Reassessment of the Role of Aromatic Amino Acid Hydroxylases and the Effect of Infection by Toxoplasma gondii on Host Dopamine

Abstract

Toxoplasma gondii infection has been described previously to cause infected mice to lose their fear of cat urine. This behavioral manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylase AAH2 gene, with no observable phenotype in parasite growth or differentiation in vitro and in vivo. Additionally, expression levels of the two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm previously described effects of parasite infection on host dopamine either in vitro or in vivo, even when AAH2 was overexpressed using the BAG1 promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or regional alterations of dopamine in the host brain, although they may affect this pathway locally. Additionally, our findings suggest alternative roles for the AHH enzymes in T. gondii, since AAH1 is essential for growth in nondopaminergic cells. The protozoan parasite Toxoplasma gondii is an obligate intracellular parasite that is capable of infecting most warmblooded animals. It is a member of the phylum Apicomplexa, which also contains Plasmodium falciparum, the causative agent of malaria. Toxoplasma gondii is one of the most widely distributed parasites in the world, both in geographic location and in the diversity of its host range (1). Its only definitive hosts are members of the genus Felis. Exclusively within enterocytes of the gut, it undergoes a sexual reproductive cycle to form environmentally resistant and infectious oocysts that are shed in cat feces (2). In all other hosts, Toxoplasma gondii infection begins as a fast-growing lytic stage called the tachyzoite. Innate and adaptive immune responses restrict the growth of tachyzoites, which can respond by differentiating into bradyzoites, a semidormant stage that exists as quiescent intracellular cysts in brain and muscle tissue. This chronic infection can persist for the lifetime of the host (3). Infections spread among incidental hosts through carnivorism, vertical transmission, and ingestion of T. gondii oocysts (4).Rodents that become infected by T. gondii exhibit an unusual behavioral response: they lose their instinctive aversion to the odor of cats and instead become mildly attracted to the scent (5-13). This behavioral manipulation appears specific to the cat (7, 8), and it has been speculated that this facilitates transmission (9). The exact mechanism of this behavioral manipulation is unknown, but parasite stimulation of host dopamine pathways in the brain has been suggested as a cause (14-16). It was observed that infection of mice by T. gondii caused a 14% increase in wholebrain dopamine levels upon establishment of chronic infection (17). Additionally, dopamine receptor antagonist drugs used for the treatment of schizophrenia block cat attraction in infected rodents (18,19). T. gondii ...

Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

“…Mice were monitored for survival daily, and surviving animals were tested serologically by enzyme-linked immunosorbent assay (ELISA) to establish that they were infected, as described previously (34). Cyst counting was performed as described previously (39). Briefly, at 30 days postinoculation, brains were harvested, and lysed by passing through 16-gauge needles in PBS.…”

Reproducing Increased Dopamine with Infection To Evaluate the Role of Parasite-Encoded Tyrosine Hydroxylase Activity

“…n a recent publication, Wang et al (1) commented on their inability to reproduce findings from a prior publication. In that study, a sophisticated gene knockout was performed, disrupting one of the two copies of the Toxoplasma gondii AAH gene to study its biological roles.…”

Reply to “Reproducing Increased Dopamine with Infection To Evaluate the Role of Parasite-Encoded Tyrosine Hydroxylase Activity”

“…However, Pru⌬ku80⌬hxg was not the only strain used in our study (3). We observed the same absence of dopamine change in vitro when using ME49 (unpublished data).…”

“…If this prediction is correct, it stands to reason that the result should still hold even if dopamine production is globally lower. The fact that we still did not see a difference in overall dopamine content despite demonstrable evidence of differentiated bradyzoite-containing vacuoles inside PC12 cells challenges this hypothesis (3).…”