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Mutagenesis, Site-Directed/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/physiology , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Idiotypes/biosynthesis , Immunoglobulin Idiotypes/genetics , Immunoglobulin Idiotypes/physiology , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/metabolism , Mice , Models, Molecular , Myeloma Proteins/genetics , Myeloma Proteins/immunology , Myeloma Proteins/metabolism , Phosphorylcholine/chemistry , Phosphorylcholine/immunology , Phosphorylcholine/metabolismABSTRACT
We recently described mutants of the murine anti-phosphocholine Ab T15, with changes in heavy chain complementarity determining region 2 (HCDR2) that caused loss of secretion. Surprisingly, the T15 HCDR2 mutations did not alter secretion when placed into the related anti-phosphocholine Ab D16, which differs from T15 only in HCDR3 and light (L) chain. Here, we exploit the differences between these two Abs to assess the basis of the secretion defect. The T15 L chain is not secreted in the absence of heavy (H) chain. In contrast, D16 L chain is secreted in the absence of H chain, as are most L chains. We co-expressed the T15 wild-type (wt) and mutant H chains with the D16 L chain, as well as with another secreted L chain, J558L. The mutant H chains were not secreted when expressed with either heterologous L chain. These results establish that the T15 L chain is not uniquely associated with the defect. The T15 and D16 Abs also differ in HCDR3 length in that D16 lacks four amino acid residues (Ser99, Ser100, Tyr100a, Trp100b) present in T15. We deleted these four residues from T15 wt and mutant H chains. Secretion of T15 wt was unaffected by the deletion, but shortening HCDR3 restored secretion in the HCDR2 mutants regardless of L chain association. Together these data demonstrate that both the HCDR2 and HCDR3 domains contain structural information that may affect the secretion competence of Abs.