(158o) Phosphatidylserine Expression as a Molecular Target in Listeria monocytogenes and Escherichia coli

Targeted delivery of antimicrobials, e.g. via encapsulation in liposomes, is often useful for mitigating noxious side effects or enhancing anti-microbial activity against difficult to treat pathogens occupying unique biological niches. In addition, targeted delivery that significantly enhanced a compound’s antimicrobial activity could make it useful against pathogens outside of its typical spectrum of activity. We have developed a novel strategy for targeting antibiotics that is highly efficacious against both Gram-positive (L. monocytogenes) and Gram-negative organisms (E. coli). This strategy takes advantage of the fact that eukaryotic and prokaryotic cells share many mechanisms of cell stress and apoptosis. One such conserved element is the expression of phosphatidylserine (PS), an anionic membrane-bound phospholipid, in response to cell stress. This expression of PS is nearly universally conserved, being demonstrated in prokaryotes and eukaryotes. Actively externalized in response to stress, PS is the natural ligand for proteins of the annexin superfamily. Interestingly, the expression of annexin superfamily members is found in both prokaryotes and eukaryotes, demonstrating the nature of important mechanisms by which cell stress is recognized, as well as signaled.

Bacterial PS expression was targeted with its ligand, annexin A5 (ANXA5). This protein was functionalized with the covalent addition of the antibiotic ampicillin and separately with the antibiotic moxifloxacin. Functionalized ANXA5 serves as a delivery vehicle, directing antibiotic to bacterial phosphatidylserine expression. Results presented here suggest that this ANXA5-ampicillin bioconjugate participates in a positive feedback loop where phosphatidylserine, the target of the delivery vehicle ANXA5, is upregulated by the chemotherapeutic payload of the bioconjugate. Importantly, the ANXA5 delivery vehicle is not toxic to bacterial cells by itself and neither is the ANXA5 bioconjugate toxic to human vascular endothelial cells. As measured by the IC50 (half maximal inhibitory concentration), conjugation to ANXA5 resulted in increasing the antibiotic activity of ampicillin against L. monocytogenes and E. coli by more than 4 and 3 orders of magnitude, respectively, compared to free ampicillin, while activity of moxifloxacin against L. monocytogenes is increased by 4 orders of magnitude. Given the conservation of phosphatidylserine expression in pathologies such as oncogenesis, and other bacterial/viral/parasitic infections, we hypothesize that a therapeutic modality targeting phosphatidylserine expression may be a viable chemotherapeutic strategy in many infectious diseases.