Adenoviral-Mediated Transfer of a Lysostaphin Gene into the Goat Mammary Gland

Abstract

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.

Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis

“…The new alternative approaches under investigation include cytokine immunotherapy, recombinant mucolytic protein therapy, viral-mediated gene transfer and genetic breeding of disease-resistant cows. Although these new approaches are promising, much work remains to be done to confirm their clinical usefulness and to evaluate their potential side effects (Sordillo et al, 1997;Gruet et al, 2001;Fan et al, 2002;Wall et al, 2005).…”

Expression of lysostaphin in HeLa cells protects from host cell killing by intracellular Staphylococcus aureus

“…In comparison to previous works, where the expression of secreted lysostaphin was studied to combat extracellular Staphylococci [6,9,21,22], the present work aimed at the development of a staphylococcal infection system that eliminates intracellular growth and persistence of S. aureus. In order to test whether the observed protection of infected HeLa-lys cells was the consequence of a drastic reduction of living intracellular S. aureus, the fate of intracellular bacteria was monitored by determining the intracellular CFU number of S. aureus.…”

“…It was successfully used to study therapeutic or prophylactic control of staphylococcal mastitis in several transgenic animal models [6,9,21]. The enzyme's specificity for S. aureus and its low cytotoxicity makes it an ideal candidate agent to study consequences of the intracellular killing of S. aureus.…”

Production of recombinant proteins in milk of transgenic and non-transgenic goats

“…Goats have already been utilized for the production of active lysostaphin in milk by intramammary infusion of an adenoviral vector. Fan et al (2002Fan et al ( , 2004 detected lysostaphin in the milk of non-transgenic goats. However, these authors did not obtain results consistent with respect to bioactivity and duration of the expression of the recombinant protein, aiming to prevent and treat the staphylococcal infections in the mammary gland.…”