Robin E. Harkness

Cloning and expression of the Haemophilus influenzae transferrin receptor genes.

The genomic transferrin receptor genes (tbpA and tbpB ) from two strains of Haemophilus influenzae type b (Hib) and two strains of non‐typable H. influenzae (NTHi) have been cloned and sequenced. The deduced protein sequences of the H. influenzae tbpA genes were 95–100% conserved and those of the tbpB genes were 66–100% conserved. The tbpB gene from one strain of NTHi was found to encode a truncated Tbp2 protein. The tbpB genes from four additional NTHi strains were amplified by the polymerase chain reaction (PCR) utilizing primers derived from the conserved N‐terminal sequences of Tbp1 and Tbp2 and were found to encode full‐length proteins. Although several bacterial species express transferrin receptors, when the Tbp1 and Tbp2 sequences from different organisms were compared, there was only limited homology. Recombinant Tbp1 and Tbp2 proteins were expressed from Escherichia coli and antisera were raised to the purified proteins. There was significant antigenic conservation of both Tbp1 and Tbp2 amongst H. influenzae strains, as determined by Western blot analysis. In a passive model of bacteraemia, infant rats were protected from challenge with Hib after transfer of anti‐rTbp2 antiserum, but not after anti‐rTbp1 antiserum.

Cross-Reaction Between Mammalian Cell Entry (Mce) Proteins of Mycobacterium tuberculosis

In addition to the previously cloned Mce1A and Mce1E genes of the Mce1 operon of Mycobacterium tuberculosis (Ahmad et al. Scand J Immunol 1999;50:510–8), Mce1B, Mce1D and Mce1F were cloned and expressed as glutathione‐S‐transferase (GST) fusion proteins in recombinant Escherichia coli. Polyclonal antibodies against a predicted B‐cell epitope of each of the Mce1 proteins of M. tuberculosis were produced by immunizing rabbits with synthetic peptides coupled to keyhole limpet haemocyanin. These antibodies reacted specifically with the corresponding fusion protein, except for GST‐Mce1F. A mouse monoclonal antibody, TB1‐5 76C, raised against a synthetic 60‐mer peptide corresponding to the residues 106–165 in the N‐terminal part of Mce1A, reacted strongly with GST‐Mce1A. The antibody cross‐reacted with GST‐Mce1F, but not with the other recombinant GST‐Mce1 fusion proteins or free GST. Bioinformatic analysis revealed only slight homology between Mce1A and Mce1F, along the length of the polypeptide chains. Higher homology was found between the residues 106–165 of Mce1A and the residues 347–406, further into the mature Mce1F polypeptide chain. There was a striking, localized homology, indicating that the epitope reacting with the monoclonal antibody TB1‐5 76C may be narrowed to the KRRITPKD region, the residues 131–138 in Mce1A corresponding to the residues 372–379 in Mce1F. This was confirmed in enzyme‐linked immunosorbent assay, showing binding of TB1‐5 76C to a 17‐mer synthetic peptide containing the KRRITPKD sequence.

Effect of lipid modification on the physicochemical, structural, antigenic and immunoprotective properties of Haemophilus influenzae outer membrane protein P6

The outer membrane lipoprotein, P6 of Haemophilus influenzae was studied to determine the importance of the native palmitoyl moiety on its physicochemical and immunological properties. A recombinant P6 (rP6) molecule devoid of lipidation signal sequence was expressed in Escherichia coli and its properties were compared to those of the palmitylated protein purified from H. influenzae. The isoelectric point of rP6 was more acidic than that of the native protein and also exhibited less secondary structure than P6 as judged by circular dichroism. However, both forms of P6 induced identical P6-specific antibody titers in guinea pigs when Freunds adjuvant was used. These antisera reacted with a panel of overlapping P6 peptides in a comparable manner and in addition, rabbit antisera raised against the P6 peptides reacted equally well with P6 and rP6. Furthermore, all human convalescent sera tested exhibited similar anti-P6 and anti-rP6 antibody titers. However, rP6 was less immunogenic than P6 when administered either without adjuvant or in alum and when tested in competitive inhibition studies with anti-P6 antibodies, was a less effective inhibitor than native P6, suggesting a diminution in some of the antigenic activity of rP6. In spite of these differences, rP6 was capable of eliciting a protective antibody response against live H. influenzae type b challenge in a modified infant rat model of bacteremia. These findings demonstrate that the non-fatty acylated rP6 could possibily be substituted for native P6 in a vaccine against H. influenzae.

Predicted molecular structure of the mammalian cell entry protein Mce1A of Mycobacterium tuberculosis

The proposed role of the mammalian cell entry protein 1A (Mce1A) of Mycobacterium tuberculosis is to facilitate invasion of host cells. The structure of Mce1A was modelled on the basis of the crystal structure of Colicin N of Escherichia coli by fold prediction and threading. Mce1A, as the model predicts, is an alpha/beta protein consisting of two major (alpha and beta) domains, connected by a long alpha helix. The model further revealed that the protein contains 12 helices, 9 strands, and 1 turn. The final model of Mce1A was verified through the program VERIFY 3D and more than 90% of the residues were in the favourable region. A mouse monoclonal antibody, TB1-5 76C, is directed to an epitope within a 60-mer peptide that has been shown to promote uptake of bacteria in mammalian cells. We show here that the epitope could be narrowed down to a core of 4 amino acids, TPKD. Upstream flanking residues, KRR also contributed to binding. Mce2A does not promote uptake in mammalian cells and sequence comparison of Mce1A and Mce2A indicates that the epitope mediates uptake. The epitope was located at the surface of the Mce1A model at the distal beta strand-loop region in the beta domain. The localization of this epitope in the model confirms its potential role in promoting uptake of M. tuberculosis in host cells.

Branhamella catarrhalis pathogenesis in SCID and SCID/beige mice

SCID and SCID/beige mice were used to study the pathogenesis of B. catarrhalis administered by intranasal, intraperitoneal or intravenous routes. Challenged adult animals did not appear overtly clinically ill. Similar symptoms were observed regardless of the challenge route, and pretreatment of mice with human transferrin did not enhance clinical virulence. Susceptibility to B. catarrhalis appeared to be age‐dependent as some mice under one week of age died following challenge. Postmortem findings included circumscribed pale foci on the liver, splenomegaly and mineralization of the myocardium. Presence of lesions did not correlate with the assessment of clinical well being, and severity of the lesions was found to be challenge strain‐dependent. Liver lesions and splenomegaly were not observed in animals challenged with heat‐killed bacteria or placebo. SCID/beige mice were more affected than SCID mice both clinically and pathologically, suggesting that natural killer cell and polymorphonuclear cell functions may be important in resolving B. catarrhalis challenge.

Immunodominant B-Cell Epitope in the Mce1A Mammalian Cell Entry Protein of Mycobacterium tuberculosis Cross-Reacting with Glutathione S-Transferase

The TB1‐5 76C monoclonal antibody raised against a synthetic 60‐mer peptide in the N‐terminal part of the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis has previously been shown to react with a linear epitope in the KRRITPKD region, residues 131–138 in Mce1A, and to cross‐react with Mce1F. Six additional monoclonal antibodies raised against the same peptide were also shown to cross‐react with Mce1F. Four of them reacted with a linear epitope in the same area, indicating that this area is immunodominant but showed distinct differrences in fine specificity. Two monoclonal antibodies did not react with synthetic peptides from this region on the solid phase in enzyme‐linked immunosorbent assay, indicating greater influence of conformation on reactivity. None of the monoclonal antibodies reacted with 14‐mer synthetic peptides from the corresponding area in Mce2A, Mce3A, Mce4A, M. avium, M. smegmatis or M. leprae. The reaction pattern of the monoclonal antibodies was analysed in relation to our model of the Mce1A molecule (AK Das et al. Biochem Biophys Res Commun 2003;302:442–7).The epitope is located on the surface of Mce1A, at the distal β‐strand‐loop region in the β‐domain supporting its potential role in promoting uptake of M. tuberculosis in host cells. Monoclonal antibody TB1‐5 19C cross‐reacted with glutathione S‐transferase of Schistosoma japonicum containing a PKE triplet. Monoclonal antibody TB1‐5 76C gave a major band at about 44 kDa in Western blotting of M. tuberculosis sonicate, whereas polyclonal rabbit anti‐Mce1A peptide antibodies reacting with the extended TTPKNPTKRRITPKDVI area of Mce1A showed a distinct band above the 160 kDa molecular mass standard.