Ticks Tick Borne Dis. 2016 Feb; 7(1):36-45.

Role of the tumor necrosis factor receptor-associated factor-type zinc finger domain containing protein 1 (TRAFD1) from the hard tick Haemaphysalis longicornis in immunity against bacterial infection

Rie Takechi1, Remil Linggatong Galay1, Tomohide Matsuo2,3, Hiroki Maeda1,2, Kodai Kusakisako1,2, Melbourne Rio Talactac1,2, Masami Mochizuki1,2, Kozo Fujisaki4, and Tetsuya Tanaka1,2*


1Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University

2Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University

3Laboratory of Parasitology, Joint Faculty of Veterinary Medicine, Kagoshima University

4Zen-noh Institute of Animal Health

*Corresponding to tetsuya@ms.kagoshima-u.ac.jp (T. Tanaka)



A tumor necrosis factor receptor-associated factor-type zinc finger domain containing protein 1 (TRAFD1) is a negative feedback regulator that controls excessive immune responses in vertebrates. The sequence of tick hemolymph TRAFD1 from the hard tick Haemaphysalis longicornis (HlTRAFD1) was analyzed after identification and cloning from the expressed sequence tag database. RT-PCR and Western blot analyses showed that HlTRAFD1 transcript and protein levels after blood feeding were present in all developmental stages, and the transcript level was consistently high in all organs examined from adult female ticks upon engorgement. Knockdown of HlTRAFD1 gene by RNA interference did not affect blood feeding or oviposition. However, HlTRAFD1 silencing affected the expression of the longicin gene, a defensin-like molecule, but not the lysozyme gene. Moreover, the survival rate of HlTRAFD1-silenced ticks was lower, and the number of Escherichia coli was higher in the hemolymph and plasmatocytes after E. coli injection compared to the control group. These results suggested that HlTRAFD1 strongly affected both the humoral and cellular immunity of ticks.

PMID: 26283173



Ticks are obligate hematophagous arthropods, and their sole source of nutrients is blood. Since ticks take in a large amount of blood for a long time, there is a high risk of encountering various pathogens (1, 2). In spite of the great damage to humans and animals worldwide, there is still no absolute control measure against ticks and tick-borne infection that is both safe and effective. Therefore, understanding of tick innate immunity components, such as the tumor necrosis factor receptor-associated factor (TRAF) family protein, related to TRAFD1 is very important in limiting pathogen multiplication in ticks. In this study, we investigated whether HlTRAFD1 has a function in tick innate immunity, particularly against bacteria.

The function of HlTRAFD1 in the hemocytes was further confirmed after injecting bacteria directly into the hemocoel of HlTRAFD1-silenced ticks. As a result, a significant difference was observed in the survival rate and the state of hemocytes of HlTRAFD1-silenced ticks and control ticks after injection of E. coli but not after injection of Staphylococcus aureus. Since HlTRAFD1 knockdown suppressed the expression of longicin but not that of lysozyme, it was thought that HlTRAFD1 does not participate in the immune response against Gram-positive bacteria. Furthermore, in the case of E. coli, there was a significant difference in the survival rate when live bacteria were used but not when heat-killed bacteria or LPS were used. In fact, there were many E. coli bacteria in the hemolymph and hemocytes, and the ratio of plasmatocytes containing E. coli was increased in HlTRAFD1-silenced ticks. Based on this result, there is a possibility that the increasing of number of E. coli inside the hemocytes was because the digestion ability of hemocytes was inhibited or because the production of antibacterial molecules was inhibited by the HlTRAFD1 gene knockdown.

Sanda et al. (3) demonstrated that TRAFD1 knockout mice exhibit enhanced induction of inflammatory cytokines and higher susceptibility to LPS and poly (I:C)-induced endotoxic shock. Toll-like receptor (TLR)-mediated activation of innate immunity, when in excess, triggers inflammatory diseases. Because dysregulation of type I or type II IFNs and of inflammatory cytokine production drives these diseases, TRAFD1 might be involved in the disease processes. Ticks might not produce inflammatory cytokine, unlike mammals; therefore, the survival rate of HlTRAFD1-silenced ticks by LPS injection was not affected, as well as in the control group.

Taken together, it was shown that expression of the HlTRAFD1 gene and HlTRAFD1 protein was increased by blood feeding, but HlTRAFD1 is not crucial for blood feeding and oviposition. Interestingly, the HlTRAFD1 gene apparently regulates the expression of the longicin gene, which is a defensin molecule, and therefore, it was thought that HlTRAFD1 had a function in the innate immunity of ticks. Moreover, the digestion capacity of hemocytes was inhibited by HlTRAFD1 knockdown; thus, it is assumed that HlTRAFD1 strongly affects both the humoral immunity and the cellular immunity of ticks (Figure 1). Further studies are needed to elucidate the HlTRAFD1 signaling pathway and the relationship between HlTRAFD1 and pathogen establishment and control.


Technical supplement:

Material introduced into ticks by injection using glass needle: After HlTRAFD1 gene silencing, adult ticks were injected with enhanced green fluorescence protein (EGFP) expressing-E. coli, and then monitored for survival rate. Hemolymph including hemocytes was collected for microscopic examination of hemocytes, and EGFP-E. coli culture of hemolymph to demonstrate bacterial viability (Figure 2).



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  2. Ocaido M, Muwazi RT, Opuda JA. (2009) Economic impact of ticks and tick-borne diseases on cattle production systems around Lake Mburo National Park in South Western Uganda. Trop Anim Health Prod 41: 731-739.
  3. Sanada T, Takaesu G, Mashima R, Yoshida R, Kobayashi T, Yoshimura A. (2008) FLN29 deficiency reveals its negative regulatory role in the Toll-like receptor (TLR) and retinoic acid-inducible gene I (RIG-I)-like helicase signaling pathway. J Biol Chem 283: 33858-33864.



This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 25292173 and 26660229.



Tetsuya Tanaka, Ph. D.

Associate Professor

Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan

Phone&Fax: +81-99-285-3570

E-mail: tetsuya@ms.kagoshima-u.ac.jp




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