Growth temperature-dependent variation in the bacteriophage-inactivating capacity and antigenicity of Yersinia enterocolitica lipopolysaccharide

Growth temperature affected the structure of Yersinia enterocolitica Ye 3827 lipopolysaccharide (LPS). Although Y. enterocolitica Ye 3827 synthesized smooth LPS when grown at a low temperature (25 degrees C), partial smooth-rough transition occurred when the bacteria were grown at the physiological temperature (37 degrees C). The structural alteration was detected by bacteriophage-inactivation assay and chemical and immunological analyses. LPS prepared from bacteria grown at 25 degrees C inactivated a number of bacteriophages that recognize the O-antigenic polysaccharide portion of LPS, whereas more than 3000 times the amount of LPS from bacteria grown at 37 degrees C was required for the same degree of inactivation. The antigenic determinant(s) responsible for the major reaction between 25 degrees C-LPS and anti-25 degree C-bacteria was located on the O-antigenic polysaccharide portion of LPS, but those responsible for the major reaction between 37 degrees C-LPS and anti-37 degrees C-bacteria were located on the R-core or inner portion of LPS.     

Susceptibility of three different strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at two different temperatures to Vibrio anguillarum and temperature effect on antibody response

Three geographically distinct-reared strains (Canadian, Icelandic, Norwegian) of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at optimal and super-optimal growth temperatures (12 and 18 degrees C respectively), were challenged with a virulent isolate of Vibrio anguillarum by injection. The halibut were injected intraperitoneally with 100 microl of the bacterial suspension (1 x 10(6) cells per fish). After challenge, temperature and strain-related differences in survival were observed. Canadian and Icelandic halibut cultured at the super-optimal temperature of 18 degrees C were significantly more susceptible to infection than those strains cultured at 12 degrees C. Total mortality at 18 degrees C for the Canadian and Icelandic strains was 56.4 and 61.85% respectively, compared to 32 and 26.6% respectively at 12 degrees C. Norwegian halibut were significantly more resistant to infection with V. anguillarum at 18 degrees C compared to the other strains, with total mortality of 13.3%. There was no significant difference in total mortality of Norwegian halibut at 18 or 12 degrees C (13.3, 25% respectively). The specificity of the antibodies in sera from challenged halibut cultured at 18 degrees C was primarily to LPS. Immunoblots showed the presence of antibodies against O-side chain antigens. This reaction was strongest in sera from the Norwegian halibut strain compared with the Canadian and Icelandic halibut, which suggests that the difference in resistance to challenge may be ascribable to the presence of antibodies to LPS. Specific antibody levels, as measured by ELISA, increased with increasing temperature and strain differences were apparent, however these did not relate to disease resistance.     

Study of Yersinia pseudotuberculosis surface antigen epitopes using monoclonal antibodies

A study of the influence of exogenous factors on the immunochemical activity of the bacterium Yersinia pseudotuberculosis and lipopolysaccharide preparations isolated from bacteria was performed using monoclonal antibodies. It was shown that the hybridomas that were obtained in this work produce antibodies against different and, most likely, species-specific epitopes associated with lipopolysaccharide O-side chains. The concentration of these epitopes increased with a decrease in the temperature, at which the bacteria were cultivated. An inhibitory effect of proteinase K, pepsin, and trypsin on the immunochemical activity of bacterial cells, determined using a solid-phase enzyme immunoassay, was demonstrated. Treatment with sodium periodate showed no uniform effect on the reactions between monoclonal antibodies and antigens (lipopolysaccharides and microbial cells), as adjudged by an immunoassay, which is most likely a consequence of the different localization of lipopolysaccharide epitopes recognized by the antibodies from four hybridomas.     

Temperature-dependent expression of flagella of Listeria monocytogenes studied by electron microscopy, SDS-PAGE and western blotting

Washed cells of Listeria monocytogenes serotype 4b, grown in broth culture at 20 degrees C and at 37 degrees C, were examined by electron microscopy for the presence of flagella. Many flagella were seen in cells grown at 20 degrees C, whereas at 37 degrees C very few were expressed. Flagella sheared from the cell surface were partially purified by differential centrifugation. Using SDS-PAGE and Western blotting two distinct protein bands were seen in this preparation, both with an apparent molecular mass of approximately 29 kDa. Further purification of these proteins was achieved by gel filtration and ion-exchange chromatography. Whole organisms grown at 20 degrees C and 37 degrees C were examined in Western blots using an affinity-purified polyclonal antibody, and a monoclonal antibody, both directed against 29 kDa putative flagellin. Bacteria grown at 20 degrees C expressed abundant flagellin, whereas only trace amounts could be detected in organisms grown at 37 degrees C. It is concluded that organisms grown at 20 degrees C both produce and assemble flagellin at the cell surface, and that flagellin production is a less marked feature of organisms grown at 37 degrees C.     

Pseudomonas aeruginosa PAO1 ceases to express serotype-specific lipopolysaccharide at 45 degrees C.

Most Pseudomonas aeruginosa strains are able to produce two distinct lipopolysaccharide (LPS) O-polysaccharide types, A-band (common-antigen) and B-band (serotype-specific) LPSs. The relative expression levels of these two LPS types in P. aeruginosa PAO1 (O5 serotype) at various growth temperatures were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining or Western blotting (immunoblotting) with monoclonal antibodies specific for each O polysaccharide. A-band and B-band LPSs were expressed concurrently when the cells grew at 15, 25, and 35 degrees C; however, growth at 45 degrees C resulted in a surface deficiency in B-band LPS as determined by immunoblotting and agglutination with B-band-specific monoclonal antibody. Transfer of these cells (expressing A-band LPS but deficient in B-band LPS) [A+B-]) to a lower temperature (at which the division time was comparable) resulted in a rapid resumption of normal A-band and B-band expression. B-band LPS was detectable by immunoblotting before measurable growth of the culture had occurred.     

Lipopolysaccharide heterogeneity in strains ofSerratia marcescens agglutinated by O14 antiserum

Lipopolysaccharides (LPS) from 71 strains ofSerratia marcescens that were agglutinated by O14 antiserum were examined by SDS-PAGE. Four major profiles were found, designated LPS1 to LPS4. These groups accounted for 51, 7, 5, and 3 strains respectively. Five strains were unclassified. Immunoblotting showed that O14 antibodies bound only to LPS1 and not to LPS2, 3, or 4. LPS1 also bound antibodies in O1, O4, O12, and O23 antisera. LPS2 reacted specifically with O8 antiserum, LPS3 with O6, and LPS4 with O2, O3, O6, O12, and O21 antisera. These reactions were not found in agglutination tests with boiled, whole-cell antigens. However, tests with autoclaved antigens (45 min at 121°C) corroborated the immunoblotting classifications; LPS1 strains belonged to serotype O14, LPS2 to serotype O8, LPS3 to serotype O6, and LPS4 to serotype O21. We conclude that there is a heat-stable antigen on many clinical strains ofS. marcescens that masks the expression of O-specific LPS antigens and which binds with nonspecific antibody in serum O14. We propose that O-antigens should be prepared from autoclaved cultures and that the H-reference strain O14H9 CDC 1783-57 (LPS2) should be reclassified as serotype O8.     

A heat-modifiable outer membrane protein carries an antigen specific for the species Yersinia enterocolitica and Yersinia pseudotuberculosis

The outer membranes of gram-negative bacteria are considered to be of importance in host-bacteria interaction, in protective immunity, and occasionally in subclassification within a species. In this study, the outer membranes of several strains of Yersinia enterocolitica and Y. pseudotuberculosis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the appearance of the major proteins depended on the temperature at which they were solubilized in SDS. A protein was identified with the use of two-dimensional gels and preparative SDS-PAGE, which was equivalent to the "heat-modifiable protein" (protein II) of other Enterobacteriaceae species. A monoclonal antibody, 4G1, was generated against an isolated preparation of this Y. enterocolitica protein. This antibody was tested with whole cell bacterial antigens of 46 individual bacterial strains. The reactive strains included only Y. enterocolitica and Y. pseudotuberculosis. In addition, the reactivity of the 4G1 monoclonal antibody preparation could be absorbed only with Y. enterocolitica and Y. pseudotuberculosis, and not with other strains of bacteria. The reactivity of this 4G1 monoclonal antibody was also tested by the Western Blot technique. Six individual strains were tested: a Y. enterocolitica serotype 0:3, a Y. enterocolitica serotype 0:9, an Escherichia coli, a Salmonella typhimurium, a Shigella flexneri, and a Klebsiella pneumoniae. The 4G1 antibody reacted with only the proteins of the two Y. enterocolitica strains. In conclusion, the equivalent of the heat-modifiable protein was present in Y. enterocolitica and Y. pseudotuberculosis. Moreover, this protein also carried a species-specific antigenic determinant.     

Mutations affecting lipopolysaccharide enhance ail-mediated entry of Yersinia enterocolitica into mammalian cells.

Two genes of Yersinia enterocolitica, inv and ail, have been identified as having a role in the bacterial adherence to and entry into mammalian cells in vitro. Expression of both genes is regulated by temperature. In stationary phase, ail gene expression is detectable only in bacteria at 37 degrees C, not at lower temperatures. An inv mutant derivative of Y. enterocolitica, which cannot enter mammalian cells when grown at 30 degrees C because of the lack of both inv and ail gene products, was mutagenized with the transposons mini-Tn10 and Tn5B50 to look for an increase in Ail-mediated cell entry. Sixteen mutants that could enter tissue culture cells after growth at 30 degrees C were selected. All of the mutants had increased cell surface Ail levels as detected by an Ail-specific monoclonal antibody. All of the ten Tn5B50 and one of the six mini-Tn10 mutants showed no increase in ail expression, but they had alterations in their lipopolysaccharide (LPS) such that no O side chains were detectable in bacteria grown at 30 degrees C. Thus, these mutants that are increased in their ability to enter cells appear to be so as a result of a change in the LPS on the surface resulting in increased levels of Ail protein able to interact with the mammalian cell surface. In the remaining mini-Tn10 mutants, LPS is normal, and the increase in cell surface Ail levels appears to be due to an increase in ail mRNA present in the cell. These mutants may therefore be affecting a repressor of ail gene expression.     

Genetic analyses of the putative O and K antigen gene clusters of pandemic Vibrio parahaemolyticus

Pandemic V. parahaemolyticus strains have rapidly changed their serotypes, but its determinants, especially K antigen, and the genes involved in serotype have been an open question. The purpose of this study was to gain insights into these points. Although V. parahaemolyticus is known to be lacking O-side chain on its lipopolysaccharide, and O antigens are thought to be represented by core OS, the genome sequence of V. parahaemolyticus O3:K6 strain RIMD2210633 suggests that this bacterium potentially synthesizes O-side chain. To explore possible relatedness between this O-side chain biosynthesis gene cluster, which is similar in the serotypes of Vibrio cholerae, and of V. parahaemolyticus, we amplified both core OS and O-side chain gene clusters of the strains belonging to various serotypes of V. parahaemolyticus by long PCR and performed PCR RFLP analyses. The results of our RFLP analyses suggest that the core OS biosynthesis gene cluster is related to the O antigens of pandemic V. parahaemolyticus and that the putative O-side chain gene cluster is related to K antigens of pandemic V. parahaemolyticus. We then determined the sequence of these regions of a pandemic O4:K68 strain, and compared it with the corresponding sequence of RIMD2210633. In addition, PCR analysis showed the putative O4 and K68 antigen gene clusters are unique to the strains belonging to the O4 and K68 serotype respectively. The data implies that the pandemic O4:K68 V. parahaemolyticus strain emerged from the pandemic O3:K6 strain by replacement of the putative O and K antigen gene clusters.     

Physical and antigenic heterogeneity in the flagellins of Listeria monocytogenes and L. ivanovii

Listeria monocytogenes serotypes 4a, 4b and 7, and L. ivanovii, all grown at 20 degrees C, were negatively stained and examined by electron microscopy. Crude extracts of the cell surface of L. monocytogenes serotypes 1/2b, 3b, 3c, 4a, 4b, 4d and 7 and of L. ivanovii (all grown at 20 degrees C) were examined by SDS-PAGE and Western blotting using (i) affinity-purified polyclonal monospecific antibody, and (ii) monoclonal antibody, each raised against 29 kDa flagellin of serotype 4b. No flagella were seen on serotype 7 by electron microscopy and no flagellin was detected in crude cell surface extracts of serotype 7 either in silver-stained gels or in Western blots. The monospecific polyclonal antibody detected flagellins of approximate molecular mass 29 kDa in each of the seven flagellate strains including L. ivanovii. The monoclonal antibody detected 29 kDa flagellin in serotypes 1/2b, 3b, 4a, 4b and 4d, but not the flagellins of serotype 3c or L. ivanovii, which had a slightly lower molecular mass. Following prolonged electrophoresis of crude flagellar extracts the 29 kDa complex was resolved into three closely migrating bands. In a heterologous system using serotype 1/2b crude flagellar extract, all three bands were detected using the polyclonal antibody whereas only two bands were detected by the monoclonal antibody. It is concluded that polyclonal anti-flagellin antibodies are not useful tools with which to distinguish serotypes of L. monocytogenes sensu lato in immunoblotting, but that differences can be determined using a monoclonal antibody directed against particular components of the flagellar complex. These differences did not fully correspond to those anticipated from results of agglutination tests.     

Enhanced cell-free protein synthesis using a S30 extract from Escherichia coli grown rapidly at 42 degrees C in an amino acid enriched medium

Growths of Escherichia coli strain A19 were investigated in a 5-L fermentor at 37 and 42 degrees C either in Pratt's medium (a standard medium for cell-free protein synthesis using its S30 extract) or in a casamino acids supplemented Pratt's medium (aa-enriched medium). Specific growth rates in Pratt's medium at 37 and 42 degrees C were 0.77 and 0.46 h(-1), respectively, whereas those in the aa-enriched medium at 37 and 42 degrees C were 0.87 and 1.49 h(-1), respectively. The extent of cell-free chloramphenicol acetyltransferase (CAT) synthesis was compared at 37 degrees C incubation (from a plasmid pK7-CAT) for S30 extracts prepared from the cells cultured in the aa-enriched medium at 37 or 42 degrees C. A 40% increase in CAT synthesis occurred when the 42 degrees C/S30 extract was used as compared with 37 degrees C/S30 extract. CAT and both the light and heavy chains (Lc and Hc) of the Fab fragment of an antibody 6D9 were synthesized at 37 degrees C in the cell-free synthesis in the presence of [(14)C]Leu. Their reaction mixtures were subjected to SDS-PAGE autoradiographic analysis. It was found that most of the synthesized proteins were in the soluble fraction when 42 degrees C/S30 extract was used, suggesting that the 42 degrees C/S30 extract contained greater amounts of various protein folding factors. A dialysis membrane minibioreactor with a reaction volume ca. 0.5 mL was handmade by the authors. The advantages of the minibioreactor are a simple configuration, a low manufacturing cost, and the capability of the dialysis membrane replacement. Increased CAT synthesis was also observed for continuous exchange cell-free (CECF) protein synthesis at 37 degrees C when the 42 degrees C/S30 extract was used in the minibioreactor. Some plausible reasons to give higher protein synthesis activity of the 42 degrees C/S30 extract are discussed.     

Characteristics of capsules in enterotoxemic Escherichia coli O139:K12 strains causing swine edema disease

The characteristics of the capsule of the enterotoxemic Escherichia coli (ETEEC) O139:K12 strains that strongly adhere to Hep-2 cells were examined. Electron microscopic studies using the freeze-substitution technique revealed that ETEEC strains had a capsule of approximately 25 nm. These strains show hydrophobic surface properties and strong adherence to human polymorphonuclear leukocytes (PMNs). In contrast, ETEEC strains RK-O139 and ED-1 show weak adherence to HEp-2 cells and fail to express the capsule layer on the cell surface. These ETEEC strains possess hydrophilic surface properties and also adhere to PMNs. The lipopolysaccharide (LPS) analysis by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that ETEEC strains had the same LPS profile and long O-side chains of LPS. Furthermore, all strains were resistant to serum killing activity. These results suggest that the capsule of ETEEC strains does not contribute as an antiphagocytic factor, but as an adherence factor to host cells.     

Heterogeneity of human melanoma-associated antigens defined by monoclonal antibodies and conventional xenoantisera

Summary Immunochemical analysis of cultured human melanoma cell detergent extracts and spent culture medium with conventional xenoantisera and monoclonal antibodies identified four types of 94,000 (94K) dalton molecules and two types of high-molecular-weight melanoma-associated antigens by the following characteristics: (1) association with other components, (2) mobility in SDS-PAGE under reducing and nonreducing conditions, (3) antigenicity, and (4) presence in spent culture medium. Conventional xenoantisera were found to contain antibody populations to antigenically distinct structures, some of which have similar apparent molecular weights. Immunodepletion studies showed that the antigenic determinant detected by the monoclonal antibody 225.28S to a high-molecular-weight melanoma-associated antigen was expressed on a subpopulation of the antigens defined by the conventional xenoantiserum #8995. These data prove that antibodies reactive with antigens of similar molecular weight cannot be assumed to identify the same structures, and indicate that tumor-associated antigens may be heterogeneous in the expression of antigenic determinants defined by monoclonal antibodies.Visiting investigator from the Veterans Administration Hospital, Minneapolis, MinnesotaVisiting investigator from Sapporo Medical College (Japan). Abbreviations used: MAA, melanoma-associated antigen; PBS, phosphate-buffered saline; NP40, nonidet P40; MoAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; 2-ME, 2-mercaptoethanol     

Monoclonal antibodies with a high degree of specificity for Listeria monocytogenes serotype 4b.

Strains of Listeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgG1) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37 degrees C). Heating L. monocytogenes cells at 80,90, or 100 degrees C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.     

Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus.

Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region.     

Pseudomonas aeruginosa lipopolysaccharide: evidence that the O side chains and common antigens are on the same molecule.

We investigated whether Pseudomonas aeruginosa produces two distinct lipopolysaccharides (LPS) containing either serologically variable O side chains or a neutral polysaccharide common antigen, designated A bands, that reacts with monoclonal antibody (MAb) E87. Immunoprecipitation of LPS and free O side chains with O-side-chain-specific antibodies or MAb E87 resulted in coprecipitation of both polysaccharides when antibody of either specificity was employed. Chromatography of LPS and free O side chains in a disaggregating deoxycholate buffer indicated the two polysaccharide antigens cochromatograph when eluates were analyzed by sensitive and specific enzyme-linked immunosorbent assay inhibitions. The LPS from a mutant of strain PAO1 that lacks polymerized O side chains but retains the common antigen eluted in fractions containing smaller LPS molecules, indicating the necessity of polymerized O side chains for elution in early fractions containing large LPS monomers. A phosphomannomutase mutant of P. aeruginosa PAO1 makes a rough LPS lacking both O side chains and common antigen but still produces a small (< 6-kDa) common antigen component detectable in cell lysates. Introduction of the cloned pmm gene into this strain restored production of a smooth LPS expressing large MAb E87-reactive common antigen. Destruction with NaOH of O side chains on recombinant LPS molecules eluting early from the molecular sieve column resulted in a shift of the MAb E87-reactive antigen to the late-eluting fractions. These results indicate that on most P. aeruginosa LPS molecules, O side chains and neutral polysaccharide common antigens are both present.     

The role of glucose in the Kluyveromyces bulgaricus flocculation phenomenon: transduction by cAMP-dependent protein kinase pathway?

The lipopolysaccharide (LPS) from eight strains of Yersinia pestis which had been cultured at 28 degrees C appeared to be devoid of an O-antigen when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LPS isolated from three of these strains which had been cultured at 37 degrees C also appeared to be devoid of an O-antigen. When the LPS from Y. pestis strain CO92 was purified and analysed by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry, the observed signals were in the mass range predicted for molecules containing lipid A plus the core oligosaccharide but lacking an O-antigen. The nucleotide sequence of Y. pestis strain CO92 revealed the presence of a putative O-antigen gene cluster. However, frame-shift mutations in the ddhB, gmd, fcl and ushA genes are likely to prevent expression of the O-antigen thus explaining the loss of phenotype.     

Monoclonal antibodies that disrupt poliovirus only at fever temperatures.

Three of thirty-six monoclonal antibodies were found to cause irreversible inactivation of type 1 poliovirus at 39 degrees C but not at 37 degrees C. Neutralization at 37 degrees C depended on aggregation and was reversible by acid-induced deaggregation; at 39 degrees C, the virions (N antigenic, 160S) were disrupted to empty capsids (H antigenic, 100S), and neutralization was irreversible. The rate of antibody-dependent conversion of N to H antigen increased steeply between 37 and 39 degrees C.     

Variation in lipid A structure in the pathogenic yersiniae

Important pathogens in the genus Yersinia include the plague bacillus Yersinia pestis and two enteropathogenic species, Yersinia pseudotuberculosis and Yersinia enterocolitica. A shift in growth temperature induced changes in the number and type of acyl groups on the lipid A of all three species. After growth at 37 degrees C, Y. pestis lipopolysaccharide (LPS) contained the tetra-acylated lipid IV(A) and smaller amounts of lipid IV(A) modified with C10 or C12 acyl groups, Y. pseudotuberculosis contained the same forms as part of a more heterogeneous population in which lipid IV(A) modified with C16:0 predominated, and Y. enterocolitica produced a unique tetra-acylated lipid A. When grown at 21 degrees C, however, the three yersiniae synthesized LPS containing predominantly hexa-acylated lipid A. This more complex lipid A stimulated human monocytes to secrete tumour necrosis factor-alpha, whereas the lipid A synthesized by the three species at 37 degrees C did not. The Y. pestis phoP gene was required for aminoarabinose modification of lipid A, but not for the temperature-dependent acylation changes. The results suggest that the production of a less immunostimulatory form of LPS upon entry into the mammalian host is a conserved pathogenesis mechanism in the genus Yersinia, and that species-specific lipid A forms may be important for life cycle and pathogenicity differences.