Hammondia hammondi , an avirulent relative of Toxoplasma gondii , has functional orthologs of known T. gondii virulence genes

Abstract

Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals, including humans. Its closest extant relative, Hammondia hammondi, has never been found to infect humans and, in contrast to T. gondii, is highly attenuated in mice. To better understand the genetic bases for these phenotypic differences, we sequenced the genome of a H. hammondi isolate (HhCatGer041) and found the genomic synteny between H. hammondi and T. gondii to be >95%. We used this genome to determine the H. hammondi primary sequence of two major T. gondii mouse virulence genes, TgROP5 and TgROP18. When we expressed these genes in T. gondii, we found that H. hammondi orthologs of TgROP5 and TgROP18 were functional. Similar to T. gondii, the HhROP5 locus is expanded, and two distinct HhROP5 paralogs increased the virulence of a T. gondii TgROP5 knockout strain. We also identified a 107 base pair promoter region, absent only in type III TgROP18, which is necessary for TgROP18 expression. This result indicates that the ROP18 promoter was active in the most recent common ancestor of these two species and that it was subsequently inactivated in progenitors of the type III lineage. Overall, these data suggest that the virulence differences between these species are not solely due to the functionality of these key virulence factors. This study provides evidence that other mechanisms, such as differences in gene expression or the lack of currently uncharacterized virulence factors, may underlie the phenotypic differences between these species. comparative genomics | gene content versus gene deployment | pathogenesis | host range

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

“…Our recently published genome sequence of an isolate of H. hammondi (HhCatGer041 [28]; GenBank accession number AVCM00000000.1) revealed a high degree of genomic synteny and conservation with T. gondii, further confirming H. hammondi as the closest known extant relative of T. gondii (28). Despite this high sequence similarity, there are dramatic phenotypic differences between H. hammondi and T. gondii.…”

“…In the ROP18 gene there is a 107-bp sequence present in T. gondii strains that actively express ROP18 (such as strain types I and II [15,23]) as well as HhROP18 (28) but absent in T. gondii strains that do not express ROP18 (such as members of the type III lineage [15,23]). The HhROP18 promoter is fully active in luciferase reporter assays and drives HhROP18 protein expression in T. gondii, and this dramatically alters virulence; this 107-bp sequence is both necessary and sufficient for ROP18 promoter-driven reporter expression (28). In the present study, we found that the 16 bp deleted in the HhROP16 promoter play an important role in the ability of the ROP16 upstream sequence to drive reporter gene expression, suggesting that this is an important core promoter sequence for ROP16.…”

“…The also could be due to distinct transcriptional profiles. Building off previous work showing that H. hammondi orthologs of the T. gondii virulence effectors ROP18 and ROP5 are functionally conserved when expressed in T. gondii (28), in the present study we examined the efficacy of the syntenic H. hammondi orthologs of GRA15 (27) and ROP16 (25). Our assay for functional conservation was 2-fold: (i) was the H. hammondi ortholog effectively expressed in T. gondii, and (ii) could the H. hammondi ortholog complement a known cell signaling phenotype?…”

“…The CACC sequence was used for directional cloning into the pENTR/D-TOPO vector (Invitrogen), and the underlined sequence in the reverse primer encodes a hemagglutinin (HA) tag and contains a stop codon. The sequence was cloned using LR Clonase recombination with the p-ATT-GRA2 destination vector (28). The p-ATT-GRA2 vector with the GRA15 insert was transfected into RH⌬HXGPRT, and resistant parasites harboring the vector, which contains an HXGPRT minigene, were selected in mycophenolic acid and xanthine as described previously (34).…”

“…We published a genomic sequence of a strain of H. hammondi isolated from a cat in Germany (HhCatGer041) and used this sequence to determine that the H. hammondi orthologs of the major T. gondii virulence factors TgROP5 and TgROP18 are functionally conserved in H. hammondi (28). Specifically, H. hammondi ROP18 and ROP5 could complement mouse virulence defects in select T. gondii strains (28). This indicates that the mere presence of virulence factors like ROP5 or ROP18 in the H. hammondi genome is not sufficient to explain the dramatic differences in mouse virulence between these two species.…”