Gunhild Jonson

Cloning and sequencing of Vibrio cholerae mannose‐sensitive haemagglutinin pilin gene: localization of mshA within a cluster of type 4 pilin genes

The mannose‐sensitive haemagglutinin (MSHA) pilus that is associated with Vibrio cholerae strains of EI Tor biotype has been shown to be a potential colonization factor and protective antigen. The gene encoding the structural subunit of MSHA pili was cloned from size‐fractionated Sacl‐cleaved chromosomal DNA in the expression phage vector lambda ZAPII. Positive clones carried a c. 5.3 kb Sacl fragment and were identified on the basis of MSHA expression and hybridization with a synthetic oligonucleotide probe based upon the N‐terminus of MshA, the structural subunit of MSHA. The mshA gene vitas localized to a 2.6kb Sall‐EcoRI fragment, which was subcloned and shown to express MshA from Its own promoter in Escherichia coli. Nucleotide sequencing of the entire fragment revealed six open reading frames (ORFs) of which four were complete. The mshA gene encodes an 18094 Da prepilin protein, which in its mature form has a size of 17 436 Da. MshA is a type 4 (N‐MePhe) pilin protein that is more homologous to pilins produced by Pseudomonas aeruginosa and Neisseria gonorrhoeae than to TcpA, the structural subunit of the toxin‐coregulated pilus of V. cholerae. The protein seems to be directly involved in receptor binding, as an in‐frame mutation in the mshA gene was found to abolish both d‐mannose‐dependent haemagglutination and binding of V. cholerae bacteria to d‐mannose‐containing agarose beads. Three additional ORFs, all in the same transcriptional orientation as mshA, were found to encode type 4 pilin‐like proteins. A potential promoter with a sequence homologous to that of cAMP‐CRP‐activated promoters in E. coli was identified upstream of ORF3, the gene preceding mshA.

Identification of a mannose-binding pilus on Vibrio cholerae El Tor

The mannose-sensitive hemagglutinin (MSHA) that is associated with Vibrio cholerae strains of El Tor biotype is identified as a pilus composed of subunits with a molecular mass of approximately 17 kDa. In immunoelectron microscopy, a monoclonal antibody against MSHA that inhibited El Tor vibrio-mediated mannose-sensitive agglutination of chicken erythrocytes or El Tor bacterial binding to mannose-coated agarose beads, bound specifically to repetitive subunits along typical fimbriae extending from the surface of El Tor vibrios. No such pili were seen on the surface of MSHA negative classical vibrios, although non-surface exposed fimbrial subunits could be demonstrated in these bacteria by immunoblotting techniques.

The maltose-inducible 43 kDa major outer membrane protein in Vibrio cholerae is immunogenic and common to different isolates

We have previously demonstrated the presence of a maltose-inducible major outer membrane protein of 43 kDa in an El Tor Inaba strain of Vibrio cholerae. The occurrence of similar proteins was examined in several isolates of V. cholerae 01. The results indicate that the 43 kDa protein is common to all of the isolates as evidenced by Western blotting analysis with antiserum raised against this protein. The 43 kDa protein was maltose-inducible in most isolates although some strains exhibited a constitutive production of the protein. This protein was present also on V. cholerae 01 organisms harvested directly from the small intestine of rabbits with experimental cholera and it gave rise to specific antibodies after immunization with in vivo grown vibrios.

Enzyme‐linked immunosorbent assay for determination of antibodies to Vibrio cholere toxin‐coregulated pili

An ELISA for determination of antibodies to V. cholerae TCP was developed. Since purified TCP preparations contained detectable amounts of LPS (as shown by ELISA and immunoelectron microscopy with anti‐LPS polyclonal serum), a capture ELISA was used. In this test the plate was coated with anti‐TCP monoclonal antibody followed by incubation with TCP fimbriae. By this procedure no LPS bound to the solid phase as shown by the loss of reactivity with anti‐LPS serum. The capture ELISA allowed sensitive and specific determination of TCP antibodies in sera of rabbits immunized with classical but not El Tor V. cholerae strains. There was good agreement between results in the TCP ELISA and reactivity with the TcpA band in immunoblot analyses when antisera raised against classical and El Tor vibrios were studied.

Immune Response to Vibrio cholerae Infection in Rabbits with Special Reference to Antibodies Against in vivo Specific Antigens

In Vibrio cholerae 01 bacteria, the causative agent of cholera, changes in the in vitro environment have been found to have marked influences both on the production of cholera toxin and of cell-surface associated structures, e.g. haemagglutinins, outer membrane proteins and a toxin-coregulated pilus (TCP)1,2. We have previously shown that the intestinal milieu provide an environment in which V. cholerae 01 of both El Tor and classical biotype expresses new surface antigens, that are not found after in vitro-growth3. We have also recently shown, by studying in vivo-grown bacteria, that both TCP and a putative adhesin on El Tor vibrios, i.e. the mannose-sensitive haemagglutinin (MSHA), are expressed by V. cholerae during infection4.