Impact of Engineered Expression of Mitochondrial Association Factor 1b on Toxoplasma gondii Infection and the Host Response in a Mouse Model

Abstract

The opportunistic intracellular parasite Toxoplasma gondii causes a lifelong chronic infection capable of reactivating in immunocompromised individuals, which can lead to life-threatening complications. Following invasion of the host cell, host mitochondria associate with the parasitophorous vacuole membrane. This phenotype is T. gondii strain specific and is mediated by expression of a host mitochondrial association-competent (HMA⁺) paralog of the parasite protein mitochondrial association factor 1 (MAF1b). Previous work demonstrated that expression of MAF1b in strains that do not normally associate with host mitochondria increases their fitness during acute infection in vivo However, the impact of MAF1b expression during chronic T. gondii infection is unclear. In this study, we assess the impact of MAF1b expression on cyst formation and cytokine production in mice. Despite generally low numbers of cysts generated by the in vitro culture-adapted strains used in this study, we find that parasites expressing MAF1b have higher numbers of cysts in the brains of chronically infected mice and that MAF1b⁺ cyst burden significantly increases during the course of chronic infection. Consistent with this, mice infected with MAF1b⁺ parasites have higher levels of the serum cytokines RANTES and VEGF (vascular endothelial growth factor) at day 57 postinfection, although this could be due to higher parasite burden at this time point rather than direct manipulation of these cytokines by MAF1b. Overall these data indicate that MAF1b expression may also be important in determining infection outcome during the chronic phase, either by directly altering the cytokine/signaling environment or by increasing proliferation during the acute and/or chronic phase.IMPORTANCE The parasite Toxoplasma gondii currently infects approximately one-third of the world's population and causes life-threatening toxoplasmosis in individuals with undeveloped or weakened immune systems. Current treatments are unable to cure T. gondii infection, leaving infected individuals with slow-growing tissue cysts for the remainder of their lives. Previous work has shown that expression of the parasite protein mitochondrial association factor 1 (MAF1b) is responsible for the association of T. gondii parasites with host mitochondria and provides a selective advantage during acute infection. Here we examine the impact of MAF1b expression during chronic T. gondii infection. We find that mice infected with MAF1b-expressing parasites have higher cyst burden and cytokine levels than their wild-type counterparts. A better understanding of the genes involved in establishing and maintaining chronic infection will aid in discovering effective therapeutics for chronically infected individuals.

Keywords: Toxoplasma gondii; chronic infection; cytokines; host-pathogen interactions; mitochondrial association.

FIG 1

MAF1b expression does not significantly alter parasite burden or mouse survival during acute infection in CBA/J mice. Female CBA/J mice were infected with 10, 100, or 1,000 parasites of TgME49:EV or TgME49:MAF1b, and acute infection was monitored by bioluminescence imaging. (A, C, and E) Quantification of bioluminescence in mice infected with 10, 100, or 1,000 parasites. There was no significant difference in bioluminescence between TgME49:EV (EV)- and TgME49:MAF1b (TgMAF1RHb1)-infected mice at any time at any dose, with the exception of the time point 7 dpi for mice infected with 1,000 parasites, where mice infected with TgME49:EV had significantly higher bioluminescence than mice infected with TgME49:MAF1b (*, P = 0.0004). (B, D, and F) Survival curves of mice from panels A, C, and E. There were no significant differences in survival at any dose. (G) Images of infected mice quantified in E on 3, 6, 9, and 12 dpi.

FIG 2

MAF1b expression increases cyst burden in chronically infected CBA/J mice. Female CBA/J mice were infected with 1,000 parasites of TgME49:EV or TgME49:MAF1b. Parasite viability was confirmed by plaque assay (experiment 1, TgME49:EV, 382/1,000, and TgME49:MAF1b, 404/1,000; experiment 2, TgME49:EV, 271/1,000, and TgME49:MAF1b, 364/1,000). Half of each group was sacrificed at 28 dpi, and the remainder were sacrificed 56 (experiment 1) or 60 (experiment 2) dpi. Brains were removed from sacrificed mice and divided sagitally. The left half of each brain was homogenized, and cysts were isolated by Percoll gradient before being stained with rhodamine-conjugated Dolichos biflorus agglutinin. Cysts were then quantified using an inverted fluorescence microscope. Mice infected with TgME49:MAF1b had a higher cyst burden than mice infected with TgME49:EV. This difference was significant 56 or 60 dpi (*, P = 0.007; ***, P = 0.0005). There were also significantly more cysts 60 dpi compared to 28 dpi in TgME49:MAF1b-infected mice in experiment 2 (**, P = 0.0012).

FIG 3

Seven cytokines are elevated during acute infection and return to near preinfection levels during chronic infection. Female C57BL/6J mice were infected with 1,000 parasites of TgME49:EV or TgME49:MAF1b. Blood samples were taken prior to infection, as well as 7, 21, 28, and 57 days postinfection and processed to obtain serum samples. Serum samples were then analyzed by Luminex for the presence of 32 mouse cytokines. For each cytokine, serum levels are given as fluorescent intensity (FI) minus background. Each point represents a single mouse: blue circles indicate mice infected with TgME49:EV, and red squares indicate mice infected with TgMe49:MAF1b. (A) FI minus background for 0, 7, 21, 28, and 57 dpi of seven cytokines elevated during acute infection, but returning to near prebleed levels during chronic infection. Time points where expression was significantly different between TgME49:MAF1b- and TgME49:EV-infected mice are indicated by asterisks. (B) A closer look at 21, 28, and 57 dpi for three of the seven cytokines that were still expressed at low levels during chronic infection. The gray dotted line indicates the average for 0 dpi.

FIG 4

Cytokines expressed during acute infection are expressed at a low level during early chronic infection and then increase in expression throughout the remainder of chronic infection. For each cytokine, serum levels are given as FI minus background. Each point represents a single mouse: blue circles indicate mice infected with TgME49:EV, and red squares indicated mice infected with TgME49:MAF1b. A time point where expression was significantly different between TgME49:EV- and TgME49:MAF1b-infected mice is indicated by an asterisk.

FIG 5

Cytokines expressed during chronic infection. For each cytokine, serum levels are given as FI minus background. Each point represents a single mouse: blue circles indicate mice infected with TgME49:EV, and red squares indicated mice infected with TgME49:MAF1b. Time points where expression was significantly different between TgME49:EV- and TgME49:MAF1b-infected mice are indicated by asterisks.

FIG 6

Cytokines with unique expression patterns. For each cytokine, serum levels are given as FI minus background. Each point represents a single mouse: blue circles indicate mice infected with TgME49:EV, and red squares indicated mice infected with TgME49:MAF1b.