Cloning and expression of a gene encoding the lytic functions of Bacillus amyloliquefaciens phage: Evidence of an auxiliary lysis system.

A bacteriophage specific to Bacillus amyloliquefaciens, a gram-positive bacterium, was isolated from a local sewage treatment center. Using a lysis assay, a gene, lys1521, was isolated and its nucleotide sequence revealed one open reading frame of 375 bp. Homology studies showed amino acid alignment similarity with gene 5A of Bacillus subtilis phages PZA and phi29. Overexpression of the cloned gene yielded a 13 kDa protein corresponding to the predicted gene product. Despite the fact that no significant homology with known cell wall lytic enzymes was apparent, the lytic profile obtained in an in vivo expression assay showed that lys1521 had cell wall hydrolysis activity. This is a significant revelation since the function of the homologous gene 5A product of phage phi29 has been suggested to be required for the in vivo elongation of phage DNA replication. The lys1521 gene could be evidence of the presence in gram-positive bacteriophages of a third lysis gene in addition to the well characterized two-step lysis system.

Selective killing of Leishmania amastigotes expressing a thymidine kinase suicide gene.

The thymidine kinase gene of Herpes simplex type-1 virus was transfected into several Leishmania species to create drug-sensitive mutants. Expression of the thymidine kinase gene is not by itself harmful to Leishmania cells but it is capable of phosphorylating ganciclovir, a nucleoside analog, into a highly toxic product. In addition to the generation of Leishmania promastigotes highly sensitive to ganciclovir, the thymidine kinase gene was expressed similarly by amastigotes engulfed either by murine or by human macrophages. Leishmania major amastigotes expressing thymidine kinase were eliminated by 85% when treated with ganciclovir. Selective killing of parasites expressing suicide genes at their infective stage could suggest novel strategies for controlling parasitic infections.

Isolation and characterization of a novel IgD-binding protein from Moraxella catarrhalis.

A novel surface protein of the bacterial species Moraxella catarrhalis that displays a high affinity for IgD (MID) was solubilized in Empigen and isolated by ion exchange chromatography and gel filtration. The apparent molecular mass of monomeric MID was estimated to approximately 200 kDa by SDS-PAGE. The mid gene was cloned and expressed in Escherichia coli. The complete mid nucleotide gene sequence was determined, and the deduced amino acid sequence consists of 2123 residues. The sequence of MID has no similarity to other Ig-binding proteins and differs from all previously described outer membrane proteins of M. catarrhalis. MID was found to exhibit unique Ig-binding properties. Thus, in ELISA, dot blots, and Western blots, MID bound two purified IgD myeloma proteins, four IgD myeloma sera, and finally one IgD standard serum. No binding of MID was detected to IgG, IgM, IgA, or IgE myeloma proteins. MID also bound to the surface-expressed B cell receptor IgD, but not to other membrane molecules on human PBLs. This novel Ig-binding reagent promises to be of theoretical and practical interest in immunological research.

Protection against Leishmania major challenge infection in mice vaccinated with live recombinant parasites expressing a cytotoxic gene.

A "suicide" system based on thymidine kinase-ganciclovir combination was developed and tested in a Leishmania major experimental model. Susceptible BALB/c mice were infected with L. major expressing the thymidine kinase gene of herpes simplex virus type 1 and treated for 2 consecutive weeks with 7.5 mg/kg/day ganciclovir at different times from the initial infection. Ganciclovir treatment at varying times after infection had different effects on the outcome of disease. A complete inhibition of intracellular parasites was obtained in mice treated 1 or 4 days after infection, whereas ganciclovir administration 2 weeks later resulted in the control of infection only when the drug was provided. Variable levels of protection, from partial to total, against challenge infection with virulent L. major were observed, depending on the timing of ganciclovir treatment. The thymidine kinase-ganciclovir approach represents an excellent experimental model to control Leishmania infection and to evaluate the immunologic response of the host.