Toxoplasma gondii bradyzoites form spontaneously during sporozoite-initiated development

Abstract

Tachyzoites (VEG strain) that emerge from host cells infected with Toxoplasma gondii sporozoites proliferate relatively fast and double their number every 6 h. This rate of growth is intrinsic, as neither the number of host cells invaded nor host cell type appears to influence emergent tachyzoite replication. Fast tachyzoite growth was not persistent, and following approximately 20 divisions, the population uniformly shifted to slower growth. Parasites 10 days post-sporozoite infection doubled only once every 15 h and, unlike emergent tachyzoites, they grew at this slower rate over several months of continuous cell culture. The spontaneous change in tachyzoite growth rate preceded the expression of the bradyzoite-specific marker, BAG1. Within 24 h of the growth shift, 2% of the population expressed BAG1, and by 15 days post-sporozoite infection, 50% of the parasites were positive for this marker. Spontaneous BAG1 expression was not observed in sporozoites or in tachyzoites during fast growth (through day 6 post-sporozoite inoculation), although these tachyzoites could be induced to express BAG1 earlier by culturing sporozoite-infected cells at pH 8.3. However, alkaline treatment also reduced the replication of emergent tachyzoites to the rate of growth-shifted parasites, supporting a link between reduced parasite growth and bradyzoite differentiation. The shift to slower growth was closely correlated with virulence in mice, as the initially fast-growing emergent tachyzoites were avirulent (100% lethal dose, >10(4) parasites), while a mutant VEG strain (MS-J) that is unable to growth shift caused 100% mortality in mice inoculated with 10 parasites. Parasites recovered from gamma interferon knockout mice inoculated with emergent tachyzoites grew at a slow rate and expressed BAG1, confirming that the replication switch occurs in animals and in the absence of a protective immune response.

FIG. 1

(A) Growth of VEG tachyzoite populations obtained following sporozoite inoculation. The day 10 population was purified after a single passage (at 5 days post-sporozoite inoculation) into a second HFF monolayer. The day 2 (closed bars) and day 10 (hatched bars) VEG parasites were allowed to invade confluent HFF monolayers for 1 h before extracellular parasites were removed. Vacuole sizes (number of parasites per vacuole) were determined every 12 h over 48 h. All growth data represent the average of at least 50 randomly selected vacuoles and are plotted on a log₁₀ scale. (B) Rapid growth of emergent tachyzoites is stable for 5 days and is not limited by the number of host cell invasions. Beginning at 48 h post-sporozoite inoculation, purified tachyzoites were inoculated into fresh HFF monolayers (1 h infection), and the average vacuole size was determined 24 h later. This process was repeated every 24 h through five host cell passages (see Materials and Methods for complete details). The vacuole size in host cell passage no. 1 represents tachyzoite growth within the sporozoite-infected host cell (24 to 48 h post-sporozoite inoculation).

FIG. 2

BAG1 expression is detected following a reduction in tachyzoite growth rate. VEG tachyzoites purified from days 4, 6, and 8 (arrows) post-sporozoite inoculation cultures were passed into fresh HFF monolayers, and 24 h later the average vacuole size and number of BAG1-positive parasites were evaluated (see Materials and Methods). Hatched bars, average vacuole size; closed squares, percentage of BAG1-positive parasites.

FIG. 3

Tachyzoites formed within sporozoite-infected cells can be induced to express BAG1. Sporozoite-infected cultures were switched to alkaline medium 12 h postinoculation. The average vacuole size (pH 8.3, closed circles) and number of BAG1-positive parasites (hatched bars) were then determined over 96 h and compared to those obtained with control cultures (pH 7 growth, closed squares). BAG1 expression was not detected in control parasites, which had completely lysed out of their host cells by 96 h post-sporozoite infection.

FIG. 4

Tachyzoites obtained from VEG sporozoite-infected cultures are avirulent in CD-1 mice. The virulence of day 3 and day 8 (post-sporozoite infection) VEG tachyzoite populations was compared to that for RH (closed squares) and MS-J (closed triangles) tachyzoite lines. CD-1 mice (groups of 5) were inoculated s.q., and mortality was monitored over 30 days (only 22 days are shown). The results obtained from a 10³ parasite dose are displayed. CD-1 mice infected with emergent VEG tachyzoites were asymptomatic, although all showed positive serology to T. gondii, indicating that they were infected.

FIG. 5

RFLP analysis of MS-J parasites. Restriction enzyme patterns of the SAG1 (DdeI digest shown, lanes 1 to 4) and SAG2 (Sau3A shown, lanes 4 to 8) loci from RH, ME49-P(lk), VEG, and the VEG mutant MS-J are shown. Genomic DNAs were PCR amplified, the products were restricted, and the fragments were resolved on 1.2% agarose gels. Lanes: 1 and 5, RH; 2 and 6, ME49-P(lk); 3 and 7, VEG; 4 and 8, MS-J. Molecular size standards are from HaeIII-digested Φx174 DNA.

FIG. 6

Tachyzoite growth differences are consistent with the time to death for infected gko mice. RH (open circles), MS-J (closed squares), and day 3 (closed triangles) and day 8 (open squares) VEG tachyzoites were inoculated (10³ parasites) s.q. into gko mice (groups of five), and mortality was monitored. Mock-infected controls (closed circles).

FIG. 7

Fast-growing, emergent tachyzoites display a slower growth rate after passage in gko mice. Day 3 and day 8 post-sporozoite infection and MS-J (VEG) tachyzoites (10³ parasites) were inoculated s.q. into gko mice. Infected brain tissue (10 days postinfection) was inoculated intraperitoneally into new gko mice, and after a period of amplification (7 days), parasites were recovered from the peritoneal exudate. Upon first passage into fresh HFF cells, vacuole sizes were determined 36 h postinoculation (see Materials and Methods for complete details). Closed bars (B), 36 h vacuole size prior to passage through mice; hatched bars (A), vacuole size post-gko passage.