Hammondia hammondi Harbors Functional Orthologs of the Host-Modulating Effectors GRA15 and ROP16 but Is Distinguished from Toxoplasma gondii by a Unique Transcriptional Profile

Abstract

Toxoplasma gondii and its nearest extant relative, Hammondia hammondi, are phenotypically distinct despite their remarkable similarity in gene content, synteny, and functionality. To begin to identify genetic differences that might drive distinct infection phenotypes of T. gondii and H. hammondi, in the present study we (i) determined whether two known host-interacting proteins, dense granule protein 15 (GRA15) and rhoptry protein 16 (ROP16), were functionally conserved in H. hammondi and (ii) performed the first comparative transcriptional analysis of H. hammondi and T. gondii sporulated oocysts. We found that GRA15 and ROP16 from H. hammondi (HhGRA15 and HhROP16) modulate the host NF-B and STAT6 pathways, respectively, when expressed heterologously in T. gondii. We also found the transcriptomes of H. hammondi and T. gondii to be highly distinct. Consistent with the spontaneous conversion of H. hammondi tachyzoites into bradyzoites both in vitro and in vivo, H. hammondi high-abundance transcripts are enriched for genes that are of greater abundance in T. gondii bradyzoites. We also identified genes that are of high transcript abundance in H. hammondi but are poorly expressed in multiple T. gondii life stages, suggesting that these genes are uniquely expressed in H. hammondi. Taken together, these data confirm the functional conservation of known T. gondii virulence effectors in H. hammondi and point to transcriptional differences as a potential source of the phenotypic differences between these species.

Secreted effectors inToxoplasma gondiiand related species: determinants of host range and pathogenesis?

“…Clearly then, the presence of a given virulence effector in the H. hammondi genome is not sufficient to explain its highly avirulent phenotype in mice . We have also shown that H. hammondi ROP18, ROP5, ROP16 and GRA15 are functional effectors when expressed in relevant T. gondii genetic backgrounds . Specifically, H. hammondi has a functional ROP18 ortholog that is highly effective at increasing the virulence of essentially ROP18‐null T. gondii strains (such as the type III strain CTG; ).…”

“…Importantly, the STAT6 induction was significantly higher than type II T. gondii expressing an additional copy of a type II ROP16 allele, providing strong evidence that the ‘active’ allele of ROP16 with respect to STAT6 activation is ancestral to the T. gondii / H. hammondi split. Interestingly, however, the H. hammondi ROP16 gene promoter had a 16 bp deletion compared to T. gondii ROP16, and this 16 bp was essential for ROP16 promoter function . It is therefore possible that in H. hammondi ROP16 is fully functional with respect to STAT6 activation, but is poorly expressed.…”

“…Hammondia hammondi ROP16 and GRA15 are also functional effectors. Specifically, expressing H. hammondi ROP16 in type II T. gondii significantly increased STAT6 phosphorylation and translocation to the nucleus (152), which is similar to the type I and III T. gondii ROP16 alleles (4,(139)(140)(141)153). Importantly, the STAT6 induction was significantly higher than type II T. gondii expressing an additional copy of a type II ROP16 allele, providing strong evidence that the 'active' allele of ROP16 with respect to STAT6 activation is ancestral to the T. gondii/H.…”

“…shown that H. hammondi ROP18, ROP5, ROP16 and GRA15 are functional effectors when expressed in relevant T. gondii genetic backgrounds (10,152). Specifically, H. hammondi has a functional ROP18 ortholog that is highly effective at increasing the virulence of essentially ROP18-null T. gondii strains (such as the type III strain CTG; (10)).…”

Importance of serological cross-reactivity amongToxoplasma gondii, Hammondiaspp.,Neosporaspp.,Sarcocystisspp. andBesnoitia besnoiti

“…Of course, one important prerequisite is that peptides applied in such tests are not specific for particular clonal-types or genotypes of T. gondii or related parasite species. In addition, potential cross-reactivities need to be addressed; for instance, synthetic peptides considered to react specifically with antibodies generated by T. gondii infections need to be tested with sera from animals exposed to H. hammondi , as this parasite has a very close genetic relationship to the former (Walzer et al 2013, 2014). The use of T. gondii -specific peptides for diagnosis in animals should also be tested with N. caninum , which may cross-react, as shown in Table 3.…”