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

Abstract

Background Ticks 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. Results Here, 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. Conclusion In 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. Electronic supplementary material The online version of this article (10.1186/s13071-019-3574-9) contains supplementary material, which is available to authorized users.

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

“…(D) The Dsor1 3’UTR was cloned in the pmirGLO/Fer-Luc2/Act-hRluc vector [51], and ISE6 cells that were soaked with the indicated dsRNA were transfected with the sensor plasmid. The ratio between firefly luc and Rluc was normalized to that of the empty sensor, and the means and standard deviations are shown in the chart (n=7).…”

“…A statistically significant increase in the IscDsor1 level was also observed upon Ago-96 knockdown, in addition to RdRP1 knockdown, suggesting that Ago-96 might also be involved in the regulation of IscDsor1 (Figure 7C). To test if RdRP1 regulates IscDsor1 through its 3'UTR, we cloned IscDsor1 3'UTR after the firefly luciferase coding region of the pmirGLO/Fer-Luc2/Act-hRluc vector (54). After depleting RdRP1 or RdRP3 in ISE6 cells, we transfected the IscDsor1 3'UTR sensor plasmid and performed dual luciferase assays to detect up-regulation of sensors upon knockdown of RdRPs (Figure 7D).…”

“…Note that many genes were dependent on the Aub-AGO3 (blue), RdRP3-Ago-16 (pink) and RdRP1-Ago-30 (green) combinations. (D) The Dsor1 3'UTR was cloned in the pmirGLO/Fer-Luc2/Act-hRluc vector [51], and ISE6 cells that were soaked with the indicated dsRNA were transfected with the sensor plasmid.…”

Tick Cell Lines in Research on Tick Control

“…They showed that silencing of FER1 expression by RNA interference led to a decrease in ISE6 cell proliferation and an increase in mortality, concluding that ISE6 cells could be a good tool to further understanding of the mechanism of FER1 action. Thereafter, Hernandez et al (2019) characterized the activity of an iron-inducible tick promoter derived from the tick Haemaphysalis longicornis, previously identified by Kusakisako et al (2018b), in ISE6 cells, which could be a valuable tool in the development of a gene-manipulation system to control ticks and TBPs. Kusakisako et al (2018a) established methods to visualize H 2 O 2 in ISE6 cells using an intracellular H 2 O 2 probe and observed that paraquat, known to induce oxidative stress in mammalian cells (Table 2), was also effective in this respect in ISE6 tick cells.…”

“…Kusakisako et al (2018a) established methods to visualize H 2 O 2 in ISE6 cells using an intracellular H 2 O 2 probe and observed that paraquat, known to induce oxidative stress in mammalian cells (Table 2), was also effective in this respect in ISE6 tick cells. Iron derived from host blood may react with oxygen in the tick's body and lead to high levels of H 2 O 2 , causing oxidative stress such as DNA strand breaks, lipid peroxidation and degradation of other molecules (Kusakisako et al, 2018a;Hernandez et al, 2019).…”