Individuals with inhibited immunity may develop lethal toxoplasmosis; thus, a safe and effective vaccine is urged to be developed. Toxoplasma gondii (T. gondii) a-amylase (a-AMY) is one of the enzymes responsible for starch digestion. In the present study, we first generated a ME49Da-amy mutant and discovered that loss of a-AMY robustly grew in vitro but contributed to significant virulence attenuation in vivo. Therefore, we established a mouse model to explore the protective immunity of Da-amy mutant against acute and chronic toxoplasmosis. The results indicated that the survival rates of short-term or long-term immunized mice re-infected with the tachyzoites of multiple T. gondii strains were nearly 100%. ME49Da-amy not only could provide protective immunity against tachyzoites infection but also could resist the infection of tissue cysts. Furthermore, we detected that ME49Da-amy vaccination could effectively eliminate the proliferation of parasites in mice and prevent the formation of cysts. The significant increases of Th1-type cytokines, Th2-type cytokines and specific total IgG and IgG subclasses (IgG2a and IgG1) confirmed efficiency of a combination of cellular and humoral immunity against infection. In conclusion, ME49Da-amy attenuated strain can produce strong immune responses to provide efficient protection against toxoplasmosis, which signifies that ME49Da-amy mutant may be a potential vaccine candidate.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that can infect almost all warm-blooded animals, causing serious public health problems. Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM), which is first identified on histones and has been proved relevant to procreation regulation, transcription activation, and cell signaling pathway. However, the biological functions of histone crotonylation have not yet been reported in macrophages infected with T. gondii. As a result, a total of 1,286 Kcr sites distributed in 414 proteins were identified and quantified, demonstrating the existence of crotonylation in porcine alveolar macrophages. According to our results, identified histones were overall downregulated. HDAC2, a histone decrotonylase, was found to be significantly increased, which might be the executor of histone Kcr after parasite infection. In addition, T. gondii infection inhibited the crotonylation of H2B on K12, contributing on the suppression of epigenetic regulation and NF-κB activation. Nevertheless, the reduction of histone crotonylation induced by parasite infection could promote macrophage proliferation via activating PI3K/Akt signaling pathway. The present findings point to a comprehensive understanding of the biological functions of histone crotonylation in porcine alveolar macrophages, thereby providing a certain research basis for the mechanism research on the immune response of host cells against T. gondii infection.

T. gondii has an efficient and flexible energy metabolism system to meet different growth needs. ATP is an energy-carrying molecule and needs to be exchanged between organelles with the assistance of transporters.

The lumen of the endoplasmic reticulum (ER) is the subcellular site where secretory protein folding, glycosylation and sulfation of membrane-bound proteins, proteoglycans, and lipids occur. The protein folding and degradation in the lumen of the ER require high levels of energy in the form of ATP. Biochemical and genetic approaches show that ATP must first be translocated across ER membrane by particular transporters before serving as substrates and energy sources in the lumenal reactions. Here we describe an ATP/ADP transporter residing in the ER membranes of T.gondii. Immunofluorescence (IFA) assay in transgenic TgANT1-HA tag revealed that TgANT1 is a protein specifically expressed in the ER. In vitro assays, functional integration of TgANT in the cytoplasmic membrane of intact E. coli cells reveals high specificity for an ATP/ADP antiport. The depletion of TgANT leads to fatal growth defects in T.gondii, including a significant slowdown in replication, no visible plaque formation, and reduced ability to invade. We also found that the amino acid mutations in two domains of TgANT lead to the complete loss of its function. Since these two domains are conserved in multiple species, they may share the same transport mechanism. Our results indicate that TgANT is the only ATP/ADP transporter in the ER of T. gondii, and the lack of ATP in the ER is the cause of the death of T. gondii.

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