A conserved apicomplexan microneme protein contributes to Toxoplasma gondii invasion and virulence

Abstract

The obligate intracellular parasite Toxoplasma gondii critically relies on host cell invasion during infection. Proteins secreted from the apical micronemes are central components for host cell recognition, invasion, egress, and virulence. Although previous work established that the sporozoite protein with an altered thrombospondin repeat (SPATR) is a micronemal protein conserved in other apicomplexan parasites, including Plasmodium, Neospora, and Eimeria, no genetic evidence of its contribution to invasion has been reported. SPATR contains a predicted epidermal growth factor domain and two thrombospondin type 1 repeats, implying a role in host cell recognition. In this study, we assess the contribution of T. gondii SPATR (TgSPATR) to T. gondii invasion by genetically ablating it and restoring its expression by genetic complementation. Δspatr parasites were ~50% reduced in invasion compared to parental strains, a defect that was reversed in the complemented strain. In mouse virulence assays, Δspatr parasites were significantly attenuated, with ~20% of mice surviving infection. Given the conservation of this protein among the Apicomplexa, we assessed whether the Plasmodium falciparum SPATR ortholog (PfSPATR) could complement the absence of the TgSPATR. Although PfSPATR showed correct micronemal localization, it did not reverse the invasion deficiency of Δspatr parasites, because of an apparent failure in secretion. Overall, the results suggest that TgSPATR contributes to invasion and virulence, findings that have implications for the many genera and life stages of apicomplexans that express SPATR.

FIG 1

Structural features of apicomplexan SPATR orthologs. (A) Schematic illustrations of SPATR from T. gondii, N. caninum, E. tenella, and P. falciparum showing domain structures and approximate relative sizes. Illustrations are based on representatives from the multiple sequence analysis shown in Fig. S1 in the supplemental material. (B) Schematic representation of TgSPATR along with multiple sequence alignments of the conserved TmEGF and TSR domains. Residues conserved among all four sequences are colored red, whereas those conserved among two or three sequences are blue. Conserved cysteine residues are denoted with an asterisk. Gaps are indicated by a hyphen. An arrow indicates the beginning of the mature TgSPATR sequence. The boxed region highlights a conserved motif that is a hallmark of the thrombomodulin EGF subfamily. Secondary structure elements predicted by structural modeling (see Fig. S2) are indicated above the TSR multiple sequence alignments. (C) Pfam hidden Markov model representative of the thrombomodulin EGF signature sequence, including the conserved motif corresponding to the boxed region in panel B. (D) Pulse-chase analysis of mouse anti-SPATR immunoprecipitations show a single band for TgSPATR through various chase times of 15, 30, 45, and 60 min. Parallel analysis of TgM2AP processing was included as a positive control. (E) Alignment of the coccidian SPATR sequences adjacent to the established cleavage site for TgSPATR (arrow). Conserved P1 and P3 residues are indicated.

FIG 2

Genetic disruption and complementation of TgSPATR. (A) The YFP-tagged TgSPATR gene was knocked out by homologous recombination with the CAT selectable marker. The HXG selectable marker in the Ku80 locus was removed by negative selection with 6-thioxanthine, followed by transfection with a construct containing a myc-tagged TgSPATR gene and the HXG marker. (B) Indirect immunofluorescence of intracellular (first four rows) and extracellular (EC; fifth row) parasites with mouse anti-TgSPATR and rabbit anti-TgMIC5 antibodies. (C) Parasite lysate (pellet) and ESA fractions from the indicated strains. ESA fractions were collected from culture supernatants after parasites were treated for 2 min at 37°C with 1% ethanol to stimulate microneme secretion. Treated parasites were pelleted to create the parasite lysates. GRA1 was immunoblotted as a loading control for each strain.

FIG 3

TgSPATR knockout parasites are defective in host cell invasion. (A) Red-green invasion assays of parasites after 20 min of incubation with HFF host cells. Parasites were stained as described in Materials and Methods. *, P < 0.05. (B) Red-green invasion assays of parasites after 0.5, 1, 2, 4, and 8 h of incubation with HFF host cells. *, P < 0.05. (C) Attachment of parasites onto glutaraldehyde-fixed host cells. HFF host cells were fixed with glutaraldehyde, blocked with ethanolamine, and exposed to parasites for 20 min before parasites were stained with anti-SAG1. BAPTA-AM-treated Δku80::HXG parasites are a negative control for attachment. ns, not significant. (D) Attachment of cytochalasin D-treated parasites to HFF host cells. Parasites were treated with cytochalasin D and allowed to attach to HFF host cells for 15 min in the continued presence of cytochalasin D before being stained with anti-SAG1. ns, not significant. (E) Enumeration of nonmotile parasites or parasites performing twirling, helical, or circular gliding by video microscopy. ns, not significant. Data in all graphs are means + standard errors of the means (SEM) from three independent experiments, each with triplicate samples, with the exception of data in panel E, which are means − standard deviations (SD) from 18 individual videos of each parasite strain in 3 independent experiments. (F) Induced egress. Parasites grown for 28 h in chamber slides were induced with A23187, and occupied versus unoccupied vacuoles were enumerated.

FIG 4

Δspatr parasites are attenuated in in vivo virulence. Mice were infected intraperitoneally with 10 tachyzoites (or the indicated number for Δspatr parasites) in 2 independent experiments, which were combined for the data. An asterisk denotes a statistically significant time until moribundity of the Δspatr strain compared to the Δku80::HXG and ΔspatrComp strains as determined using the Kaplan-Meier estimator (P < 0.05). Twenty percent of Δspatr strain-infected mice survived to the end point.

FIG 5

TgSPATR domain deletion mutants are mislocalized. (A) Schematic illustrations of individual domain deletions of EGF, TSR, TSR1, and TSR2. (B) Indirect immunofluorescence localization of intracellular parasites using mouse anti-myc and rabbit anti-M2AP. (C) Induced (1% ethanol for 2 min) and constitutive (20 min) ESA fractions of all domain deletions. BAPTA-AM treatment for 10 min blocked the majority of secretion of a control MIC protein, TgMIC2, in both induced and constitutive secretion but not of the domain deletion mutants. Blots probed with mouse anti-myc or mouse anti-MIC2. (D) Red-green invasion assays of parasites after 20 min of incubation with HFF host cells. Parasites were stained as described in Materials and Methods. Data are means + SEM from two independent experiments, each with triplicate samples. The ΔEGF strain was not included in the secretion or invasion assays because it showed protein arrest in the ER.

FIG 6

Secretion of PfSPATR is not detectable, and PfSPATR cannot functionally complement TgSPATR. (A) Myc-tagged PfSPATR is trafficked to the micronemes based on colocalization with TgM2AP. (B) Expression of PfSPATR in the Δspatr strain is detected in pellets but not ESA in immunoblot analysis with anti-myc antibody. ΔspatrComp lysate and ESA are included as controls. The top blot was stripped and reprobed with anti-MIC2 to control for the presence of ESA products in the PfSPATR lanes. B-AM, BAPTA-AM. (C) Red-green invasion assay of PfSPATRmyc relative to Δku80::HXG. Data are means + SEM from three independent experiments, each with triplicate samples.