• Barriers to Infection: Tick Immune Effectors and Vectored Bacteria

      Pelc, Rebecca Sellers; Ceraul, Shane M.; 0000-0001-5080-4945 (2015)
      Ticks are hematophagous arthropods which transmit bacterial, viral, and protozoan pathogens. The interaction of a tick's innate immune response with transmitted pathogens is directly linked to its vector competence. The American dog tick, Dermacentor variabilis vectors the etiologic agents of Rocky Mountain spotted fever (Rickettsia rickettsii) and tularemia (Francisella tularensis). Previous studies demonstrated that transcript abundance of two defensins from D. variabilis increases in response to the model Spotted Fever Group organism Rickettsia montanensis. Based on this observation we hypothesized that these defensins function against tick-borne pathogens to limit bacterial load, enabling D. variabilis to serve as vector and reservoir. This study demonstrates that these phylogenetically distinct peptides are also distinct in their antimicrobial mechanism of action and specificity for transmitted bacterial species. Defensin-2 associated with R. montanensis bacteria in vitro and in vivo, inducing cytoplasmic leakage and reducing host burden. Meanwhile, defensin-1 treatment did not reduce host burden of R. montanensis but reduced CFUs of the model organism Francisella novicida in vitro. This reduction in viability correlated with hyperpolarization, potassium efflux, and ATP reduction in the bacteria, factors important for maintaining homeostasis. Additionally, lipid A mutants of F. novicida demonstrated altered susceptibility to and activation of defensin-1 implicating the importance of bacterial membrane components in a successful tick immune response. Finally, this work developed a method of using translation occluding Peptide Nucleic Acids (PNA) to inhibit Rickettsial protein expression. Targeting OmpB and RickA, factors established as important to infection, reduced expression and bacterial burden of host cells. This strategy provides a new tool to further examine the tick-pathogen relationship. Taken together, this work uses a biologically relevant model to examine how the extrinsic incubation period allows for the perpetuation of tick-borne disease in nature.
    • XIAP-p47 Pairing Activates the Immune Deficiency Pathway in the Lyme Disease Tick Ixodes scapularis

      McClure Carroll, Erin; Pedra, Joao H. F. (2019)
      Globally, vector-borne diseases account for 17% of all infectious diseases. Most vectors are blood-feeding arthropods, which transmit bacterial, viral, and parasitic diseases to humans and animals. The tick Ixodes scapularis transmits seven pathogens, including Borrelia burgdorferi, the agent of Lyme disease. Lyme disease is the most important vector-borne disease in the United States and causes an estimated 329,000 infections annually. Best described in the model organism Drosophila melanogaster, the arthropod immune deficiency (IMD) pathway responds to microbial infection through activation of Relish, a nuclear factor (NF)-κB family transcription factor. In I. scapularis ticks, the E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) regulates the IMD pathway through ubiquitylation. Yet, the tick genome notably lacks homologs to genes encoding key IMD pathway proteins as described in Drosophila. How XIAP activates the IMD pathway in response to microbial infection is poorly characterized and targets of XIAP-mediated ubiquitylation remain unknown. In this study, we identified the XIAP enzymatic substrate p47 as a positive regulator of the I. scapularis IMD network. XIAP polyubiquitylates p47 in a lysine (K)63-dependent manner and interacts with the ubiquitin-like (UBX) domain of p47. p47 also binds to Kenny (IKKγ/NF-κB essential modulator [NEMO]), the regulatory subunit of the inhibitor of NF-κB kinase (IKK) complex. Replacement of the amino acid lysine with arginine in the p47 linker region completely abrogated molecular interactions with Kenny. Furthermore, reduction of p47 transcription levels through RNA interference in I. scapularis limited Kenny accumulation, reduced phosphorylation of IKKβ (IRD5), and impaired cleavage of the NF-κB molecule Relish. Accordingly, disruption of p47 expression increased microbial colonization of the tick-transmitted spirochete B. burgdorferi and the rickettsial agent Anaplasma phagocytophilum. In summary, we demonstrated that XIAP ubiquitylates p47 in a K63-dependent manner, culminating in Relish activation and antimicrobial responses. Manipulating immune signaling cascades in I. scapularis may lead to innovative approaches to reducing the burden of tick-borne diseases.