Proline dehydrogenase, a rate-limiting catabolic enzyme, affecting the growth and pathogenicity of Toxoplasma gondii tachyzoites by regulating the proline metabolism and mitochondrial function of the parasite

Abstract

Background: The pathogenicity of Toxoplasma gondii is closely associated with its intracellular lytic cycle in host cells. Currently, the mechanisms by which T. gondii completes the lytic cycle remain unclear. The proline metabolism has been reported to be crucial for intracellular growth of pathogens by providing energy and nutrients. However, it remains unclear whether the intracellular growth and pathogenicity of T. gondii are related to proline metabolism.

Methods: The gene-edited strains of proline dehydrogenase (Tgprodh) were constructed by using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. The effects of the Tgprodh gene on the growth in vitro and pathogenicity in vivo of the tachyzoites for T. gondii were studied through proliferation, plaque, invasion, egress and virulence assays. The effects of the Tgprodh gene on mitochondrial function were studied by using reactive oxygen species (ROS), mitochondrial membrane potential (∆Ψm), adenosine triphosphate (ATP) assay kits, mitochondrial DNA (mtDNA) copy numbers, transmission electron microscopy (TEM) analysis, and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The effects of the Tgprodh gene on proline metabolism were studied by using L-proline (L-Pro), L-glutamic acid (L-Glu), L-glutamine (L-Gln) assay kits, and RT-qPCR.

Results: TgPRODH, the first rate-limiting enzyme in proline metabolism, was identified to be encoded by T. gondii and localized in the cytoplasm of T. gondii. Deletion of the Tgprodh gene resulted in significant growth inhibition in vitro and reduced pathogenicity in vivo of T. gondii. Further study found that deletion of the Tgprodh gene caused damage to the mitochondrial morphology, decreased membrane potential, mtDNA copy numbers, and the production of ATP and ROS. The expression of genes for maintaining mitochondrial integrity was downregulated in the Tgprodh-knockout strain of T. gondii, while complementation of the Tgprodh gene restored these defects in this parasite. Meantime, the deletion of the Tgprodh gene resulted in the accumulation of proline, reduced the contents of glutamate and glutamine, and affected the expression of genes related to proline catabolism in T. gondii.

Conclusions: The present study found the significance of the Tgprodh gene for the intracellular growth and pathogenicity of T. gondii through regulating mitochondrial function and the proline metabolism and provided a novel insight to reveal intracellular survival strategies of T. gondii.

Keywords: Tgprodh; Toxoplasma gondii; Growth; Mitochondrial function; Proline metabolism.

Fig. 1

Sequence alignment, structure, and subcellular localization of TgPRODH were shown in T. gondii. a, Sequence alignment of TgPRODH, NcPRODH, TcPRODH, and TbPRODH. Three arginine (*) and two glutamic acid (#) sites were identified in TgPRODH. b, The proline oxidase active domains of TgPRODH predicted by using the SMART database (http://smart.embl-heidelberg.de/). Two proline oxidase active sites, located at 21 aa–381 aa and 388 aa–464 aa, were identified. c, The protein level of TgPRODH was detected by using western blot. The endogenous labeling strain (Tgprodh-HA) was used as the experimental group, while the RHku80 and RH wild strains were used as the control groups. The PVDF membrane was sectioned into two segments. One section was incubated with HA mouse monoclonal antibody, and the other was incubated with GAPDH rabbit monoclonal antibody. d, Location of TgPRODH in T. gondii by using immunofluorescence with mouse anti-HA (red), rabbit anti-TgCDPK1 (green), rabbit anti-NcPRODH (green), and MitoTraker (red). The Tgprodh-HA strain was used as the experimental group, and the RHku80 strain was used as the control groups. The scale bar is 20 μm

Fig. 2

Deletion of Tgprodh gene resulted in the growth inhibition of T. gondii in vitro and reduced pathogenicity in vivo. a, The intracellular proliferation differences of the tachyzoites. b, The growth abilities of the tachyzoites were evaluated by using plaque assays. c, The sizes of the plaques for the tachyzoites were analyzed by Image J software. d, Comparison of the virulence of the tachyzoites to mice. e, The average survival time of mice infected with the tachyzoites. f, The tissue loads of mice infected with the tachyzoites. The differences were analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Deletion of Tgprodh gene resulted in mitochondrial damage in T. gondii. a, Electron microscopy analysis of tachyzoites in T. gondii. The mitochondria of tachyzoites in the RH strain showed a typical lasso shape (blue arrow). b, The ROS levels of tachyzoites. c, The ATP content of the tachyzoites. d, The mitochondrial membrane potential of tachyzoites. e, The mitochondrial DNA copy number of tachyzoites. f, The mRNA levels of genes related to mitochondrial integrity in the tachyzoites. Three independent experiments were conducted, and the differences were analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Deletion of Tgprodh gene resulted in disorder of proline metabolism in T. gondii. a, The concentration of l-proline in the tachyzoites. b, The concentration of l-glutamic acid in the tachyzoites. c, The concentration of l-glutamine in the tachyzoites. d, e, The mRNA levels of enzymes involved into the proline catabolism in the tachyzoites. f, The mRNA levels of enzymes involved into the proline anabolism in the tachyzoites. Three independent experiments were conducted, and the differences were analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Fig. 5

A hypothesized mechanism diagram showed impacts of the Tgprodh gene on the mitochondria and proline metabolism in T. gondii. On the one hand, disruption of the Tgprodh gene caused damage to the mitochondrial morphology, decreased membrane potential and mtDNA copy numbers, reduced the production of ATP and ROS, and downregulated the gene expression for maintaining mitochondrial integrity of T. gondii. On the other hand, disruption of the Tgprodh gene downregulated the mRNA levels of proline catabolic enzymes (Tgp5cdh, Tggs, Tgssadh, Tgpd-he1β, and Tg2OG-Fe(II)_oxy), led to accumulation of proline, and decreased the contents of L-Glu and L-Gln in T. gondii, and then, L-Glu was unable to enter the TCA cycle. Conversely, to obtain nutrients and energy from proline metabolism to maintain the intracellular growth of tachyzoites, the Tgprodh-deficient strain may have utilized the L-Glu, L-Gln, and ornithine in the cytoplasm of T. gondii to synthesize l-proline by upregulating the mRNA levels of proline anabolism enzymes (Tgpycr1, Tgpycr2, Tgoat, Tgg5k, and Tgγ-gpr). The Tgprodh gene is important for intracellular growth of tachyzoites by regulating the proline metabolism and mitochondrial function of the parasite