A bioluminescent HL-60 cell line to assay anti-leukaemia therapeutics under physiological conditions.

Screens for compounds and proteins with anti-cancer activity employ viability assays using relevant cancer cell lines. For leukaemia studies, the human leukaemia cell line, HL-60, is often used as a model system. To facilitate the discovery and investigation of anti-leukaemia therapeutics under physiological conditions, we have engineered HL-60 cells that stably express firefly luciferase and produce light that can be detected using an in vivo imaging system (IVIS). Bioluminescent HL-60luc cells could be rapidly detected in whole blood with a sensitivity of approximately 1000 viable cells/200 microl blood. Treatment of HL-60luc cells with the drug chlorambucil revealed that the bioluminescent viability assay is able to detect cell death earlier than the Trypan blue dye exclusion assay. HL-60luc cells administered intraperitoneally (i.p.) or intravenously (i.v.) were visualized in living mice. The rapidity and ease of detecting HL-60luc cells in biological fluid indicates that this cell line could be used in high-throughput screens for the identification of drugs with anti-leukaemia activity under physiological conditions.

Aggregatibacter actinomycetemcomitans LtxC is required for leukotoxin activity and initial interaction between toxin and host cells.

Aggregatibacter actinomycetemcomitans is a human pathogen that produces the RTX toxin (repeats in toxin), leukotoxin (LtxA). Based on other RTX toxin systems, the product of ltxC, the first gene of the ltx operon, is predicted to be involved in fatty acid modification of LtxA. To determine the function of ltxC in A. actinomycetemcomitans, we generated an ltxC mutation in the highly leukotoxic strain JP2N using random mutagenesis. The toxin from the ltxC mutant (LtxA(ltxC)) was expressed and secreted into the cell culture supernatant but could not lyse human leukocytes or erythrocytes. Mass spectrometric analysis of LtxA(ltxC) and LtxA from strain JP2N (LtxA(wt)) revealed two peptides that differed and this data suggests that two internal lysine residues of LtxA from the wild-type strain are modified. In blocking experiments, pre-treatment of cells with LtxA(ltxC) was unable to prevent LtxA(wt) from killing cells. Furthermore, in contrast to LtxA(wt), LtxA(ltxC) did not cause an increase in intracellular calcium levels in human leukocytes. Taken together, our data show that ltxC is required for full activity and modification of LtxA in A. actinomycetemcomitans and that modification is important for initial binding of toxin to host cells, as defined by an increase in intracellular calcium levels.

Interaction between leukotoxin and Cu,Zn superoxide dismutase in Aggregatibacter actinomycetemcomitans.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative oral pathogen that is the etiologic agent of localized aggressive periodontitis and systemic infections. A. actinomycetemcomitans produces leukotoxin (LtxA), which is a member of the RTX (repeats in toxin) family of secreted bacterial toxins and is known to target human leukocytes and erythrocytes. To better understand how LtxA functions as a virulence factor, we sought to detect and study potential A. actinomycetemcomitans proteins that interact with LtxA. We found that Cu,Zn superoxide dismutase (SOD) interacts specifically with LtxA. Cu,Zn SOD was purified from A. actinomycetemcomitans to homogeneity and remained enzymatically active. Purified Cu,Zn SOD allowed us to isolate highly specific anti-Cu,Zn SOD antibody and this antibody was used to further confirm protein interaction. Cu,Zn SOD-deficient mutants displayed decreased survival in the presence of reactive oxygen and nitrogen species and could be complemented with wild-type Cu,Zn SOD in trans. We suggest that A. actinomycetemcomitans Cu,Zn SOD may protect both bacteria and LtxA from reactive species produced by host inflammatory cells during disease. This is the first example of a protein-protein interaction involving a bacterial Cu,Zn SOD.