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

Several divergent Cryptosporidium parvum subtypes have emerged in people 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 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 most polymorphic virulence-associated cgd8_5420 gene encoding a hypothetical 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 d) and IIoA15G1 (>57.5 ± 0.9 d) was significantly longer than for IIaA17G2R1 (5.5 ± 0.9 d; p < 0.001). One of the mice infected with IIpA11 died on day 33 post infection. The genomes of IIaA17G2R1, IIoA15G1, and IIpA11 had 203, 46839, and 47,122 single nucleotide polymorphisms, respectively, compared to C. parvum IOWA II. In contrast, only 3,361 nucleotide differences were found between IIoA15G1 and IIpA11, with several genes encoding invasion-associated mucin glycoproteins and cgd8_5420 encoding a secretory protein being highly polymorphic. The latter is mainly expressed in trophozoites, merozoites, and macrogametes. Deletion of this gene reduced the intensity of IIpA11 infection and increased the survival of infected mice. Therefore, the emerging IIoA15G1 and IIpA11 subtypes have divergent genomes compared to common IIa subtypes 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.