Trichostrongylus colubriformis extract upregulates TNF-alpha receptor expression and enhances TNF-alpha sensitivity of L929 cells.

Many pathogens have developed strategies to avoid the host's immune system and hence improve their long-term survival. These strategies include antigenic variation, mimicry of host regulatory proteins and production of immunoregulatory molecules. The ruminant gastrointestinal nematode Trichostrongylus colubriformis produces several factors with homology to human immunoregulatory proteins. However, direct immunomodulation by T. colubriformis proteins has not yet been unequivocally demonstrated. Results in the present paper demonstrate that soluble T. colubriformis factors promote proliferation of the TNF-susceptible mouse fibrosarcoma cell line L929, while inhibiting proliferation of all other cell types tested. In addition, T. colubriformis homogenate enhanced the susceptibility of L929 cells to the cytotoxic action of ovine TNF-alpha. Within 1 h of exposure, T. colubriformis factors bind L929 cells in a stable fashion, yet it takes up to 24 h for the cells to become sensitised to TNF-alpha. Interestingly, the increase of both TNF-alpha sensitivity and proliferation of treated L929 cells correlated with an upregulation in expression of TNF-alpha p55 and p75 receptors.

A single amino-acid change between the antigenically different extracellular serine proteases V2 and B2 from Dichelobacter nodosus.

Dichelobacter nodosus (Dn), the causative organism of ovine footrot, secrets three distinct types of extracellular serine proteases which have been implicated in virulence. Southern analyses have shown that the proteases are encoded by three separate genes, and the genes encoding an acidic protease V5 and a basic protease have already been characterised from virulent Dn strain 198. The gene encoding the third protease type, as represented by acidic protease V2, was isolated from an EcoRI-BamHI library of strain 198 genomic DNA by probing with a polymerase chain reaction (PCR) fragment generated with oligodeoxyribonucleotides based on protease V2 amino acid (aa) sequences. A further clone from an RsaI library was isolated to complete the 5' region of the gene to yield an ORF of 1803 bp encoding a protein precursor of 601 aa. The acidic protease V2 gene, aprV2, shows the same precursor structure as the bprV and aprV5 genes with 72% and 69% similarity at the nucleotide (nt) level and with 73% and 69% similarity at the aa level, respectively. As monoclonal antibodies consistently distinguish the virulent (V) and benign (B) forms of this protease, the gene encoding the acidic protease B2 from benign Dn strain 305 was isolated using the PCR and characterized to investigate the molecular basis for this difference in antigenicity. A 2-bp substitution in a single codon was identified which appeared to be responsible for a change of epitope.

Nucleotide and deduced protein sequence of the extracellular, serine basic protease gene (bprB) from Dichelobacter nodosus strain 305: comparison with the basic protease gene (bprV) from virulent strain 198.

In earlier studies, it appeared that benign strains of the Gram-negative, obligate anaerobe, Dichelobacter nodosus, were devoid of the extracellular, serine basic protease (pI approximately 9.5) of virulent strains. However, Southern and PCR analysis have shown a homologous gene (bprB) in the representative benign strain 305. The deduced amino acid sequence of the prepro- and mature protease regions of bprB confirmed this homology and showed 97% sequence identity with the bprV precursor from virulent strain 198. Identity in the carboxy-terminal extension region was 90%. Expression studies in Escherichia coli transformed with bprB, showed that the gene was capable of the production of an active protease. A protease, albeit with a lower iso-electric point (approximately 8.6), was isolated from D. nodosus culture supernatants and shown to cross-react with antibodies raised against the more basic protease from strain 198. The amino acid sequence encoded by the strain 305 gene revealed two additional acidic residues consistent with a lowered iso-electric point and supported the conclusion that bprB and bprV produce equivalent basic proteases.

Cloning, sequence and expression of the gene (aprV5) encoding extracellular serine acidic protease V5 from Dichelobacter nodosus.

The acidic protease V5-encoding gene (aprV5) from Gram- Dichelobacter nodosus virulent strain 198 was isolated from a cosmid bank by activity screening and sequenced. The 2371-bp nucleotide (nt) sequence contained an open reading frame coding for a protein precursor of 595 amino acid (aa) residues composed of a signal peptide, a pro-region, a mature active protease of 347 aa and a C-terminal extension region of 120 aa. The deduced aa sequence of the pre-pro-mature protease regions showed about 65% similarity to that of D. nodosus basic protease while the C-terminal extension region showed only about 26% similarity. The aprV5 gene, without its C-terminal extension region, was cloned and expressed in Escherichia coli. The acidic protease B5-encoding gene (aprB5) from non-virulent strain 305 was also cloned and sequenced. The aprB5 nt sequence showed 99% homology to that of aprV5 with two single-aa changes occurring in the precursor.

Amino acid sequence of extracellular acidic protease V5 of Dichelobacter nodosus, the causative organism of ovine footrot.

Dichelobacter nodosus, a Gram-negative obligate anaerobe and the causative organism of ovine footrot, secretes a family of extracellular serine proteases with pI's in the range of 5.2 to 5.6 and a serine basic protease with a pI of approximately 9.5. The primary structure of acidic protease V5 (pI approximately 5.2) from D. nodosus virulent strain 198 was determined by direct amino acid sequencing. This protease consists of a single polypeptide chain of 347 amino acids, contains two disulfide bonds and has a M(r) of 35960. Comparison of the D. nodosus acidic protease V5 sequence with that of other serine proteases showed that it is a member of the subtilisin family of proteases with strong conservation of identity around the catalytic residues. The sequence of protease V5 showed 64% identity to D. nodosus basic protease (pI approximately 9.5) and 53% identity to the extracellular serine protease of Xanthomonas campestris, a plant pathogen but only 25-35% identity to other proteases of the subtilisin family. The D. nodosus proteases are similar in length to X. campestris protease (but some 70 residues shorter than the subtilisins) and they share two conserved disulfide bonds with the X. campestris protease, a feature not observed for other members of the subtilisin family.