Roger D. Pearson

Synthesis and properties of crosslinked recombinant pro-resilin

Resilin is a member of a family of elastic proteins that includes elastin, as well as gluten, gliadin, abductin and spider silks. Resilin is found in specialized regions of the cuticle of most insects, providing low stiffness, high strain and efficient energy storage; it is best known for its roles in insect flight and the remarkable jumping ability of fleas and spittle bugs. Previously, the Drosophila melanogaster CG15920 gene was tentatively identified as one encoding a resilin-like protein (pro-resilin). Here we report the cloning and expression of the first exon of the Drosophila CG15920 gene as a soluble protein in Escherichia coli. We show that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking. This observation validates the role of the putative elastic repeat motif in resilin function. The resilience (recovery after deformation) of crosslinked recombinant resilin was found to exceed that of unfilled synthetic polybutadiene, a high resilience rubber. We believe that our work will greatly facilitate structural investigations into the functional properties of resilin and shed light on more general aspects of the structure of elastomeric proteins. In addition, the ability to rapidly cast samples of this biomaterial may enable its use in situ for both industrial and biomedical applications.

Characterization of a Major Peritrophic Membrane Protein, Peritrophin-44, from the Larvae of Lucilia cuprina cDNA AND DEDUCED AMINO ACID SEQUENCES

The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: effects of induced immunity on tick populations.

Abstract Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: effects of induced immunity on tick populations. International Journal for Parasitology16: 27–34. Injection of extracts derived from adult female ticks induced partial immunity to B. microplus in both Bos tawus and Bos taurus × Bos indicus breeds. Cattle showed a variable response to vaccination with the complete extract; 7/15 showed good resistance to ticks, 5/15 were intermediate and the remaining three animals showed poor resistance. The immunity induced was still evident after 14 weeks of daily challenge with 1000 larvae and tick populations on vaccinated cattle were reduced by 70% compared to matched controls during this period. In another experiment, cattle were challenged twice with 20,000 larvae and tick populations on the vaccinated group were reduced by over 90% compared to a matched unvaccinated group. Following vaccination, serum antibodies to soluble extracts of adult ticks were detected by gel diffusion and radio-immunoassay but antibody levels in individual cattle were not correlated with their immunity to ticks. Protective antigens were found both in the supernatant and the pellet derived from tick extracts. Insoluble complexes formed by reacting serum from successfully vaccinated cattle with soluble crude tick extracts were not protective.

Larvicidal activity of lectins on Lucilia cuprina: mechanism of action

Larvae of the blowfly Lucilia cuprina (Wied.) (Diptera: Calliphoridae) were grown in vitro on a serum‐free medium in the presence of a number of lectins. Lectins with specificities for β‐(1,4)‐N‐acetylglucosamine (wheat germ lectin) and α‐D‐mannopyranosyl and (α‐D‐glucopyranosyl residues (lentil lectin and Con A) caused strong concentration‐dependent inhibition of the growth of the larvae and substantial mortality. Wheat germ lectin had the strongest effects, showing 50% inhibition of larval growth at a concentration of 2 μM and 100% mortality at 25 μM. Other lectins with different sugar specificities had much less effect. The mechanism of the larvicidal action(s) of wheat germ lectin, lentil lectin and Con A was investigated. There were at least three effects of these lectins on L. cuprina larvae. First, these lectins bound to and reduced the permeability of the peritrophic membrane of the larvae. Second, they reduced ingestion of diet medium by larvae. Third, the lectins also bound to the apical membranes of larval gut epithelial cells although there were no obvious signs of damage to these cells. It is concluded that the combination of these effects probably results in the starvation of the larvae. The implications of these results in terms of possible control strategies for L. cuprina are discussed.

Vaccination againstBoophilus microplus: Localization of antigens on tick gut cells and their interaction with the host immune system

Cattle have been vaccinated againstBoophilus microplus with antigens derived from partially fed female ticks. The immune response of the host lyses the gut cells of adult ticks, causing a reduction in the number, weight and reproductive capacity of engorging ticks. This response is different from the immunity that cattle acquire after repeated tick infestation. Evidence is presented that the antigens used in vaccination are located on the plasma membrane of the gut cells and it is unlikely that these antigens are secreted into the host during feeding. Vaccination using such ‘concealed’ antigens may not encounter the mechanisms of immune evasion that parasites usually demonstrate.In-vitro assays suggest that vaccination immunity is not dependent on the need to stimulate cell-mediated responses. Immunoglobulin G alone, or with the aid of complement, is enough to damage tick gut.The normal function of the one protein antigen isolated so far is unknown but we speculate that it serves some vital function on the cell plasma membrane.

Identification of immune genes and proteins involved in the response of bovine mammary tissue to Staphylococcus aureus infection

BackgroundMastitis in dairy cattle results from infection of mammary tissue by a range of micro-organisms but principally coliform bacteria and Gram positive bacteria such as Staphylococcus aureus. The former species are often acquired by environmental contamination while S. aureus is particularly problematic due to its resistance to antibiotic treatments and ability to reside within mammary tissue in a chronic, subclinical state. The transcriptional responses within bovine mammary epithelial tissue subjected to intramammary challenge with S. aureus are poorly characterised, particularly at the earliest stages of infection. Moreover, the effect of infection on the presence of bioactive innate immune proteins in milk is also unclear. The nature of these responses may determine the susceptibility of the tissue and its ability to resolve the infection.ResultsTranscriptional profiling was employed to measure changes in gene expression occurring in bovine mammary tissues sampled from three dairy cows after brief and graded intramammary challenges with S. aureus. These limited challenges had no significant effect on the expression pattern of the gene encoding β-casein but caused coordinated up-regulation of a number of cytokines and chemokines involved in pro-inflammatory responses. In addition, the enhanced expression of two genes, S100 calcium-binding protein A12 (S100A12) and Pentraxin-3 (PTX3) corresponded with significantly increased levels of their proteins in milk from infected udders. Both genes were shown to be expressed by mammary epithelial cells grown in culture after stimulation with lipopolysaccharide. There was also a strong correlation between somatic cell count, a widely used measure of mastitis, and the level of S100A12 in milk from a herd of dairy cows. Recombinant S100A12 inhibited growth of Escherichia coli in vitro and recombinant PTX3 bound to E. coli as well as C1q, a subunit of the first component of the complement cascade.ConclusionThe transcriptional responses in infected bovine mammary tissue, even at low doses of bacteria and short periods of infection, probably reflect the combined contributions of gene expression changes resulting from the activation of mammary epithelial cells and infiltrating immune cells. The secretion of a number of proinflammatory cytokines and chemokines from mammary epithelial cells stimulated by the bacteria serves to trigger the recruitment and activation of neutrophils in mammary tissue. The presence of S100A12 and PTX3 in milk from infected udder quarters may increase the anti-bacterial properties of milk thereby helping to resolve the mammary tissue infection as well as potentially contributing to the maturation of the newborn calf epithelium and establishment of the newborn gut microbial population.

Lucilia cuprina: Inhibition of larval growth induced by immunization of host sheep with extracts of larval peritrophic membrane

A culture system has been established to produce gram amounts of peritrophic membrane from larvae of the sheep blowfly, Lucilia cuprina. Peritrophic membrane obtained from this culture has been used to immunize sheep. The immunization produced an immune response which resulted in the average weight of larvae on immunized sheep being only 50% of that of larvae grown on control sheep (P < 0.05). Fractionation of the components of the peritrophic membrane followed by immunization trials showed that the protective antigen fraction comprised material that could only be solubilized by harsh agents such as 4 M-urea. Even after solubilization by 4 M-urea, the protective antigens were able to produce a protective immune response which reduced growth of larvae on immunized sheep to 55% of larvae grown on control sheep (P < 0.05). This immune response which reduced growth of the larvae did not cause gross morphological damage to the larvae.

Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA

Two chymotrypsin‐like proteases were purified from the secretory and excretory material of first‐instar larvae of Lucilia cuprina. The hydrolysis of N‐succinyl‐L‐phenylalanine‐nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor‐Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease {Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis‐causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.

Bovine Muc1 inhibits binding of enteric bacteria to Caco-2 cells

Inhibition of bacterial adhesion to intestinal epithelial receptors by the consumption of natural food components is an attractive strategy for the prevention of microbial related gastrointestinal illness. We hypothesised that Muc1, a highly glycosylated mucin present in cows’ milk, may be one such food component. Purified bovine Muc1 was tested for its ability to inhibit binding of common enteric bacterial pathogens to Caco-2 cells grown in vitro. Muc1 caused dose-dependent binding inhibition of Escherichia coli, Salmonella enterica serovar Typhimurium (S. Typhimurium), Staphylococcus aureus and Bacillus subtilis. This inhibition was more pronounced for the Gram negative compared with Gram positive bacteria. It was also demonstrated that Muc1, immobilised on a membrane, bound all these bacterial species in a dose-dependent manner, although there was greater interaction with the Gram negative bacteria. A range of monosaccharides, representative of the Muc1 oligosaccharide composition, were tested for their ability to prevent binding of E. coli and S. Typhimurium to Caco-2 cells. Inhibition was structure dependent with sialic acid, L(-) fucose and D(+) mannose significantly inhibiting binding of both Gram negative species. N-acetylglucosamine and N-acetylgalactosamine significantly inhibited binding of E. coli whilst galactose, one of the most abundant Muc1 monosaccharides, showed the strongest inhibition against S. Typhimurium. Treatment with sialidase significantly decreased the inhibitory properties of Muc1, demonstrating the importance of sialic acid in adhesion inhibition. It is concluded that bovine Muc1 prevents binding of bacteria to human intestinal cells and may have a role in preventing the binding of common enteropathogenic bacteria to human intestinal epithelial surfaces.

Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro.

The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Lucilia cuprina was assayed in vivo and in vitro. One in vivo assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in vivo technique, in which larvae were allowed to develop to second instar in small aluminium rings attached to the sheep, indicated no reduction in larval growth or survival. Larvae of Lucilia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media. No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments. ELISA antibody levels against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in vitro was significant. The immunoglobulin fraction isolated from sera of infested sheep significantly retarded larval growth when incorporated with normal serum in growth media. These results are consistent with an effect of specific anti-larval antibody produced by sheep in response to infestation.