BacTx(R) technology, developed by Immunetics for detection of bacteria, employs a unique and innovative assay chemistry to detect Gram-positive and Gram-negative bacteria. The technology is based on components of the innate immune system of the tobacco hornworm (Manduca sexta), in particular the prophenoloxidase cascade, in which serine proteases cleave prophenoloxidase to phenoloxidase, which subsequently oxidizes chromogen to generate a colored reaction product. The BacTx(R) Bacterial Detection Kit received 510(k) clearance from the FDA (CBER) in June 2012 as a quality control test for bacterial contamination in platelets. For M. sexta and other Lepidoptera, the most potent elicitor of the prophenoloxidase cascade is peptidoglycan, which is a major structural component of bacterial cell walls. In this application we propose to biochemically characterize the relationship between the presence of specific bacterial cell wall features and evasion of detection by pathogen recognition proteins present in M. sexta hemolymph. The role of these features in inhibiting prophenoloxidase activation will be investigated, with the long term goal of potentially enhancing assay performance and opening up new applications of BacTx(R) technology, including potential use for diagnosis of sepsis, meningitis, and nosocomial infections. Due to the striking similarities between the insect innate immune system and human complement system, this research may also prove significant to elucidating the relationship between inflammation due to bacterial infection and debilitating disorders such as cancer, autoimmune diseases, and septic shock.
Bacterial contamination of platelets is considered the greatest infectious risk of blood product transfusion today at about 1 in 2000 platelet units, which is several orders of magnitude greater than that of HIV. The FDA- cleared BacTx(R) Assay is a screening test for detection of bacterial contamination in platelet units that takes advantage of the innate immune system of an insect (Manduca sexta) to detect peptidoglycan, a component of all bacterial cell walls. We propose to characterize the binding specificities of Peptidoglycan Recognition Proteins (PGRPs) from Manduca and analyze their role in BacTx(R) Assay analytical sensitivity, with the long term goal of adding recombinant or mutated PGRPs to the assay to increase sensitivity, so that the assay can be used for additional applications beyond platelet testing.