Characterization of two ferritins from hard tick, Haemaphysalis longicornis

Title Characterization of two ferritins from hard tick, Haemaphysalis longicornis
Lecturer Dr. Tetsuya Tanaka (Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University)
Language English
Date&Time 02/15/2024 (Thu) 14:00~15:00
Venue Large seminar room (C109)

 Ticks are obligate hematophagous parasites, utilizing blood from their hosts for survival and reproduction. Their feeding behaviour makes them capable of transmitting several infectious diseases. Ticks require large amounts of blood, exposing them to great free iron. Iron is both essential and toxic for living organisms. Different organisms have several mechanisms for iron metabolism. In ticks, however, iron metabolism remains to be unclear. Here, we identified a new secreted ferritin (HlFER2) from the hard tick, Haemaphysalis longicornis, and characterized it with the previously identified intracellular ferritin (HlFER1).
 RT-PCR analysis showed that Hlfer1 was similarly expressed in different organs of adult ticks and developmental stages, while Hlfer2 showed weak expression in some organs and developmental stages. Through western blot analysis using specific anti-ferritin mouse sera, a similar pattern of expression in different organs and developmental stages was observed for both ferritins. However, only HlFER2 was detected in the egg and hemolymph. The indirect immunofluorescent antibody technique revealed the localization of both ferritins in the midgut, salivary glands, and ovary of partially fed adults. Silencing of ferritin genes through RNA interference reduced the ticks' capacity to engorge, high mortality after blood meal fully, and decreased egg laying capacity. Silencing was confirmed through RT-PCR and Western blot analysis. Most of the Hlfer1 and Hlfer2-silenced ticks died without laying eggs, and laid eggs failed to hatch. These results suggest that FER1 and FER2 are crucial to successful tick feeding and reproduction.
 Ticks are vectors of multiple pathogens and may be exposed to bacterial pathogens when they feed on a septicemic host. Therefore, tick innate immunity is crucial in limiting pathogen multiplication to ensure tick survival. Iron withholding through iron-binding proteins, such as transferrin and lactoferrin, is a known component of the innate immunity of most organisms. This mechanism limits the access of bacteria to iron, suppressing their multiplication. However, the function of the iron-binding protein ferritin is still unknown. Here, we examined the role of HlFERs in immunity against bacterial infection. After Hlfer silencing, adult ticks were injected with live or heat-killed enhanced green fluorescence protein (EGFP) expressing Escherichia coli, and then monitored for survival rate. Hemolymph, including hemocytes, was collected for microscopic examination, and an E. coli culture was used to demonstrate bacterial viability. Hlfer-silenced ticks had a significantly lower survival rate after live E. coli injection than control. Microscopic examination of hemocytes showed that plasmatocytes from Hlfer-silenced ticks had more bacteria in the cytoplasm, particularly at 72 hours after bacterial injection, compared to control ticks. Furthermore, the culture of hemolymph from Hlfer-silenced ticks yielded more bacterial colonies than control ticks. These results indicate that the iron storage function of FERs is essential in limiting bacterial multiplication and is crucial to tick immunity.

Contact RNA Molecular Medicine
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