Cathepsin L—a novel cysteine protease from Haemaphysalis flava Neumann, 1897
Ixodid ticks are ectoparasites responsible for the transmission of a large number of bacterial, viral, and protozoan pathogens to animals and humans. As long-term blood-pool feeders, the digestion of host blood is critical to their development as well as to the establishment of the sexual cycle of hemoparasites such as Babesia parasites, the agents of human and animal babesiosis. Previous studies have demonstrated that cysteine proteases are involved in blood digestion, embryogenesis, and pathogen transmission in other species of ticks, but their characteristics and functions are still unidentified in Haemaphysalis flava. Here, we describe the characterization of a cysteine protease HfCL from H. flava. We show that HfCL belongs to the L-like papain family of proteases, exhibits high expression in nymphs and adults, and localizes to both the midgut and salivary glands. Biochemical assays using purified recombinant enzyme reveal that rHfCL can hydrolyze the fluorogenic substrate Z-phe-Arg-MCA with optimal activity detected at pH 6. Furthermore, the short-term growth assay indicates that rHfCL can inhibit the intraerythrocytic development of Babesia microti and Babesia gibsoni in vitro.
KeywordsCathepsin L Papain Haemaphysalis flava Babesia microti Babesia gibsoni
- H. flava
- B. microti
- B. gibsoni
open reading frame
tris base-boric acid-EDTA
rapid-amplification of cDNA ends
quantitative real-time PCR
polymerase chain reaction
bull serum albumin
sodium dodecyl sulfate polyacrylamide gel electrophoresis
phosphate buffered solution
tris buffer saline Tween 20
red blood cells
l-trans-epoxysuccinyl-leucylamido 4-guanidino butane
indirect immunofluorescence assay
We thank all the research team members in our lab for assistance. The authors would like to thank Professor Choukri Ben Mamoun (Yale University, USA) for revising manuscript.
YS performed the study and wrote the manuscript. JZ and LH conceived the study. ZN, JG, and QL collected samples. LH critically revised the manuscript. YS and LY performed the experiments and data analyses. All authors have read and approved the final manuscript.
This study was supported by the National Basic Science Research Program (973 program) of China (Grant No. 2015CB150300), the National Key Research and Development Program of China (Grant No. 2017YFD0501201), the National Natural Science Foundation of China (Grant No. 31772729), and the Natural Science Foundation of Hubei Province (Grant No. 2017CFA020).
Compliance with ethical standards
Ethics approval and consent to participate
The care and maintenance of experimental rabbit, mice, and dog in this study was approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, and all experiments were performed according to the regulations for the administration of affairs concerning experimental animals of Hubei Province, P.R. China consent for publication.
The authors declare that they have no competing interests.
Consent for publication
HfCL mRNA sequence was available in GenBank (accession no. MG914066).
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