ABSTRACT
Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis (xiap) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks, which currently limit efficient and scalable molecular genetic screens in vivo.IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis.
Subject(s)
Anaplasma phagocytophilum , Borrelia burgdorferi , Ixodes , Rickettsia , Animals , Humans , Borrelia burgdorferi/genetics , Anaplasma phagocytophilum/genetics , Cell Line , MammalsABSTRACT
BACKGROUND: The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility. METHODS: Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. RESULTS: Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis. CONCLUSIONS: Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.
Subject(s)
Anaplasma phagocytophilum , Arthropod Proteins , Extracellular Vesicles , Ixodes , rab27 GTP-Binding Proteins , Animals , Humans , Mice , Anaplasma phagocytophilum/physiology , Ixodes/cytology , Ixodes/metabolism , Ixodes/microbiology , Mammals , Extracellular Vesicles/metabolism , rab27 GTP-Binding Proteins/metabolism , Arthropod Proteins/metabolismABSTRACT
Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.
Subject(s)
Anaplasma phagocytophilum , Arthropods , Borrelia burgdorferi , Ixodes , Lyme Disease , Animals , Hemocytes , Ixodes/microbiology , Borrelia burgdorferi/physiologyABSTRACT
Arthropod-borne pathogens are responsible for hundreds of millions of infections in humans each year. The blacklegged tick, Ixodes scapularis, is the predominant arthropod vector in the United States and is responsible for transmitting several human pathogens, including the Lyme disease spirochete Borrelia burgdorferi and the obligate intracellular rickettsial bacterium Anaplasma phagocytophilum, which causes human granulocytic anaplasmosis. However, tick metabolic response to microbes and whether metabolite allocation occurs upon infection remain unknown. Here we investigated metabolic reprogramming in the tick ectoparasite I. scapularis and determined that the rickettsial bacterium A. phagocytophilum and the spirochete B. burgdorferi induced glycolysis in tick cells. Surprisingly, the endosymbiont Rickettsia buchneri had a minimal effect on bioenergetics. An unbiased metabolomics approach following A. phagocytophilum infection of tick cells showed alterations in carbohydrate, lipid, nucleotide and protein metabolism, including elevated levels of the pleiotropic metabolite ß-aminoisobutyric acid. We manipulated the expression of genes associated with ß-aminoisobutyric acid metabolism in I. scapularis, resulting in feeding impairment, diminished survival and reduced bacterial acquisition post haematophagy. Collectively, we discovered that metabolic reprogramming affects interspecies relationships and fitness in the clinically relevant tick I. scapularis.