Huang Yue, Pei Shifeng, Lv Xin, Yang Fuxian, Gong Xiaoqing, Li Na, Guo Yaqiong, Feng Yaoyu, Xiao Lihua
State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou, China.
Guangdong Laboratory for Lingnan Modern Agriculture, Center for Emerging and Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
PLoS Negl Trop Dis. 2025 Jan 13;19(1):e0012777. doi: 10.1371/journal.pntd.0012777. eCollection 2025 Jan.
The determinants of differences in host infectivity among Cryptosporidium species and subtypes are poorly understood. Results from recent comparative genomic studies suggest that gains and losses of multicopy subtelomeric genes encoding insulinase-like proteases (INS-19 and INS-20 in Cryptosporidium parvum and their orthologs in closely related species) may potentially contribute to these differences.
METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the expression and biological function of the INS-19 and INS-20 of C. parvum. CRISPR/Cas9 was used to endogenously tag both genes with the hemagglutinin epitope. Immunofluorescence analysis revealed that INS-19 and INS-20 are expressed at different developmental stages of the pathogen. Although knockout of either had no detectable effect on the in vitro growth of C. parvum, knockout of INS-20, deletion of its multiple domains, or mutation of the active motif in the functional domain reduced the intensity of C. parvum infection in IFN-γ knockout mice. Consistent with this, mice infected with the INS-20-deleted mutant had reduced intestinal damage and parasite burden.
CONCLUSIONS/SIGNIFICANCE: These results suggest that INS-19 and INS-20 have stage-specific expression with distinct biological functions, and that the presence of the INS-20 in zoonotic C. parvum contributes to its infectivity and fitness in mice.
隐孢子虫物种和亚型之间宿主感染性差异的决定因素尚不清楚。最近的比较基因组研究结果表明，编码胰岛素酶样蛋白酶的多拷贝亚端粒基因（微小隐孢子虫中的INS-19和INS-20及其密切相关物种中的直系同源基因）的获得和丢失可能是造成这些差异的潜在原因。
方法/主要发现：在本研究中，我们调查了微小隐孢子虫INS-19和INS-20的表达及生物学功能。使用CRISPR/Cas9将血凝素表位内源性标记到这两个基因上。免疫荧光分析显示，INS-19和INS-20在病原体的不同发育阶段表达。虽然敲除其中任何一个对微小隐孢子虫的体外生长均无明显影响，但敲除INS-20、删除其多个结构域或在功能结构域中使活性基序发生突变，均会降低微小隐孢子虫在干扰素γ基因敲除小鼠中的感染强度。与此一致的是，感染INS-20缺失突变体的小鼠肠道损伤和寄生虫负荷均降低。
结论/意义：这些结果表明，INS-19和INS-20具有阶段特异性表达及不同的生物学功能，并且人畜共患的微小隐孢子虫中INS-20的存在有助于其在小鼠中的感染性和适应性。
