ISE6 Cells Research Articles

Biochemical characterization of ClpB and DnaK from Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an intracellular tick-transmitted bacterial pathogen that infects neutrophils in mammals and causes granulocytic anaplasmosis. In this study, we investigated the molecular chaperones ClpB and DnaK from A. phagocytophilum. In Escherichia coli, ClpB cooperates with DnaK and its co-chaperones DnaJ and GrpE in ATP-dependent reactivation of aggregated proteins. Since ClpB is not produced in metazoans, it is a promising target for developing antimicrobial therapies, which generates interest in studies on that chaperone's role in pathogenic bacteria. We found that ClpB and DnaK are transcriptionally upregulated in A. phagocytophilum 3-5 days after infection of human HL-60 and tick ISE6 cells, which suggests an essential role of the chaperones in supporting the pathogen's intracellular life cycle. Multiple sequence alignments show that A. phagocytophilum ClpB and DnaK contain all structural domains that were identified in their previously studied orthologs from other bacteria. Both A. phagocytophilum ClpB and DnaK display ATPase activity, which is consistent with their participation in the ATP-dependent protein disaggregation system. However, despite a significant sequence similarity between the chaperones from A. phagocytophilum and those from E. coli, the former were not as effective as their E. coli orthologs during reactivation of aggregated proteins in vitro and in supporting the survival of E. coli cells under heat stress. We conclude that the A. phagocytophilum chaperones might have evolved with distinct biochemical properties to maintain the integrity of pathogenic proteins under unique stress conditions of an intracellular environment of host cells.

Validation of heat-inducible Ixodes scapularis HSP70 and tick-specific 3xP3 promoters in ISE6 cells

Ixodes scapularis is an important vector of many pathogens, including the causative agent of Lyme disease. The gene function studies in I.scapularis and other ticks are hampered by the lack of genetic tools, including an inducible promoter for temporal control over transgene-encoding protein or double-stranded RNA. We characterized an intergenic sequence upstream of a heat shock protein 70 (HSP70) gene that can drive Renilla luciferase and mCherry expression in the I.scapularis cell line ISE6 (IsHSP70). In another construct, we replaced the Drosophila melanogaster minimal HSP70 promoter of the 3xP3 promoter with a minimal portion of IsHSP70 promoter and generated an I.scapularis-specific 3xP3 (Is3xP3) promoter. Both IsHSP70 and Is3xP3 have a heat-inducible expression of mCherry fluorescence in ISE6 cells with an approximately 10-fold increase in the percentage of fluorescent cells upon 2h heat shock. These promoters described will be valuable tools for gene function studies.

Subolesin knockdown in tick cells provides insights into vaccine protective mechanisms

Ticks as obligate blood-feeding arthropod vectors of pathogenic viruses, bacteria, protozoa and helminths associated with prevalent tick-borne diseases (TBDs) worldwide. These arthropods constitute the second vector after mosquitoes that transmit pathogens to humans and the first vector in domestic animals. Vaccines constitute the safest and more effective approach to control tick infestations and TBDs, but research is needed to identify new antigens and improve vaccine formulations. The tick protein Subolesin (Sub) is a well-known vaccine protective antigen with a highly conserved sequence at both gene and protein levels in the Ixodidae and among arthropods and vertebrates. In this study, transcriptomics and proteomics analyses were conducted together with graph theory data analysis in wild type and Sub knockdown (KD) tick ISE6 cells in order to identify and characterize the functional implications of Sub in tick cells. The results support a key role for Sub in the regulation of gene expression in ticks and the relevance of this antigen in vaccine development against ticks and TBDs. Proteins with differential representation in response to Sub KD provide insights into vaccine protective mechanisms and candidate tick protective antigens.

Genetic manipulation of an Ixodes scapularis cell line.

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.

The role of Rab27 in tick extracellular vesicle biogenesis and pathogen infection

BackgroundThe 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.MethodsUsing 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.ResultsOur 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.ConclusionsRab27 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.Graphical

A novel eukaryotic RdRP-dependent small RNA pathway represses antiviral immunity by controlling an ERK pathway component in the black-legged tick.

Small regulatory RNAs (sRNAs) are involved in antiviral defense and gene regulation. Although roles of RNA-dependent RNA Polymerases (RdRPs) in sRNA biology are extensively studied in nematodes, plants and fungi, understanding of RdRP homologs in other animals is still lacking. Here, we study sRNAs in the ISE6 cell line, which is derived from the black-legged tick, an important vector of human and animal pathogens. We find abundant classes of ~22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs). RdRP1-dependent sRNAs possess 5'-monophosphates and are mainly derived from RNA polymerase III-transcribed genes and repetitive elements. Knockdown of some RdRP homologs misregulates genes including RNAi-related genes and the regulator of immune response Dsor1. Sensor assays demonstrate that Dsor1 is downregulated by RdRP1 through the 3'UTR that contains a target site of RdRP1-dependent repeat-derived sRNAs. Consistent with viral gene repression by the RNAi mechanism using virus-derived small interfering RNAs, viral transcripts are upregulated by AGO knockdown. On the other hand, RdRP1 knockdown unexpectedly results in downregulation of viral transcripts. This effect is dependent on Dsor1, suggesting that antiviral immunity is enhanced by RdRP1 knockdown through Dsor1 upregulation. We propose that tick sRNA pathways control multiple aspects of immune response via RNAi and regulation of signaling pathways.

A Quantum Vaccinomics Approach for the Design and Production of MSP4 Chimeric Antigen for the Control of Anaplasma phagocytophilum Infections.

Anaplasma phagocytophilum Major surface protein 4 (MSP4) plays a role during infection and multiplication in host neutrophils and tick vector cells. Recently, vaccination trials with the A. phagocytophilum antigen MSP4 in sheep showed only partial protection against pathogen infection. However, in rabbits immunized with MSP4, this recombinant antigen was protective. Differences between rabbit and sheep antibody responses are probably associated with the recognition of non-protective epitopes by IgG of immunized lambs. To address this question, we applied quantum vaccinomics to identify and characterize MSP4 protective epitopes by a microarray epitope mapping using sera from vaccinated rabbits and sheep. The identified candidate protective epitopes or immunological quantum were used for the design and production of a chimeric protective antigen. Inhibition assays of A. phagocytophilum infection in human HL60 and Ixodes scapularis tick ISE6 cells evidenced protection by IgG from sheep and rabbits immunized with the chimeric antigen. These results supported that the design of new chimeric candidate protective antigens using quantum vaccinomics to improve the protective capacity of antigens in multiple hosts.

Extended characterisation of five archival tick-borne viruses provides insights for virus discovery in Australian ticks

BackgroundA subset of Australians who have been bitten by ticks experience a complex of chronic and debilitating symptoms which cannot be attributed to the known pathogenic species of bacteria present in Australia. As a result, there has been a renewed effort to identify and characterise viruses in Australian terrestrial ticks. Recent transcriptome sequencing of Ixodes and Amblyomma ticks has revealed the presence of multiple virus sequences. However, without virus isolates our ability to understand the host range and pathogenesis of newly identified viruses is limited. We have established a successful method for high-throughput virus discovery and isolation in mosquitoes using antibodies to double-stranded RNA. In this study we sought to characterise five archival tick-borne viruses to adapt our virus discovery protocol for Australian ticks.MethodsWe performed virus characterisation using a combination of bioinformatic sequence analysis and in vitro techniques including replication kinetics, antigenic profiling, virus purification and mass spectrometry.ResultsOur sequence analysis of Nugget virus, Catch-me-Cave virus and Finch Creek virus revealed marked genetic stability in isolates collected from the same location approximately 30 years apart. We demonstrate that the Ixodes scapularis-derived ISE6 cell line supports replication of Australian members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families, including Saumarez Reef virus (SREV), a flavivirus isolated from the soft tick Ornithodoros capensis. While antibodies against double-stranded RNA could be used to detect replication of a tick-borne reovirus and mosquito-borne flavivirus, the tick-borne flaviviruses Gadgets Gully virus and SREV could not be detected using this method. Finally, four novel virus-like sequences were identified in transcriptome sequencing of the Australian native tick Ixodes holocyclus.ConclusionsGenetic and antigenic characterisations of archival viruses in this study confirm that three viruses described in 2002 represent contemporary isolates of virus species first identified 30 years prior. Our findings with antibodies to double-stranded RNA highlight an unusual characteristic shared by two Australian tick-borne flaviviruses. Finally, comparative growth kinetics analyses of Australian tick-borne members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families in ISE6 and BSR cells will provide a useful resource for isolation of Australian tick-borne viruses using existing cell lines.Graphical

Association between Growth Rate and Pathogenicity of Spotted Fever Group Rickettsia

Rickettsia parkeri and Rickettsia amblyommatis are spotted fever group Rickettsia (SFGR) associated with Amblyomma ticks. R. parkeri is a recognized human pathogen that causes an eschar-associated febrile illness, while R. amblyommatis has not been confirmed as a causative agent of human disease. We hypothesized that the rate of replication is one of the factors contributing to rickettsial pathogenicity. In this study, growth and infectivity of R. parkeri and R. amblyommatis in mammalian (Vero E6) and tick-derived (ISE6) cell lines were assessed and compared over a 96-hour time course of infection using quantitative real-time polymerase chain reaction and microscopy. The pathogenic R. parkeri displayed a significantly higher level of infection in both Vero E6 and ISE6 cells than R. amblyommatis at 72 hours post-inoculation (hpi). Distinct growth profiles between rickettsial species with known and uncertain pathogenicity were identified. R. parkeri burdens were significantly greater than those of R. amblyommatis from 24 to 96 hpi. The relative fold changes of load were significantly higher in the pathogenic agent than in R. amblyommatis from 48 hpi onward and reached the maximum fold increase of ~2002- and ~296-fold in Vero E6 cells and ~1363- and ~161-fold in ISE6 cells, respectively, at 96 hpi. The results from the present study demonstrate that growth rate is associated with the pathogenicity of rickettsiae. Understanding SFGR growth characteristics in mammalian and tick cells will provide insight into rickettsial biology and pathogenesis.

Cationic Glycopolyelectrolytes for RNA Interference in Tick Cells.

The black-legged tick (Ixodes scapularis) is the primary vector for bacteria that cause Lyme disease (Borrelia burgdorferi), where numerous glycosylated tick proteins are involved at the interface of vector-host-pathogen interactions. Reducing the expression of key tick proteins, such as selenoprotein K (SelK), through RNA interference is a promising approach to reduce pathogen transmission, but efficient delivery of nucleic acids to arthropods has proven challenging. While cationic glycopolymers have been used as nonviral gene delivery vehicles in mammalian cells, their use in arthropod or insect gene transfection has not been established. In this study, statistical acrylamide-based cationic glycopolymers with glucose or galactose pendant groups were synthesized by reversible addition-fragmentation chain transfer polymerization, and the effects of the saccharide pendant group and cationic monomer loading on polymer cytotoxicity, RNA complexation, and SelK gene knockdown in ISE6 cells were evaluated. All polymers exhibited low cytotoxicity, yet RNA/copolymer complex cell uptake and gene knockdown were highly dependent on the saccharide structure and the N:P (amino to phosphate groups) ratio.

Molecular characterization of “Candidatus Anaplasma testudinis”: An emerging pathogen in the threatened Florida gopher tortoise (Gopherus polyphemus)

Members of the family Anaplasmataceae are obligate intracellular bacteria that replicate within membrane bound vacuoles in the cytoplasm of cells in vertebrate and invertebrate hosts. This study reports a putative new Anaplasma species in gopher tortoises in Florida. Two Florida gopher tortoises (Gopherus polyphemus) presented at the University of Florida Veterinary Hospital with anemia and intracytoplasmic vacuoles filled with bacteria within erythrocytes. The bacteria within these parasitophorous vacuoles were morphologically similar to Anaplasma marginale. We inoculated ISE6 cells with blood from one tortoise and isolated bacterial colonies consistent with A. marginale. Molecular characterization targeting Anaplasmataceae 16S rRNA sequences indicated that the clinical isolate, named here provisionally as “Candidatus Anaplasma testudinis”, grouped within the genus Anaplasma on a separate clade, most closely related to the A. marginale, Anaplasma ovis and Anaplasma centrale group. We next screened archived red blood cells from 38 wild gopher tortoises with documented clinical anemia. Fourteen of the 38 wild tortoises, representing 5 of 11 geographical locations were PCR-positive for Anaplasmataceae spp. Sequencing analysis revealed 16S rRNA sequence identical to “Ca. A. testudinis”. The clinical presentation of significant anemia associated with “Ca. A. testudinis” in a threatened species could have conservation implications. Importantly, the availability of a clinical isolate will aid further studies to develop diagnostic tests and to investigate potential tick vectors and infectivity for other wildlife and domestic animal species.

Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick—Pathogen Interactions

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick–pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick–pathogen interactions.

An Ixodes scapularis glutathione S-transferase plays a role in cell survival and viability during Langat virus infection of a tick cell line

Ticks are important vectors of diseases affecting both humans and animals. To be an efficient vector, ticks have to survive infection by pathogens such as the Langat virus (LGTV). One method utilized by ticks is their complex antioxidant mechanism. Included in the vast antioxidant processes are several enzymes involved in redox homeostasis. The ubiquitous glutathione S-transferases (GSTs) belong to the antioxidant family of enzymes. In this study, we evaluated the role of a GST during LGTV infection.ISE6 cells were infected with LGTV with a multiplicity of infection (MOI) of 0.01 and observed daily. The infection success was monitored via indirect immunofluorescent antibody test (IFAT) for LGTV for up to 4 days. The gene expression of IsGST1 was determined by real-time polymerase chain reaction (PCR) using IsGST1 gene–specific primers. Knockdown of the IsGST1 gene with subsequent LGTV infection was also performed. Afterward, ISE6 cell mortality and viability were checked daily until the fourth day. The virus titer from supernatants of IsGST1-knockdown cells was quantified using a focus-formation assay.IFAT data showed that LGTV infects ISE6 cells in a time-dependent manner with increasing infection from day 0 to day 4. The IsGST1 genes showed an increasing expression until day 2 of infection, while decreased expression was observed from day 3 to day 4 post-infection. Knockdown of the IsGST1 resulted in increased mortality on the third day of infection, while the cell viability was also negatively affected by the knockdown of the IsGST1 genes from day 0 to day 4 post-infection. Knockdown of the IsGST1 genes also resulted in a decreased viral titer from the supernatants of the ISE6 cells infected with LGTV.Based on the results, GSTs are possibly utilized both by cells and the virus for mutual survival and proliferation.

Discovery of Exosomes From Tick Saliva and Salivary Glands Reveals Therapeutic Roles for CXCL12 and IL-8 in Wound Healing at the Tick-Human Skin Interface.

Ticks secrete various anti-coagulatory, anti-vasoconstrictory, anti-inflammatory, and anti-platelet aggregation factors in their saliva at the bite site during feeding to evade host immunological surveillance and responses. For the first time, we report successful isolation of exosomes (small membrane-bound extracellular signaling vesicles) from saliva and salivary glands of partially fed or unfed ixodid ticks. Our data showed a novel role of these in vivo exosomes in the inhibition of wound healing via downregulation of C-X-C motif chemokine ligand 12 (CXCL12) and upregulation of interleukin-8 (IL-8). Cryo-electron microscopy (cryo-EM) analysis revealed that tick saliva and salivary glands are composed of heterogeneous populations of in vivo exosomes with sizes ranging from 30 to 200 nm. Enriched amounts of tick CD63 ortholog protein and heat shock protein 70 (HSP70) were evident in these exosomes. Treatment of human skin keratinocytes (HaCaT cells) with exosomes derived from tick saliva/salivary glands or ISE6 cells dramatically delayed cell migration, wound healing, and repair process. Wound healing is a highly dynamic process with several individualized processes including secretion of cytokines. Cytokine array profiling followed by immunoblotting and quantitative-PCR analysis revealed that HaCaT cells treated with exosomes derived from tick saliva/salivary glands or ISE6 cells showed enhanced IL-8 levels and reduced CXCL12 loads. Inhibition of IL-8 or CXCL12 further delayed exosome-mediated cell migration, wound healing, and repair process, suggesting a skin barrier protection role for these chemokines at the tick bite site. In contrast, exogenous treatment of CXCL12 protein completely restored this delay and enhanced the repair process. Taken together, our study provides novel insights on how tick salivary exosomes secreted in saliva can delay wound healing at the bite site to facilitate successful blood feeding.

Global Transcription Profiles of Anaplasma phagocytophilum at Key Stages of Infection in Tick and Human Cell Lines and Granulocytes.

The incidence of human diseases caused by tick-borne pathogens is increasing but little is known about the molecular interactions between the agents and their vectors and hosts. Anaplasma phagocytophilum (Ap) is an obligate intracellular, tick-borne bacterium that causes granulocytic anaplasmosis in humans, dogs, sheep, and horses. In mammals, neutrophil granulocytes are a primary target of infection, and in ticks, Ap has been found in gut and salivary gland cells. To identify bacterial genes that enable Ap to invade and proliferate in human and tick cells, labeled mRNA from Ap bound to or replicating within human and tick cells (lines HL-60 and ISE6), and replicating in primary human granulocytes ex vivo, was hybridized to a custom tiling microarray containing probes representing the entire Ap genome. Probe signal values plotted over a map of the Ap genome revealed antisense transcripts and unannotated genes. Comparisons of transcript levels from each annotated gene between test conditions (e.g., Ap replicating in HL-60 vs. ISE6) identified those that were differentially transcribed, thereby highlighting genes associated with each condition. Bacteria replicating in HL-60 cells upregulated 122 genes compared to those in ISE6, including numerous p44 paralogs, five HGE-14 paralogs, and 32 hypothetical protein genes, of which 47% were predicted to be secreted or localized to the membrane. By comparison, 60% of genes upregulated in ISE6 encoded hypothetical proteins, 60% of which were predicted to be secreted or membrane associated. In granulocytes, Ap upregulated 120 genes compared to HL-60, 33% of them hypothetical and 43% of those predicted to encode secreted or membrane associated proteins. HL-60-grown bacteria binding to HL-60 cells barely responded transcriptionally, while ISE6-grown bacteria binding to ISE6 cells upregulated 48 genes. HL-60-grown bacteria, when incubated with ISE6 cells, upregulated the same genes that were upregulated by ISE6-grown bacteria exposed to uninfected ISE6. Hypothetical genes (constituting about 29% of Ap genes) played a disproportionate role in most infection scenarios, and particular sets of them were consistently upregulated in bacteria binding/entering both ISE6 and HL-60 cells. This suggested that the encoded proteins played central roles in establishing infection in ticks and humans.

The identification of tick autophagy-related genes in Ixodes scapularis responding to amino acid starvation

Ticks are obligate hematophagous arthropods and must tolerate starvation during off‐host periods. Macroautophagy (hereafter autophagy) is a well-conserved self-eating mechanism of cell survival and is essential for recycling cellular contents during periods of starvation, stress, and injury in organisms. Although the genome sequence of Ixodes scapularis (Say) is available, the characteristics and functions of autophagy-related gene families remain largely unknown. To advance our understanding of autophagy in I. scapularis, we used comprehensive genomic approaches to identify Atg genes. Homologues of 14 Atg genes were identified, and their protein motif compositions were predicted. Phylogenetic analysis indicated that ATGs in I. scapularis were evolutionarily closely related to their homologues in Haemaphysalis longicornis and Rhipicephalus microplus ticks. Expression patterns of Atg genes differed across tick developmental stages. Immunofluorescence results by monodansylcadaverine (MDC) staining indicated that autophagy was activated after amino acid starvation treatments in I. scapularis embryo-derived cell lines ISE6 and IDE8. Subsequently, the expression of key Atg genes involved in autophagy pathway in both cell lines were examined. In ISE6 cells, the expression levels of three Atg genes (Atg4B, Atg6 and Atg8A) increased significantly after amino acid starvation; similarly, four Atg genes (Atg4A, Atg4B, Atg6 and Atg8B) were upregulated in IDE8 cells in response to starvation. In parallel, the MDC and lysotracker staining results indicated that autophagy was triggered after amino acid starvation treatments in R. microplus embryo-derived cell line BME26. Our observations showed that Atg family genes are highly conserved in ticks and function in autophagy pathway induced by amino acid starvation. These results also provide valuable insight for further autophagy-related research as a new strategy for blocking the transmission of tick-borne pathogens.

Characterization of an iron-inducible Haemaphysalis longicornis tick-derived promoter in an Ixodes scapularis-derived tick cell line (ISE6)

BackgroundTicks are important vectors of disease-causing pathogens. With the rise of resistance to chemical acaricides, alternative methods in tick control are warranted. Gene manipulation has been successful in controlling mosquitoes and mosquito-borne diseases and is now looked upon as a candidate method to control ticks and tick-borne pathogens. Our previous study has identified the actin and ferritin promoter regions in the Haemaphysalis longicornis tick.ResultsHere, the ferritin-derived promoter from the H. longicornis tick was characterized in silico, and the core promoter sequences and some of its important components were identified. Several truncations of the promoter region were created and inserted to a reporter plasmid to determine the important components for its activity. The activities of the truncated promoters on the Ixodes scapularis tick cell line (ISE6) were measured via a dual luciferase assay using experimental and control reporter genes. To induce the promoter’s activity, transfected ISE6 cells were exposed to ferrous sulfate. The 639 nucleotides truncated promoter showed the highest activity on ISE6 cells when exposed to 1 mM ferrous sulfate.ConclusionIn this study, we characterized an iron-inducible tick promoter that could be a valuable tool in the development of a gene-manipulation system to control ticks and tick-borne pathogens.

Tissue-specific signatures in tick cell line MS profiles

BackgroundThe availability of tick in vitro cell culture systems has facilitated many aspects of tick research, including proteomics. However, certain cell lines have shown a tissue-specific response to infection. Thus, a more thorough characterization of tick cell lines is necessary. Proteomic comparative studies of various tick cell lines will contribute to more efficient application of tick cell lines as model systems for investigation of host-vector-pathogen interactions.ResultsThree cell lines obtained from a hard tick, Ixodes ricinus, and two from I. scapularis were investigated. A cell mass spectrometry approach (MALDI-TOF MS) was applied, as well as classical proteomic workflows. Using PCA, tick cell line MS profiles were grouped into three clusters comprising IRE/CTVM19 and ISE18, IRE11 and IRE/CTVM20, and ISE6 cell lines. Two other approaches confirmed the results of PCA: in-solution digestion followed by nanoLC-ESI-Q-TOF MS/MS and 2D electrophoresis. The comparison of MS spectra of the cell lines and I. ricinus tick organs revealed 29 shared peaks. Of these, five were specific for ovaries, three each for gut and salivary glands, and one for Malpighian tubules. For the first time, characteristic peaks in MS profiles of tick cell lines were assigned to proteins identified in acidic extracts of corresponding cell lines.ConclusionsSeveral organ-specific MS signals were revealed in the profiles of tick cell lines.

Novel insights into endogenous RNA viral elements in Ixodes scapularis and other arbovirus vector genomes.

Many emerging arboviruses are not transmitted by traditional mosquito vectors, but by lesser-studied arthropods such as ticks, midges, and sand flies. Small RNA (sRNA) silencing pathways are the main antiviral defence mechanism for arthropods, which lack adaptive immunity. Non-retroviral integrated RNA virus sequences (NIRVS) are one potential source of sRNAs which comprise these pathways. NIRVS are remnants of past germline RNA viral infections, where viral cDNA integrates into the host genome and is vertically transmitted. In Aedes mosquitoes, NIRVS are widespread and produce PIWI-interacting RNAs (piRNAs). These are hypothesised to target incoming viral transcripts to modulate viral titre, perhaps rendering the organism a more efficient arbovirus vector. To explore the NIRVS landscape in alternative arbovirus vectors, we validated the NIRVS landscape in Aedes spp. and then identified novel NIRVS in six medically relevant arthropods and also in Drosophila melanogaster. We identified novel NIRVS in Phlebotomus papatasi, Culicoides sonorensis, Rhipicephalus microplus, Anopheles gambiae, Culex quinquefasciatus, and Ixodes scapularis. Due to their unexpected abundance, we further characterised NIRVS in the blacklegged tick I. scapularis (n = 143). Interestingly, NIRVS are not enriched in R. microplus, another hard tick, suggesting this is an Ixodes-specific adaptation. I. scapularis NIRVS are enriched in bunya- and orthomyxo-like sequences, reflecting that ticks are a dominant host for these virus groups. Unlike in mosquitoes, I. scapularis NIRVS are more commonly derived from the non-structural region (replicase) of negative-sense viruses, as opposed to structural regions (e.g. glycoprotein). Like other arthropods, I. scapularis NIRVS preferentially integrate into genomic piRNA clusters, and serve as a template for primary piRNA production in the commonly used embryonic I. scapularis ISE6 cell line. Interestingly, we identified a two-fold enrichment of non-long terminal repeat (non-LTR) retrotransposons, in genomic proximity to NIRVS, contrasting with studeis in Ae. aegypti, where LTR retrotransposons are instead associated with NIRVS formation. We characterised NIRVS phylogeny and integration patterns in the important vector, I. scapularis, revealing they are distinct from those in Aedes spp. Future studies will explore the possible antiviral mechanism conferred by NIRVS to I. scapularis,which may help the transmission of pathogenic arboviruses. Finally, this study explored NIRVS as an untapped wealth of viral diversity in arthropods.

Induction of intracellular ferritin expression in embryo-derived Ixodes scapularis cell line (ISE6)

Iron is a very important nutrient for cells; however, it could also cause fatal effects because of its capability to trigger oxidative stress. Due to high exposure to iron from their blood diet, ticks make use of several mechanisms to cope up with oxidative stress. One mechanism is iron sequestration by ferritin and its control protein (IRP). Since the IRP activity is dependent on the ferrous iron concentration, we tried to induce intracellular ferritin (FER1) protein expression by exposing Ixodes scapularis embryo-derived cell line (ISE6) to different concentrations of ferrous sulphate at different time points. We were able to induce FER1 protein after exposure to 2 mM of ferrous sulphate for 48 h, as observed in both Western blotting and indirect immunofluorescent antibody tests. This could indicate that the FER1 produced could be a product of the release of IRPs from the FER1 mRNA leading to its translation. The RNA interference of FER1, through the transfection of dsRNA, led to an increase in mortality and decrease in the cellular proliferation of ISE6 cells. Overall, ISE6 cells could be a good tool in further understanding the mechanism of FER1 action, not just in Ixodes ticks but in other tick species as well.