Heat Shock Proteins DnaK Research Articles

Hrp12, a novel heat-responsive, tissue-specific, phosphorylated protein isolated from mouse liver.

Previous studies from this laboratory identified a 28-kd nonreducible protein, liver-derived immunoinhibitory factor (LDIF) from the mouse liver. Isolation of this protein resulted in the co-purification of another unique protein called heat responsive protein 12 kd (Hrp12). In contrast to LDIF, Hrp12 was totally reducible to a protein of 12 kd suggesting a dimer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) purification, followed by sequencing of an in situ cyanogen bromide digest of membrane bound Hrp12, yielded an internal 20-amino acid polypeptide. Degenerate oligonucleotides made from this peptide were used to screen a murine liver complementary DNA (cDNA) library. A 1240-bp cDNA clone was obtained with an internal 521-bp open reading frame (ORF). Sequence analysis of the 173-amino acid ORF of mouse Hrp12 showed a high degree of homology with a 99 amino acid rat liver-kidney perchloric acid-soluble protein (LKPS) and a 136-amino acid perchloric acid soluble rat protein (PSP). Transcripts for Hrp12 were mainly restricted to the liver and kidney in mouse and man. The protein was estimated to be approximately 0.8% of the total liver-soluble cytosolic protein. A zoo-blot probed at moderate stringency with labeled cDNA revealed a strong conservation of the gene in all of the mammalian species tested. Analysis of the protein structure of Hrp12 revealed motifs predicted to be targets for protein kinase C (PKC). More importantly, purified mouse Hrp12 could be phosphorylated in vitro with PKC. The protein had significant similarity to DnaK heat shock protein (Hsp)70 and contained a 54-amino acid stretch with sequence similarity to Hsp90. This prompted us to investigate the heat shock response of Hrp12. Isolated hepatocytes and hepatoma cells were exposed to different heat shock temperatures (39.5 degrees C, 42.5 degrees C, and 44.5 degrees C); and then total RNA was extracted and Northern analysis carried out. The message for this novel protein responded atypically to heat shock. Although the steady-state level of the message increased after heat shock, a marked oscillatory pattern was superimposed on it. In contrast, the steady-state levels of Hsp90 and Hsp70 messenger RNA (mRNA) were found to respond to heat shock in the expected manner. Finally, the amount of Hrp12 protein was also found to increase after heat shock in a manner that was consistent with heat-responsive proteins.

Heat shock regulation of sigmaS turnover: a role for DnaK and relationship between stress responses mediated by sigmaS and sigma32 in Escherichia coli.

The cellular level of the rpoS-encoded sigmaS subunit of RNA polymerase increases in response to various stress situations that include starvation, high osmolarity, and shift to acid pH, and these different stress signals differentially affect rpoS translation and/or sigmaS stability. Here we demonstrate that sigmaS is also induced by heat shock and that this induction is exclusively due to an interference with sigmaS turnover. Some sigmaS-dependent genes exhibit similar heat shock induction, whereas others are not induced probably because they need additional regulatory factors that might not be present under conditions of heat shock or exponential growth. Despite its induction, sigmaS does not seem to contribute to heat adaptation but may induce cross-protection against different stresses. While sigmaS is not involved in the regulation of the heat shock sigma factor sigma32, the heat shock protein DnaK has a positive role in the posttranscriptional control of sigmaS. The present evidence suggests that DnaK is involved in the transduction of two of the signals that result in reduced sigmaS turnover, i.e., heat shock and carbon starvation. Heat shock induction of sigmaS also clearly indicates that a cessation of growth or even a reduction of the growth rate is not a prerequisite for the induction of sigmaS and sigmaS-dependent genes and underscores the importance of sigmaS as a general stress sigma factor.

Attributes of the Heat Shock Response in Three Species of Dairy Lactobacillus

Summary Lactobacillus acidophilus, L. casei , and L. helveticus are industrially important bacteria for the manufacture of fermented dairy foods. Despite widespread commercial use, there is limited knowledge of basic physiological responses by these bacteria to dairy processing conditions. This study investigated the heat shock (HS) response in L. acidophilus NCFM, L. casei LC301, and L. helveticus LH212. Thermotolerance experiments showed HS improved the ability of log phase L. acidophilus NCFM, L. casei LC301, and L. helveticus LH212 cells to withstand a 20 min high temperature incubation by approximately 27-, 5- and 11-fold, respectively. Two-dimensional polyacrylamide gel electrophoresis showed HS induced synthesis of several proteins in each Lactobacillus species, and Western blots revealed these molecules included homologs to the universally conversed heat shock proteins DnaK, GroEL, ClpB, and GrpE. DnaJ was also detected, but expression of this protein was not stimulated by HS in any of the Lactobacillus species tested.

DnaK Heat Shock Protein of Escherichia coli Maintains the Negative Supercoiling of DNA against Thermal Stress

Plasmid DNA in exponentially growing Escherichia coli immediately relaxes after heat shock, and the relaxed state of DNA rapidly reverts to the original state with exposure to conditions of heat shock. We have now obtained genetic and biochemical evidence indicating that DnaK heat shock protein of E. coli, a prokaryotic homologue of hsp70, is involved in this re-supercoiling of DNA. As re-supercoiling of DNA did not occur in an rpoH amber mutant, it seems likely that heat shock proteins are required for this reaction. Plasmid DNA in a dnaK deletion mutant relaxed excessively after temperature shift-up, and the re-supercoiling of DNA was not observed. DNAs incubated with a crude cell extract prepared from the dnaK mutant were more relaxed than seen with the extract from its isogenic wild-type strain, and the addition of purified DnaK protein to the mutant extract led to an increase in the negative supercoiling of DNA. Moreover, reaction products of purified DNA gyrase more negatively supercoiled in the presence of DnaK protein. Based on these results, we propose that DnaK protein plays a role in maintaining the negative supercoiling of DNA against thermal stress.

The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis

The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp. lactis has been studied. A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5. Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells. Among these are the major heat shock proteins DnaK and GroEL. In conjunction with a previous report (Hartke et al. 1994), the results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons. Both systems are independent of de novo protein synthesis.

Bifunctional fusion proteins of calmodulin and protein A as affinity ligands in protein purification and in the study of protein-protein interactions.

An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand. The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system. The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA). The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein. In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine. Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein. Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK. While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0). In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance. The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column. This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions. This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions. Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.

Overproduction of the Brucella melitensis heat shock protein DnaK in Escherichia coli and its localization by use of specific monoclonal antibodies in B. melitensis cells and fractions

The Brucella melitensis dnaK gene was amplified by the polymerase chain reaction using primers chosen according to the published sequence of B. ovis and cloned in multiple copy plasmids enabling expression under the control of the P lac promoter. Monoclonal antibodies (mAb) obtained by immunizing mice with B. melitensis B115 cell wall (CW) fraction or by infecting mice with virulent B. melitensis strain H38 and recognizing a 73-kDa band in immunoblotting of the B. melitensis CW fraction reacted with the cloned dnaK gene product and were thus shown to be specific for the heat shock protein DnaK. The anti-Dnak protein mAbs did not react with Escherichia coli control cells or cell lysates and could therefore be specific to Brucella DnaK protein epitopes. These mAbs were further used to study overproduction of the DnaK protein. B. melitensis DnaK overproduction in E. coli resulted in a defect in cell septation and formation of cell filaments. Immunogold labelling with the mAbs and electron microscopy localized the DnaK protein inside as well as outside the E. coli cells, probably resulting from lysis due to toxicity of the overproduced DnaK protein. These results indicated that overproduction of the B. melitensis DnaK protein in E. coli had similar physiological consequences as that of E. coli overproduced in E. coli. The DnaK protein localization in B. melitensis cells was essentially cytoplasmic, as shown by immunoelectron microscopy. Heat shock treatment of these cells resulted in increased binding of mAbs and labelling in the cytoplasm. However, in subcellular fractions the DnaK protein was predominantly found in the cell envelope fraction of B. melitensis, which could perhaps be due to interaction of the DnaK protein with membrane proteins.

Synthesis rates of cellular proteins involved in translation and protein folding are strongly altered in response to overproduction of basic fibroblast growth factor by recombinant Escherichia coli.

The cellular response to temperature-induced production of human basic fibroblast growth (bFGF) factor by recombinant E. coli (bacteriophage lambda PRPL promoter/cI857 repressor expression system) was studied by one- and two-dimensional gel electrophoresis. Temperature shift from 30 to 42 degrees C caused the induction of heat-shock protein synthesis and the repression of synthesis of ribosomal proteins and the protein folding catalyst trigger factor. Compared to control cells, carrying the expression vector without structural bFGF gene cells, producing the heterologous protein exhibited a stronger increase in the synthesis rate of heat-shock proteins ClpB (HtpM), DnaK, HtpG, GroEL, GrpE, and IbpB (HtpE) in response to temperature upshift. Unexpectedly, formation of the chaperone heat-shock protein GroES was not detected after temperature shift to 42 degrees C in cells producing bFGF. In addition to amplified heat-shock protein formation, the syntheses of ribosomal proteins and of the protein folding catalyst trigger factor were more severely repressed after temperature upshift in cells producing bFGF. In conclusion, the normal cellular stress response caused by the high inducing temperature was strongly amplified by heterologous protein synthesis. In particular, syntheses of proteins involved in translation and protein folding were affected by the overproduction of the heterologous protein.

Thermostability of lactate dehydrogenase LDH-A4 isoenzyme: Effect of heat shock protein DnaK on the enzyme activity

Cells exposed to temperature a few degrees higher than their growth temperature synthesize heat shock proteins (hsp) which may then compose even 20% of total protein content. This paper examined the in vitro protective effect of heat shock protein DnaK (70 kDa) from Escherichia coli against the heat inactivation of lactate dehydrogenase isoenzyme LDH-A4. The LDH-A4 isoenzyme was purified from fish skeletal muscle using the affinity chromatography on Oxamate-agarose. The enzyme was then heated in the absence and the presence of DnaK protein in a water bath at either 51 or 55°C. The LDH activity was determined by measuring the change in absorbency at 340 nm min−1 at 30°C. The addition of DnaK protein to the LDH-A4 isoenzyme before heat treatment can protect enzyme activity against mild thermal inactivation. Incubation of the LDH-A4 isoenzyme at 51°C in the presence of DnaK protein stimulates its activity by about 30%. The presence of 2 mM ATP can raise LDH activity by another 10%. No significant recovery was observed when DnaK protein was added to LDH at 25°C following earlier inactivation. The maximal activities (Vmax) in the presence of DnaK protein are almost twice those without DnaK protein in the case of heat-treated LDH-A4 isoenzyme at 51°C. The observed protection of LDH-A4 activity increased with the increasing DnaK protein concentration in the incubation medium. Results suggested that the presence of DnaK protein can protect LDH-A4 from heat inactivation. This action may be important as a part of cellular chaperone machinery capable of repairing heat-induced protein damage. It may have a fundamental role in the acquisition of the thermotolerance to stress temperatures.

The recA gene of Lactococcus lactis: characterization and involvement in oxidative and thermal stress.

The role of recA in Lactococcus lactis, a microaerophilic fermenting organism, was examined by constructing a recA-disrupted strain. This single alteration had a surprisingly pleiotropic effect. In addition to its roles in homologous recombination and DNA repair, recA is also involved in responses to oxygen and heat stresses. We found that oxygen stress induced by aeration causes reductions in growth and stationary-phase survival of the recA strain. Toxicity is a consequence of hydroxyl radical production via the Fenton Reaction and is alleviated by catalase or Ferrozine addition. These results suggest that oxygen radicals are not efficiently eliminated and accumulate in lactococcal cultures, and that RecA is needed to deal with the damage they incur. Unexpectedly, thermal stress arrested growth of the recA strain. Immunological data indicate that the recA mutant is deficient in heat-shock proteins DnaK, GroEL, and GrpE. Poor growth at elevated temperature is therefore due to a diminished heat-shock response in the recA strain. In contrast, levels of a novel heat-shock protein, HfIB, are elevated. In Escherichia coli, HfIB downregulates the heat-shock response by promoting degradation of the transcription factor sigma 32. We propose that recA regulates the heat-shock response via HfIB. This work provides the first evidence showing that two major pathways of stress response, induced by heat shock and DNA damage, are interactive.

Specificity of the immune response to Haemophilus ducreyi

The specificity of the antibody response toHaemophilus ducreyiin sera from patients attending a sexually transmitted disease clinic in South Africa has been studied using immunoblotting. Patients with chancroid were shown to have higher levels of IgG (mean 0.74, SD 0.34) toH. ducreyithan those with no history of chancroid (mean 0.34, SD 0.19). The pattern of the antibody specificity was highly variable between patients with culture proven chancroid but there was no observed strain specificity. In comparison, the patterns obtained using sera from patients without known exposure toH. ducreyishowed less variation between patients and were of less intensity at the dilution used. Sera from patients with chancroid recognised epitopes on proteins that varied in molecular weight between strains, particularly of 60–66kDa (10 of 36 patients) and 25–27kDa (8 of 36 patients). In addition epitopes were recognised on the GroEL and/or DnaK heat shock proteins in 13 of 36 sera tested. There was no apparent change in the epitopes recognised on proteins between the homologous and heterologous strains. Patterns of antibody specificity in sequential sera only varied in one of six patients tested.

Regulation of the Caulobacter crescentus dnaKJ operon.

The bacterial heat shock proteins DnaK and DnaJ are members of a class of molecular chaperones that are required for a wide variety of cellular functions at normal growth temperatures. In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock. Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2. Transcription initiating at the sigma 70-like promoter is under strict temporal control, whereas transcription initiating at the heat shock promoter at 30 degrees C is not. Transcription of dnaKJ occurs during a short period in the cell cycle, concomitant with the onset of DNA replication. Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site. Transcripts initiating from either the P1 or the P2 promoter have an RNA leader sequence with a high probability of forming an extensive secondary structure. Deletion of this leader sequence resulted in an increased rate of expression in both transcriptional and translational fusions. Although the temporal control of expression at physiological temperatures is not affected by the presence or absence of the leader sequence, changes in mRNA secondary structure may contribute to the modulation of DnaK and DnaJ levels at normal temperatures and during heat shock.

Studies of the specificity and cross-reactions of antibodies to lipid A found in juvenile arthritis.

This work was started to determine whether the immunoglobulin G (IgG) reactions with monophosphoryl lipid A (MPL) found in children with arthritis were due to contaminants, a specific site on lipid A, or polyspecific binding. Different lots of MPL were examined by electrophoresis and immunoblot. Competitive inhibition of enzyme-linked immunosorbent assays (ELISAs) by analogs of MPL and biologic materials of clinical interest was used to determine the specificity of the binding site and potential cross-reactions. IgG in all patient sera tested reacted with a single band just < 6.5 kDa on immunoblots of all lots of MPL tested. The ELISAs were inhibited best by analogs of lipid A with an exposed diglucosamine core and intact polar domains. The anti-MPL was also inhibited by fetal bovine collagen types I and II and in some instances by cardiolipin, but not by keratan sulfate, proteoglycan, or DnaK heat shock protein. Lot variation was a persistent technical problem, but no protein contaminant could be demonstrated in any lot. The ELISA and immunoblot results confirmed each other. Immunoblots detected a single band of MPL reactive with IgG. This antibody remains of interest because of its disease association and correlations and because it cross-reacts with collagen and cardiolipin.

Identification of phosphoproteins in Escherichia coli.

The substrates of ion- and lipid-stimulated protein kinase activity in extracts of Escherichia coli were purified by chromatography. Subsequent N-terminal sequencing suggests that these substrates include the following: a novel 80 kDa protein co-purifying with RNA polymerase but partially homologous to elongation factor G; a protein with an apparent molecular weight of 65 kDa identified as the ribosomal protein S1; and a 32 kDa protein identified as succinyl CoA synthetase, a key enzyme in the tricarboxylic acid cycle. The phosphorylation of these three proteins was markedly stimulated by the addition of manganese, and occurred on threonine, serine or tyrosine residues as indicated by the stability of the phosphoresidues during acid treatment. In addition, a calcium-stimulated protein of 70 kDa was identified as the heat-shock protein DnaK, and a 17 kDa lipid-stimulated phosphoprotein as nucleotide diphosphate kinase.

Basic features of the Streptococcus thermophilus heat shock response

The heat shock response was investigated in the thermophilic acid bacterium Streptococcus thermophilus. The heat resistance (58°C, 30 min) of log-phase cells grown at 42°C was enhanced by pretreatment at 52°C for 15 or 30 min. Concurrently to this acquired thermotolerance, two-dimensional gel electrophoresis indicates that the cells induced the synthesis of at least 22 heat shock proteins after temperature upshift. Furthermore, following SDS-PAGE, Western blotting, and immunological analysis, six proteins were found to be antigenically related to the Escherichia coli heat shock proteins DnaK, DnaJ, GroEL, GrpE, and La and to the Bacillus subtilis σ43 factor Among these six proteins, two related to DnaK and GroEL, are clearly overexpressed during this stress. It is concluded that S. thermophilus possesses a heat shock response similar to that known to occur in mesophilic microorganisms.

Growth phase-dependent variations in the outer membrane protein profile of Brucella melitensis

Changes in Brucella cell envelope protein profiles were investigated with batch cultures of B melitensis strain 16M in a 2-litre fermenter. Analysis of expression of outer membrane proteins (OMP) (apparent molecular masses of 10, 16.5, 19, 25–27, 31–34, 36–38 and 89 kDa) and heat-shock protein DnaK (73 kDa) was performed with monoclonal antibodies (mAb) and immunoblotting techniques. Synthesis of the 89-kDa OMP and the heat-shock protein DnaK was invariant during B. melitensis growth. Expression of the 10-, 19- and 36–38-kDa minor OMPs was never detected. Variations in profiles of some OMPs, i.e. 25–27-kDa and 31–34-kDa major proteins and 16.5-kDa minor protein, occurred during growth stages, principally at the end of the exponential growth phase. These variations consisted of shifts in apparent molecular masses for the 25–27-kDa and 31–34-kDa OMPs and of peptidoglycan association for the 16.5-kDa OMP. Therefore, whereas the strong association of major OMPs with peptidoglycan was confirmed, results suggested that the 16.5-kDa minor OMP is also a peptidoglycan-associated protein.

Thermally‐induced cell lysis in Escherichia coli K12

Escherichia coli cells exposed to high temperatures exhibit a progressive loss of viability. We observed two mechanisms of cell death induced by lethal temperatures: with and without lysis. The number of cells lysed by heat decreased at later stages of the growth curve, when cells were pre-treated at lower temperatures for 10 minutes and when cells were pre-treated with novobiocin, nalidixic acid and cadmium chloride. Cell lysis was similar in wild type, rpoH, groE and dnaK mutant cells as well as in cells which overproduce heat shock proteins GroE or DnaK. Results using cells aligned for cell division and cells growing at 42 degrees C, 45 degrees C and 47 degrees C suggest that cells near division are more sensitive to lysis and that a high concentration of heat-shock proteins increases their resistance to lysis.

Regulation of 70-kDa heat-shock-protein ATPase activity and substrate binding by human DnaJ-like proteins, HSJ1a and HSJ1b.

The DnaJ family of molecular chaperones is characterized by the presence of a highly conserved 70-amino-acid J domain. Escherichia coli DnaJ interacts with the 70-kDa heat-shock protein (DnaK), in vitro, to stimulate the 70-kDa heat-shock protein ATPase activity and modify substrate binding. The conservation of the interaction of DnaJ-like proteins with the 70-kDa heat-shock proteins has been demonstrated for the yeast protein YDJ1, a protein that shows full domain conservation with E. coli DnaJ. Human neurone-specific DnaJ-like proteins, HSJ1a and HSJ1b, possess a J domain and a glycine/phenylalanine-rich region in common with E. coli DnaJ, although the overall amino acid identity is less than 23%. We have investigated, in vitro, the interaction of HSJ1a and HSJ1b with the mammalian brain constitutive 70-kDa heat-shock protein (hsc70). The weak intrinsic ATPase activity of the constitutive 70-kDa heat-shock protein is enhanced more than fivefold by stoichiometric amounts of both HSJ1a and HSJ1b. This enhancement is mediated by an increase in the rate of bound ATP hydrolysis, whereas the rate of ADP release is unaffected. HSJ1 proteins appear to regulate the affinity of the 70-kDa constitutive heat-shock protein for the permanently unfolded substrate, carboxymethylated alpha-lactalbumin. A recent report [Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J. & Fink, A. L. (1993) Nature 365, 664-666] has suggested that substrate release by 70-kDa heat-shock proteins requires a conformational change in these proteins induced by K+ in concert with ATP binding. In the presence of ATP, HSJ1 proteins reduce 70-kDa constitutive heat-shock protein/carboxymethylated alpha-lactalbumin complex formation both in the presence and absence of K+. This suggests that HSJ1 proteins induce a conformational change in the 70-kDa constitutive heat-shock protein that can mimic the effect mediated by K+ and therefore modulate 70-kDa heat-shock protein substrate release by another mechanism rather than merely stimulating the 70-kDa heat-shock protein ATPase activity. As HSJ1 proteins have limited similarity to DnaJ, we suggest that this action is being mediated by the J domain alone, and that this modulation of 70-kDa heat-shock-protein substrate binding will be common to all proteins that contain a J domain.

Brucella melitensis cell envelope protein and lipopolysaccharide epitopes involved in humoral immune responses of naturally and experimentally infected sheep.

Cell envelope fraction (CEF) of Brucella melitensis B115 was used to investigate antibody responses of B. melitensis naturally and strain H38 experimentally infected sheep by immunoblotting, indirect enzyme-linked immunosorbent assay (ELISA) (I-ELISA), and competitive ELISA (C-ELISA) with monoclonal antibodies (MAbs). MAbs used were directed to outer membrane proteins with molecular masses of 10, 16.5, 19, 25 to 27, 31 to 34, 36 to 38, and 89 kDa; to the heat shock protein DnaK, to O-polysaccharide (O-PS) common (C) and M epitopes; and to rough lipopolysaccharide (R-LPS) epitopes. In immunoblotting, all infected sheep sera tested recognized a large number of protein bands, including the above-cited proteins and other proteins for which MAbs have not been defined. The antibody response pattern was different from one animal to another, even within the experimentally infected sheep which were infected under the same experimental conditions. A number of protein bands were recognized by the sheep sera prior to experimental infection and by other uninfected sheep sera. The antibody reactivity to these antigens and others might explain the nonspecific antibody reactivity of sera in I-ELISA with CEF. C-ELISA confirmed also the individual variability of the antibody responses of infected sheep to protein antigens. Antibody responses to O-PS C and M epitopes were detected in all experimentally infected sheep and in half of the naturally infected sheep, but these responses were also heterogeneous in relation to their intensities. Antibody responses to R-LPS epitopes detected by use of C-ELISA with the anti-R-LPS MAbs were low or negative in most of the infected animals. Despite antibody response heterogeneity for CEF antigens, immunoblot and C-ELISA results indicated that, among the CEF antigens, the O-PS epitopes (C and M epitopes) and epitopes of the major 25- to 27- and 31- to 34-kDa outer membrane proteins seem to be the most promising for detecting B. melitensis infection in sheep.

Characterization of a linear epitope on Chlamydia trachomatis serovar L2 DnaK-like protein.

A cytoplasmic 75-kDa immunogen from Chlamydia trachomatis serovar L2 has previously been characterized as being similar to the Escherichia coli heat shock protein DnaK. We have localized a linear epitope for one monoclonal antibody specific for C. trachomatis DnaK. By use of a recombinant DNA technique, the epitope was limited to 14 amino acids. With synthetic peptides, the epitope was further limited to eight amino acids. Six of these amino acids are conserved in bovine HSP70, which has a known three-dimensional structure. The amino acid sequence homologous to the epitope is located in a linear part of the HSP70 molecule known as connect II.