Involvement of a variant secretory protein in virulence of emerging Cryptosporidium parvum subtypes

Several divergent Cryptosporidium parvum subtypes have emerged in humans in recent years but their infectivity, pathogenicity and genetic characteristics are unclear. In the present study, IFN-γ knockout C57BL/6 (GKO) mice were infected with the novel IIoA15G1 and IIpA11 subtypes of C. parvum and compared with the common IIaA17G2R1 subtype. The genomes of these isolates were sequenced and compared with each other. Further gene tagging and deletion were performed on the virulence-associated polymorphic cgd8_5420 gene encoding a secretory protein using the CRISPR/Cas9 technology. IIpA11 and IIoA15G1 were highly infectious in GKO mice, with an ID50 of 2.4 and 3.6 oocysts, respectively. The duration of oocyst shedding for IIpA11 (> 58.0 ± 1.4 days) and IIoA15G1 (> 57.5 ± 0.9 days) was significantly longer than for IIaA17G2R1 (5.5 ± 0.9 days; P < 0.001). The genomes of IIaA17G2R1, IIoA15G1, and IIpA11 had 203, 46,839, and 47,122 single nucleotide polymorphisms, respectively, compared to C. parvum IOWA II. In contrast, minor sequence differences were found between IIoA15G1 and IIpA11, mainly in several genes encoding invasion-associated mucin glycoproteins and cgd8_5420 encoding a secretory protein. The latter is mainly expressed in trophozoites, merozoites, and macrogamonts. Deletion of this gene reduced the intensity of IIpA11 infection and increased the survival of infected mice. The emerging IIoA15G1 and IIpA11 subtypes have divergent genomes and are highly infectious and pathogenic in GKO mice. Several secretory proteins, including a variant protein encoded by the subtelomeric cgd8_5420 gene, are associated with differences in virulence between the two subtypes.

近年来，人类中出现了多种分歧的隐孢子虫小孢子变种，但其感染性、致病性和遗传特征尚不明确。在本研究中，对IFN-γ敲除的C57BL/6（GKO）小鼠感染了新的IIoA15G1和IIpA11亚型的隐孢子虫，并与常见的IIaA17G2R1亚型进行了比较。对这些分离株的基因组进行了测序并相互比较。进一步利用CRISPR/Cas9技术对与致病性相关的多态性cgd8_5420基因进行了基因标记和删除，该基因编码一种分泌蛋白。IIpA11和IIoA15G1在GKO小鼠中表现出高度传染性，分别以2.4和3.6个子孢子为半数感染剂量（ID50）。IIpA11（>58.0 ± 1.4天）和IIoA15G1（>57.5 ± 0.9天）的子孢子排出持续时间显著长于IIaA17G2R1（5.5 ± 0.9天；P < 0.001）。与隐孢子虫IOWA II相比，IIaA17G2R1、IIoA15G1和IIpA11的基因组分别具有203、46,839和47,122个单核苷酸多态性。相反，IIoA15G1和IIpA11之间发现了微小的序列差异，主要在于编码与侵袭相关的粘蛋白糖蛋白和编码分泌蛋白的cgd8_5420基因。后者主要在滋养体、裂殖体和大配子体中表达。删除此基因降低了IIpA11感染的强度并增加了感染小鼠的存活率。新出现的IIoA15G1和IIpA11亚型具有分歧的基因组，在GKO小鼠中具有高度传染性和致病性。包括由亚端粒cgd8_5420基因编码的变异蛋白在内的多种分泌蛋白与两个亚型之间致病性差异相关。