Development of non-antibiotic-resistant, chromosomally based, constitutive and inducible expression systems for aroA-attenuated Salmonella enterica Serovar Typhimurium.

Live-vaccine delivery systems expressing two model antigens from Mycoplasma hyopneumoniae, F2(P97) (Adh) and NrdF, were constructed using Salmonella enterica serovar Typhimurium aroA (STM-1), and immunogenicity in mice was evaluated. Recombinant plasmid-based expression (PBE) and chromosomally based expression (CBE) systems were constructed. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter-trapping technique whereby the promoterless operon was stably integrated into the chromosome of STM-1, and the expression of antigens was assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained by using the in vivo-induced sspA promoter and recombining the expression construct into aroD. Dual expression of the antigens was detected in all systems, with PBE producing much larger quantities of both antigens. The stability of antigen expression after in vivo passage was 100% for all CBE strains recovered. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice, and significant systemic immunoglobulin M (IgM) and IgG responses against both antigens were detected among all CBE groups. No significant immune response was detected using PBE strains. Expression of recombinant antigens in S. enterica serovar Typhimurium aroA from chromosomally located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response.

Differences in macrophage activation by bacterial DNA and CpG-containing oligonucleotides.

Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN >44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

Investigation of ansB and sspA derived promoters for multi- and single-copy antigen expression in attenuated Salmonella enterica var. typhimurium.

Five candidate promoters were examined to determine their utility in directing immunogenic levels of expression of the C fragment from tetanus toxin in attenuated S. enterica used as an oral vaccine in mice. Promoters derived from the genes encoding the stringent starvation protein (sspA) from E. coli and S. enterica, but not ansB derived promoters, expressed immunogenic levels of C fragment from multi-copy plasmids in attenuated S. enterica in vivo and, following oral immunization, induced high titre specific anti-tetanus toxoid serum antibodies. We also demonstrate that not only the choice of promoter, replicon and growth conditions but also how expression constructs are assembled in the chosen plasmid is critical for the successful development of plasmid-based antigen delivery systems using attenuated S. enterica. In addition, the S. enterica sspA promoter is able to elicit anti-tetanus toxoid antibodies in mice when the psspA-tetC expression cassette is integrated in single copy on the S. enterica chromosome.

Plasmid-encoded Tet B tetracycline resistance in Haemophilus parasuis.

The complete sequence of two plasmids, pHS-Tet (5.1 kb) and pHS-Rec (9.5 kb), isolated from Haemophilus parasuis strain HS1543 has been obtained. Plasmid pHS-Tet contains four open reading frames including a tet(B) tetracycline resistance gene which unusually did not have an associated tetR repressor gene. From a total of 45 H. parasuis isolates surveyed (15 international reference strains, 15 field isolates selected for their genetic diversity, and 15 recent Australian field isolates), 2 tetracycline-resistant field isolates (HS226 and HS1859) were identified. Analysis of three additional isolates from the same disease outbreak as strain HS1859 revealed a further tetracycline-resistant H. parasuis strain (HS1857, serovar 8) and a tetracycline-resistant Actinobacillus pleuropneumoniae strain (HS1861). An approximately 10.6-kb plasmid was identified in field isolate HS226 and outbreak strains HS1857, HS1859, and HS1861. Southern hybridization analysis of these plasmids showed that the Tet B determinant was present, and restriction digest comparisons suggest that these plasmids are related. This is believed to be the first report of native H. parasuis plasmids and Tet B-mediated tetracycline resistance in this microorganism.

Analysis of the role of pglI in pilin glycosylation of Neisseria meningitidis.

Pilin is the major subunit of the essential virulence factor pili and is glycosylated at Ser63. In this study we investigated the gene pglI to determine whether it is involved in the biosynthesis of the pilin-linked glycan of Neisseria meningitidis strain C311#3. A N. meningitidis C311#3pglI mutant resulted in a change of apparent molecular weight in SDS-PAGE and altered binding of antisera, consistent with a role in the biosynthesis of the pilin-linked glycan. These data, in conjunction with homology with well-characterised acyltransferases suggests a specific role for pglI in the biosynthesis of the basal 2,4-diacetamido-2,4,6-trideoxyhexose residue of the pilin-linked glycan.

Genetic analysis of a plasmid encoding haemocin production in Haemophilus paragallinarum.

The full sequence of plasmid p250, isolated from Haemophilus paragallinarum strain HP250, has been obtained. The plasmid contains seven ORFs: a putative integrase, a putative replication protein (repB) and five ORFs similar to those from the haemocin (bacteriocin) hmcDCBAI operon from Haemophilus influenzae. Of 19 other non-plasmid-containing H. paragallinarum strains screened (11 serovar reference strains and 8 field isolates), 17 strains produced haemocin and were resistant to killing by strain HP250. These strains, unlike strain HP250, have a chromosomally encoded haemocin operon. A number of other members of the family Pasteurellaceae were tested for haemocin sensitivity. Pasteurella avium, Pasteurella volantium and Pasteurella species A, all non-pathogenic bacteria found in the respiratory tract of chickens suffering from respiratory diseases, were sensitive to H. paragallinarum haemocin. However, amongst the pathogenic Pasteurellaceae, 50 % of P. multocida isolates and all five isolates of Pasteurella haemolytica tested were sensitive to the haemocin. Given the prevalence of haemocin production in H. paragallinarum strains, it may play a role in aiding colonization by inhibiting other Gram-negative bacteria that are associated with the respiratory tract in chickens. The origin of replication from plasmid p250 has been used to generate an Escherichia coli-H. paragallinarum shuttle vector which may be useful in genetically manipulating H. paragallinarum.

Molecular analysis of a haemagglutinin of Haemophilus paragallinarum.

The gene encoding a haemagglutinin of H. paragallinarum, hagA, has been identified and the full-length nucleotide sequence determined. A approximately 39 kDa protein, recognized by an anti-haemagglutinin monoclonal antibody, mAb4D, was purified from H. paragallinarum strain 0083 and the N-terminal sequence obtained. The full-length nucleotide sequence was obtained by inverse PCR and the deduced amino acid sequence of the protein encoded was shown to be similar to other outer-membrane proteins of closely related organisms in the HAP group (Haemophilus, Actinobacillus, Pasteurella), especially the P5 protein of Haemophilus influenzae. The hagA gene was cloned into a His-tag expression vector and overexpressed in Escherichia coli strain M15(pREP4). The identity of the purified recombinant protein as a H. paragallinarum haemagglutinin was confirmed by haemagglutination of chicken red blood cells and reactivity, in a Western blot, with the monoclonal antibody specific for the serovar A haemagglutinin.