Elucidating the function of a novel antibacterial amidase in Ixodes scapularis



 DESCRIPTION (provided by applicant): Despite the importance of ticks as disease vectors, the immune system of these organisms remains poorly understood. We have discovered a novel antibacterial enzyme found in the hard tick Ixodes scapularis that was horizontally acquired from a bacterium early in the evolution of ticks and mites. This enzyme, which we term Dae2 (domesticated amidase effector 2), is related to a family of peptidoglycan-degrading toxins transferred between bacteria during interbacterial competition. Preliminary in vitro data show that Dae2 is indeed an antibacterial enzyme, and that it retains the DD-endopeptidase peptidoglycan-degrading activity of its characterized bacterial homologs. Additionally, we demonstrate Dae2 is expressed in the midgut and salivary glands of I. scapularis, and that dae2 knockdown reduces the ability of I. scapularis to control replication of the Lyme disease agent Borrelia burgdorferi. In this grant, we propose to define the function and physiological mechanism of action of Dae2 in I. scapularis using in vitro and in vivo approaches. In the first aim of this proposal, we will establish the target range and mechanism of action of Dae2 antibacterial activity in vitro. To accomplish this, we will measure purified Dae2 activity against each major peptidoglycan type. Furthermore, we will test the direct antibacterial capacity of Dae2 against diverse species representing tick-associated microbes in the presence and absence of accessory immune factors present in tick salivary gland and midgut extracts. The second aim of our work is to define the function of Dae2 in vivo. Taking advantage of RNAi-based knockdown strategies, we will determine the contribution of Dae2 to structuring the commensal microbial community of I. scapularis, and define the role of Dae2 in mediating the tick innate immune response to a bacterial pathogen. In total, our studies will provide key insights into the role of Dae2 in the innate immune system of I. scapularis.