Proteomic profile of Toxoplasma gondii stress granules by high-resolution mass spectrometry.

Ribonucleoprotein granules are bio-condensates that form a diverse group of dynamic membrane-less organelles implicated in several cellular functions, including stress response and cellular survival. In Toxoplasma gondii, a type of bio-condensates referred to as stress granules (SGs) are formed prior to the parasites' egress from the host cell and are implicated in the survival and invasion competency of extracellular tachyzoites. We used paraformaldehyde to fix and cross-link SG proteins to allow purification by centrifugation and analysis by mass spectrometry. We profiled protein components of SGs at 10 and 30 min post-egress when parasite's invasion ability is significantly diminished. Thirty-three proteins were identified from 10 min SGs, and additional 43 proteins were identified from 30 min SGs. Notably, common SG components such as proteins with intrinsically disordered domains were not identified. Gene ontology analysis of both 10 and 30 min SGs shows that overall molecular functions of SGs' proteins are ATP-binding, GTP-binding, and GTPase activity. Discernable differences between 10 and 30 min SGs are in the proportions of translation and microtubule-related proteins. Ten-minute SGs have a higher proportion of microtubule-related proteins and a lower proportion of ribosome-related proteins, while a reverse correlation was identified for those of 30 min. It remains to be investigated whether this reverse correlation contributes to the ability of extracellular tachyzoites to reinvade host cells.

Formation of mRNP granules in Toxoplasma gondii during the lytic cycle.

Two poly(A) binding proteins (PABPs) of Toxoplasma gondii, were identified and characterized. They were named TgPABPC and TgPABPN as they were found to localize in the cytoplasm and nucleus respectively. TgPABPC, which colocalizes with mRNA granules, is therefore used as a cellular marker of mRNP granules. We detected that the formation of mRNP granules was independent of polymerized microtubules, and that the granules were distributed stochastically within the cytosol. Formation of mRNP granules was found to occur prior to parasite egress when a Ca2+ ionophore is used to induce egress. It was also found that maturation of mRNP granules could be described as a two-phase process. First, prior to host cell lysis, mRNP granules were formed rapidly within the cytosol. Second, the mRNP granule load was reduced within 10 min post egress. To investigate the link between translational state and mRNP granule formation, treatments with salubrinal, nutrient deprivation, and pH stress were used. While salubrinal induced granule formation in tachyzoites, nutrient starvation and pH stress showed no induction effect on mRNP granule formation. Interestingly, salubrinal treatment in bradyzoites did not induce RNP granule formation, thus suggesting that mRNP granule formation is not a ubiquitous response or directly related to translational repression. Instead, mRNP granule formation is likely a response to the rapid increase in non-translating RNA brought on by sudden changes in translational state.

Toxoplasma ubiquitin-like protease 1, a key enzyme in sumoylation and desumoylation pathways, is under the control of non-coding RNAs.

Sumoylation and desumoylation are reversible pathways responsible for modification of protein structures and functions by the reversible covalent attachment of a small ubiquitin-like modifier (SUMO) peptide. These pathways are important for a wide range of cellular processes and require a steady supply of SUMO, which is generated by an enzymatic reaction catalysed by the ubiquitin-like protease (Ulp) family. Here we show by functional complementation analysis that the Ulp1 of Toxoplasma gondii (TgUlp1) can rescue a growth-deficient phenotype of a yeast-Ulp1 knockout. Recombinant TgUlp1 is an active enzyme capable of removing SUMO from a sumoylated substrate. Using a clonal transgenic strain of T. gondii expressing an epitope-tagged version of TgUlp1, we detected that the expression of TgUlp1 is modulated by Tg-miR-60, the most abundant species of micro RNA found in the T. gondii type 1 strain. The introduction of Tg-miR-60 inhibitor caused an increase in TgUlp1 expression and its enzymatic activity, as well as affecting the parasite's growth fitness. Moreover, we discovered a polyadenylated antisense RNA transcribed from the TgUlp1 locus, referred to as TgUlp1-NAT1 (TgUlp1-natural antisense transcript 1). Both Tg-miR-60 and TgUlp1-NAT1 confer a regulatory function by down-regulating the expression of TgUlp1 and affecting the sumoylation and desumoylation pathways in T. gondii.

Toxoplasma gondii infection induces the formation of host's nuclear granules containing poly(A)-binding proteins.

To study the mechanism by which human host cells respond to an infection of Toxoplasma gondii, we monitored the level of poly(A)-binding protein (PABP), an indicator of translation. Here, we report an observation of the relocalization of PABPs in human host cells upon T. gondii infection. Notably, the aggregates of PABPs formed upon infection are mainly found in the nucleus, which is a different response from that found after exposure to heat shock. Pyrimethamine treatment of the infected monolayers inhibits the multiplicity of the parasite and reverses the relocalization of PABP aggregates. This active interaction between the infected mammalian host cells and T. gondii appears to be different from that caused by viral infection.

Antisense technologies in the studying of Toxoplasma gondii.

This review covers a brief history of antisense RNAs and its applications, and summarizes the current stage of antisense technologies used in Toxoplasma gondii, a fascinating model organism with a unique characteristic blend of genetic regulatory systems normally found in plants or animals. Based on the current knowledge of regulatory RNAs and non-coding RNA (ncRNA), the antisense technologies are reviewed according to the classification of ncRNAs, which are roughly categorized into small, ranging from ~20-200 nucleotides in length, and long >200 nucleotides. Techniques utilizing small regulatory RNAs such as siRNA, miRNA, antagomirs, ribozymes and morpholino oligomers are discussed along with long non-coding RNA (lncRNA) including antisense and double stranded. These antisense technologies can be used in forward and reverse genetics studies. The future of technologies is limitless, particularly by combining these technologies with conventional methods, and should allow for ever greater understanding of gene regulation of the organism and related pathogenic microorganisms.