Functional analysis of Haemaphysalis longicornis HSP20 in regulating protein–protein interaction with pathogenic Rickettsia

AbstractBackground: Haemaphysalis longicornis, a globally distributed obligate hematophagous ectoparasite, can transmit multiple zoonotic pathogens, posing a significant threat to public health and animal husbandry. Rickettsia heilongjiangensis is one of the pathogens carried by H. longicornis and can cause various diseases in humans. sHSPs are key molecular chaperones involved in biological stress responses and pathogen–host interactions. Following experimental infection with R. heilongjiangensis, the expression of multiple HSP genes–including HSP20, HSP70, and HSP90 was upregulated, yet the specific role of HSP20 in tick–pathogen interactions remains unclear.Methods: In this study, RT–qPCR was employed to clone the full–length sequence of the HSP20 gene from H. longicornis Bioinformatics tools were used to analyze its sequence features and cross–species genetic conservation. The expression profile of the HSP20 gene across different developmental stages (egg, larva, nymph, and adult) was examined by qPCR. RNAi was applied to silence the HSP20 gene, with interference efficiency (85% inhibition) verified by qPCR, followed by quantitative analysis of R. heilongjiangensis proliferation in ticks. GST Pull–Down assays, mass spectrometry, and yeast two–hybrid experiments were combined to screen and identify R. heilongjiangensis proteins that interact with HSP20.Results: Sequence analysis confirmed that HSP20 is a non–transmembrane small heat shock protein containing a conserved αB–crystallin domain and exhibits high genetic conservation among different tick species. Expression profiling showed that the HSP20 gene was most highly expressed during the egg and larval stages, suggesting a potential role in early tick development. RNAi–mediated silencing of the HSP20 gene significantly promoted the proliferation of R. heilongjiangensis in ticks, indicating that HSP20 inhibits the replication of this rickettsia. Moreover, interaction assays confirmed that HSP20 specifically binds to the 50S ribosomal protein of R. heilongjiangensis, although the interaction strength is weak.Conclusion: HSP20 in H. longicornis is a conserved small heat shock protein with stage–specific expression during development. It can regulate the proliferation of R. heilongjiangensis through interaction with the pathogen’s 50S ribosomal protein. Although the strength and underlying molecular mechanisms of this interaction require further validation and exploration, this study clarifies the regulatory role of HSP20, providing new insights into deciphering the tick–rickettsia molecular network and identifying potential control targets for tick–borne rickettsioses.