Hemagglutinin Production Research Articles

Lethal infection by Bordetella pertussis mutants in the infant mouse model

Different aspects of lethal infection of infant mice with Bordetella pertussis were examined. Mutants deficient in vir-regulated genes were tested for the ability to cause a lethal infection in the infant mouse model. Adenylate cyclase toxin-hemolysin and pertussis toxin were required to cause a lethal infection at low doses. Mixed infection caused by challenging the mice with an equal number of pertussis toxin and adenylate cyclase toxin-hemolysin mutants at a dose at which neither alone was lethal was also unable to cause a lethal infection. Production of the filamentous hemagglutinin and the dermonecrotic toxin was not required to cause a lethal infection. Nine other mutants in vir-regulated genes whose phenotypes have yet to be determined were also tested. Only two of these mutants were impaired in the ability to cause a lethal infection. Expression of fimbriae does not appear to affect the dose required to cause a lethal infection; however, fimbrial expression was correlated with the later stages of a nonlethal, persistent infection. Growth of the bacteria in MgSO4, a condition which reversibly suppresses expression of the genes required for virulence, did not alter the ability of the bacteria to cause a lethal infection. Auxotrophic mutants deficient in leucine biosynthesis were as virulent as the parental strain; however, mutants deficient in methionine biosynthesis were less virulent. A B. parapertussis strain was much less effective in promoting a lethal infection than any of the wild-type B. pertussis strains examined. A persistent infection in the lungs was observed for weeks after challenge for mice given a sublethal dose of B. pertussis, and transmission from infected infants to the mother was never observed.

Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes

Mutants of Bordetella pertussis deficient in virulence-associated factors were identified by using the transposon Tn5 lac. Tn5 lac is a derivative of Tn5 which generates promoter fusions for beta-galactosidase. Tn5 lac insertions in the vir-regulated genes of B. pertussis were identified by selecting for kanamycin-resistant mutants that expressed beta-galactosidase when the vir-regulated genes were expressed but not when the vir-regulated genes were turned off. Fourteen different mutations in vir-regulated genes were identified. Two mutants were deficient in the production of the filamentous hemagglutinin, two mutants were deficient in the production of adenylate cyclase toxin and hemolysin, and one mutant was deficient in the production of dermonecrotic toxin. One insertion mapped adjacent to the pertussis toxin gene, but the mutant produced pertussis toxin. The phenotypes of the remaining eight mutants were not determined, but the mutants did not appear to be deficient in the production of the 69,000-dalton outer membrane protein (agglutinogen 3) or the capsule. Screening for mutations in either of the fimbrial genes proved to be problematic since the parental strain was found to switch from a fimbriated to a nonfimbriated state at a high frequency, which was suggestive of the metastable expression of pili in other bacteria. We used Southern blot analysis with a 30-mer specific for the fimbrial sequences. No bands with the predicted increase in size due to the 12 kilobases from Tn5 lac were observed, which suggests that none of these genes were mutated. Southern blot analysis also revealed that seven of the eight unidentified mutations mapped to different restriction fragments, which suggests that they could be deficient in as many as seven different genes.

Rapid detection of influenza virus infections in human fetal lung diploid cell cultures

Haemadsorbing foci were found in human fetal lung (HFL) diploid cell cultures 12 h after inoculation with influenza viruses A and B. The size and number of the foci were maximal after 48 h of incubation, being limited by production of an unidentified inhibitor. By contrast, inoculation with parainfluenza virus type 3 led to haemadsorption which increased during 10 days of incubation. For the detection of influenza viruses A and B maximum sensitivity was achieved by changing the medium, the day before use to one that was serum free. The number of foci at 15.5 h post-infection and infectivity for primary African green monkey kidney (AGMK) cultures were similar. Virus infectivity and production of haemagglutinin in HFL cells were entirely cell-associated; they were not affected by treatment with trypsin. Nevertheless, influenza viruses A and B antigens were identified in the infected cells by means of immunofluorescence at 15.5 h and virus was recovered by passage of frozen and thawed cells in AGMK cultures. For rapid routine diagnosis of viral infections, the early haemadsorption test was shown to have the same sensitivity as immunofluorescence tests on specimens and virus detection by the shell-vial technique but was cheaper and simpler to perform.

The effect of cyclodextrin on lipopolysaccharide production in cultures of Bordetella pertussis

The effect of adding 500 micrograms of (2,6-0-dimethyl) beta-cyclodextrin (Me-beta-CD) per ml of Stainer-Scholte (SS) medium in two-day shaker flask cultures of Bordetella pertussis on the production of lipopolysaccharide (LPS) was investigated. The amount of LPS per 10(9) cells found in the supernatants of these cultures was either somewhat reduced or unaffected by comparison with the amounts in cultures grown in SS-medium alone. In addition, the time course of LPS release from cultures of B. pertussis strain 3843 cells during a 96-h growth period in normal and Me-beta-CD-enriched SS medium is described. By using the enriched medium bacterial growth, the production of filamentous haemagglutinin (FHA) and of pertussis toxin (Pt) and the levels of haemagglutination and lymphocytosis-promoting activity were enhanced to various degrees. Measurements made on sedimented whole and on sonicated B. pertussis cells grown in the two media showed no differences in LPS content. The reasons for the reduced/unaffected LPS production are discussed. It has been suggested that an interaction between hydrophobic cavities of the Me-beta-CD molecules and the 'lipid A' part of LPS reduces the reactivity of LPS in the Limulus Amoebocyte Lysate (LAL) assay. This possibility, however, was rejected as the reactivity of Me-beta-CD-spiked purified B. pertussis strain 3803 LPS, compared with unspiked samples, remained unchanged.

Vibrio cholerae non-O1: production of cell-associated hemagglutinins and in vitro adherence to mucus coat and epithelial surfaces of the villi and lymphoid follicles of human small intestines treated with formalin.

Clinically isolated Vibrio cholerae non-O1 strains produced more cell-associated hemagglutinins (HAs) on colonization factor antigen agar after ca. 3 h than after ca. 20 h of incubation at 37 degrees C. A high cell-associated HA producer variant of strain TVN-318, grown for 3 h at 37 degrees C, was entrapped in a native mucus coat covering the human ileal mucosa and displayed a striking ability to adhere to the surface of a Formalin-treated mucus coat, in contrast to a poor cell-associated HA producer variant of TVN-318, grown for 20 h at 37 degrees C. Adherence to the Formalin-treated human mucus coat was confirmed with all of the strains tested. V. cholerae non-O1 strains also possessed the ability to adhere to the epithelial surfaces of Formalin-treated human and rabbit ileal or jejunal villi, as well as human lymphoid follicles, in proportion to cell-associated HA levels. The epithelial surface of the lymphoid follicles provided most of the adherence sites for V. cholerae non-O1 strains under the test conditions. We conclude that a mucus coat covering the human small intestinal mucosa is a primary adherence target for V. cholerae non-O1 strains in human intestinal infections and that cell-associated HAs have at least a partial role in the adherence of V. cholerae non-O1 strains to the human small intestine, suggesting a potential role for V. cholerae non-O1 strains in an oral live vaccine.

Correlation between electrophoretic types B1 and B2 of carboxylesterase B and host-dependent factors in Escherichia coli septicaemia.

Electrophoretic types B1 and B2 of carboxylesterase B produced by strains of Escherichia coli isolated from 100 septicaemia cases were correlated with alpha-haemolysin and mannose resistant haemagglutinin (MRHA) production and with clinical data including eventual underlying diseases, origin of septicaemia and evolution. Electrophoretic type B2 was phenotypically linked with alpha-haemolysin and MRHA production. The proportion of type B2 isolates varied significantly with occurrence of an underlying illness (45% for patients without an underlying disease and 22% for compromised patients) and with the site of origin of the septicaemia (40% for those of urinary origin and 18% for infection of digestive origin). In the former infections, type B2 isolates were obtained in the majority from male patients while type B1 isolates predominated in women. The septicaemias associated with type B1 were characterized by a lower proportion of isolates producing alpha-haemolysin and MRHA and by a greater frequency of septic shock and death than those associated with type B2. These facts emphasize the importance of host-dependent factors in E. coli septicaemia.

Late intervention therapy using immunomodulators and antiserum in a of model Flavivirus infection : D. H. Coppenhaver, I. P. Singh, J. Poast, P. Sriyuktasuth, M. Sarzotti, S. Baron. Dept. of Microbiology, University of Texas Medical Branch, Galveston, Texas, USA 77550

Several Togaviridae of the alphavirus and flavivirus genera agglutinate trypsinized human group 0 erythrocytes (THOE) (Shortridge and Hu, 1976). Haemagglutinin titers of Semliki Forest virus (SFV) and Japanese encephalitis virus (JEV) measured with THOE were equivalent to, if not higher than, those obtained with Embden gander erythrocytes, even with unextracted haemagglutinin. Results obtained with THOE in JEV haemagglutination-inhibition tests on sera taken from a previously infected individual over a 20-yr period were similar to those measured during the initial JEV infection. The inhibition of SFV haemagglutinin production as measured with THOE was a very sensitive bioassay for chicken interferon: interferon titers were 6- to 10-fold higher than those obtained with the vesicular stomatitis virus plaque-reduction method. The generally greater availability of human erythrocytes (including those stabilized with gluteraldehyde), the simplicity of the trypsin treatment, and the possibility of using unextracted haemagglutinin recommend this technique for use with haemagglutinating Togaviridae.

Influence of insulin and 12- O-tetradecanoylphorbol-13-acetate (TPA) on influenza virus multiplication

Insulin and 12- O-tetradecanoylphorbol-13-acetate (TPA) interfere with the multiplication of fowl plague virus, an influenza A virus, in primary chick embryo cells. Specifically the production of the viral glycoproteins hemagglutinin and neuraminidase are affected by the drugs. A decrease or omission of glucose from the culture medium enhances this effect, which is in agreement with the idea that these drugs act on virus replication via a shortage of glucose in the host cell. Virus replication in cells of different organs is affected to different extents by insulin and TPA.

Differences in adhesiveness among type 1 fimbriate strains of Enterobacteriaceae revealed by an in vitro HEp2 cell adhesion model.

Ten type 1 fimbriate strains of Enterobacteriaceae were examined in an in vitro adhesion assay with HEp2 epithelial cells. The range of HEp2 cell adhesiveness, which was characteristic for each strain, was affected by motility, type 1 fimbriation and production of mannose sensitive haemagglutinin. Nevertheless, not all type 1 fimbriate strains adhered well in this model. The findings are discussed with regard to the possibility that different type 1 fimbriate enterobacteria, though all are mannose sensitive, recognize different mannose-containing receptors present or available on the surfaces of the HEp2 cells.

Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin.

Recombinant plasmids encoding a Vibrio cholerae haemagglutinin were isolated from the highly virulent V. cholerae strain C5 by cosmid cloning. Both Escherichia coli HB101 containing the recombinant plasmids and V. cholerae C5 were able to agglutinate a variety of erythrocytes from human and animal origin; this haemagglutination was not inhibited by D-mannose or L-fucose. Subcloning of the recombinant cosmid DNA revealed that a 1.3 kb DNA fragment was sufficient for haemagglutinin production in E. coli HB101. Under direction of this 1.3 kb Vibrio DNA fragment, two proteins were made in E. coli minicells, of 27 and 10 kDa. Haemagglutinin-encoding sequences were not detected in every V. cholerae strain.

Filamentous hemagglutinin has a major role in mediating adherence of Bordetella pertussis to human WiDr cells.

[35S]methionine-labeled Bordetella pertussis adhered to monolayers of WiDr cells, an epitheliumlike cell line from a human intestinal carcinoma. Adherence was proportional to the density of the WiDr cells and to the concentration of B. pertussis in the assay. Adherence of virulent phase I strains Tohama phase I, 114, and BP338 was much greater than adherence of avirulent strains Tohama phase III and 423 phase IV. Mutants deficient in the production of the filamentous hemagglutinin (FHA) were hemagglutination negative and adhered to WiDr cells much less efficiently than the parent strains. Preincubation of B. pertussis cells with FHA increased their hemagglutination activity and adherence to WiDr cells. Goat antibody to FHA inhibited, in a dose-dependent manner, the adherence of strain Tohama I but not the adherence of FHA-deficient mutant Tohama 325. At similar protein concentrations, normal goat antibody, goat antibody to pertussis toxin, or the Fab fragments of goat antibody to serotype 2 fimbriae had no effect on adherence. Also, an FHA-positive strain without fimbriae showed high adherence, while a fimbriated FHA-deficient mutant adhered poorly. Our data indicate that FHA plays a major role in adherence of B. pertussis to human WiDr cells. Fimbriae do not appear to mediate attachment of B. pertussis to WiDr cells.

Protection of Mice from Lethal Influenza: Evidence that Defective Interfering Virus Modulates the Immune Response and Not Virus Multiplication

Intranasal administration of defective interfering (DI) influenza virus (A/WSN) ensured the survival of 80% of C3H mice otherwise lethally infected with WSN by the intranasal route, whereas a control group which received beta-propiolactone-inactivated DI virus in place of DI virus died at 7.4 days post-infection. DI virus-treated mice developed significantly less lung consolidation than controls although qualitatively the cellular pathology in the two groups was indistinguishable. Surprisingly, in view of the accepted mode of action of DI virus interference, multiplication of infectious virus in the lung, production of viral haemagglutinin (HA) antigen and neuraminidase, and the distribution and amount of viral antigen in cells as shown by immune labelling were unaffected by the presence of active DI virus. Furthermore, assays of lung extracts showed that DI virus was not stimulating significantly greater amounts of interferon than the control inactivated DI virus. An alternative explanation arises from the fact that the pathology of influenza in inbred mice is immune (T lymphocyte)-mediated. Thus, since there is no evidence that DI virus affects virus multiplication we suggest that DI virus is responsible for ameliorating the damaging host responses. Another aspect of the immune response modulated by DI virus was the enhancement of local haemagglutination-inhibiting (HI) antibody in the lung, with peak increases of up to 10-fold over the relevant controls being demonstrated at 5 days after infection. This antibody was presumably complexed to HA antigen in the lung as activity was only demonstrated after elution at low pH. It had no detectable neutralizing activity (less than 10% HI: neutralization ratio of convalescent serum) which accounts for the coexistence of local antibody and virus infectivity. Mice infected with virus alone or which received beta-propiolactone-inactivated DI virus in addition to a lethal dose of WSN did not develop significant amounts of lung antibody. No differences were seen in serum HI titres. The increased level of antibody could not be attributed to the presence of greater amounts of HA antigen in lungs of mice treated with DI virus, as ELISA showed no significant difference from control preparations. The possibility that the two modulated immune responses are linked through HI antibody blocking access of T cells to cell membrane-borne HA antigen is discussed.

Suppressive effect of mizoribine on humoral antibody production in DBA/2 mice.

The mode of action of mizoribine (MZR) as a B cell inhibitor was studied using DBA/2 mice. Its in vitro administration significantly delayed the primary response in hemagglutinin production against sheep erythrocytes by suppressing the IgM antibody formation. In vitro plaque-forming cell (PFC) response against both T-dependent and T-independent antigens, such as TNP-SRBC and TNP-Brucella abortus, was dose-dependently suppressed by MZR. Since PFC formation by the T-depleted fraction of splenocytes was likewise suppressed, MZR may inhibit humoral antibody response by directly affecting the B cells (and/or macrophages) as well as by modulating the regulatory T cells. MZR may only act on a certain stage of the cell cycle of B lymphocytes following antigenic stimulation. It may not interfere with the initial antigen recognition or with mature B cells.

Fimbrial and non-fimbrial haemagglutinins in Enterobacter aerogenes.

Ten strains of Enterobacter aerogenes were examined for their ability to produce haemagglutinins and fimbriae. Nine strains formed a mannose-sensitive (MS) haemagglutinin associated with thin (4 nm) non-channelled fimbriae. These thin fimbriae of E. aerogenes were antigenically different from the thin fimbriae of other fimbriate strains of Enterobacter and Klebsiella and probably represent a new kind of fimbria not previously described in Enterobacteriaceae. Eight of these same nine strains also formed a non-fimbrial mannose-resistant, proteus-like (MR/P) haemagglutinin. The formation of thin fimbriae associated with MS haemagglutinin and of non-fimbrial MR/P haemagglutinin are properties not associated with other strains of Enterobacter and Klebsiella. E. aerogenes strain NCIB11460 was unusual among the strains examined in this series in that it alone produced mannose-resistant, Klebsiella-like (MR/K) haemagglutinin and type-3 fimbriae which, as judged by immunoelectronmicroscopy, were antigenically like those of type-3 fimbriate Klebsiella strains. The identifying characters of this exceptional strain of E. aerogenes are discussed in detail. All ten strains also produced thick fimbriae which by immunoelectronmicroscopy behaved like the type-1 fimbriae of Klebsiella strains. However, correlation between their presence and the production of MS haemagglutinin in E. aerogenes was not established. The findings are discussed in the light of the present difficult taxonomic status of E. aerogenes within the tribe Klebsielleae.

A new culturing method for production of filamentous hemagglutinin of Bordetella pertussis

The addition of cyclodextrins, especially heptakis (2,6- O-dimethyl)β-cyclodextrin (MeβCD), to synthetic medium, such as Stainer-Scholte medium (D.W. Stainer and M.J. Scholte, J. Gen. Microbiol. 63: 211–220) greatly stimulated the production of filamentous hemagglutinin (F-HA) by Bordetella pertussis Tohama phase I under shaking conditions. Purified F-HA from this culturing system was demonstrated immunochemically and morphologically to be identical with that from static culture.

Pertussis toxin and extracytoplasmic adenylate cyclase as virulence factors of Bordetella pertussis.

A wild-type strain of Bordetella pertussis and a series of transposon Tn5-induced mutants deficient in the production of various factors believed to play a role in pertussis (whooping cough) were tested for virulence in infant mice. The 50% lethal dose of the wild-type strain in these animals was 2 X 10(3) bacteria. A mutant deficient in the production of the filamentous hemagglutinin was almost as virulent as the wild type. Avirulent phase mutants (i.e., deficient in the production of all toxins), a pertussis-toxin mutant, and a double mutant deficient in both hemolysin and adenylate cyclase were severely impaired in the ability to cause pertussis. These data underscore the role of pertussis toxin in the disease and provide the first direct evidence that adenylate cyclase and hemolysin are important in the pathogenesis of the infection.

Spontaneous phase variation in Bordetella pertussis is a multistep non-random process.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi-synthetic solid media. The frequency of these variants was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non-random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.

Modulation of IgE Response by Phytohemagglutinin

The effect of the administration of phytohemagglutinin (PHA) on the IgE response was studied. RF mice were immunized with OA in aluminum hydroxide gel and PHA was injected 1 or 2 days before the immunization in doses of 80 or 1 microliter. Results show that PHA injected on day -1 induces suppression and PHA administered on day -2 induces some stimulation in the IgE response. This effect is independent of the PHA doses used and does not affect the anti-OA hemagglutinin production.

Grouping in Japanese Quail: 2. Suppression of Humoral Immunity

Four-week-old Japanese quail were placed in either stable (isolate) or unstable (resident-visitor) groupings in this study. The visitor quail were transferred daily to cages containing unfamiliar residents, and the isolate quail were handled daily. The immune responsiveness to sheep red blood cells (SRBC), as indicated by production of hemagglutinins of the visitors and residents, was significantly less than that of the isolates, and the visitors were found to have less antibody than the residents. In subsequent experiments adrenocorticotropic hormone (ACTH) was used to compare its effect on immune responsiveness to that caused by grouping. The ACTH caused a suppression in humoral immune response, but only 8 IU ACTH/100 g body weight consistently suppressed antibody levels, indicating that the quail immune system is moderately resistant to this hormone. At the end of the experimental periods the bursa of Fabricius, spleen, and adrenal glands were removed. The relative weights of the organs suggested that grouping and ACTH did not induce the classical symptoms of physiological stress. However, stress was not eliminated as a possible cause of the immunosuppressive effects of grouping and ACTH found in this study, because there was significant body weight loss in unstable groupings and in ACTH-treated birds.

Inhibition of feline leukemia virus replication by human leukocyte interferon

The replication of feline leukemia virus (FeLV) is inhibited by treatment of cat cell cultures with crude human leukocyte interferon (HuIFN-α) as evidenced by titration of the infectious progency. The inhibition can be demonstrated in three different cell lines in which the production of hemagglutinin by encephalomyocarditis (EMC) virus, and plaque formation by vesicular stomatitis virus (VSV) are also inhibited by the HuIFN-α. The dose dependency of the inhibition of EMC virus by the HuIFN-α is similar to that obtained with feline interferon in each of the three cell lines. VSV and EMC virus are less than 10 times more sensitive than FeLV to the inhibitory action of HuIFN-α if responses to a single interferon treatment are compared for each of the viruses tested in the most sensitive cell line, FEA. The interferon effect on FeLV is more pronounced when it is added within one day after the inoculation of the cells rather than applied before cell infection. The induction of focus formation by FeLV can also be inhibited by HuIFN-α in cat cells (CCC-81) which contain the murine sarcoma virus genome.