m6A RNA methylation facilitates pre-mRNA 3'-end formation and is essential for viability of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3'-boundary of mRNAs. Knockdown of the m6A writer components METTL3 and WTAP resulted in diminished m6A marks and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3'-end processing of pre-mRNAs. Loss of METTL3, WTAP, or YTH1 led to a defect in transcript 3'-end formation. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3'-ends.

Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii.

Translational control mechanisms modulate microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5'-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5'-cap is impaired by insertion of a 5'-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we show that a trans -acting RNA-binding protein called BFD2/ROCY1 is necessary for cap-independent translation of BFD1 through its binding to the 5'-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress, but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.

mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency.

The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and latent cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5'-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogeneous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence. IMPORTANCE: Toxoplasma gondii is an opportunistic pathogen important to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation is required. Here we show that the mRNA cap-binding protein, eIF4E1, regulates the encystation process. Encysted parasites reduce eIF4E1 levels, and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.

mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency.

The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and dormant cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5'-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence.

Plant growth and resistance promoted by Streptomyces spp. in tomato.

Plant Growth Promoting Rhizobacteria (PGPR) represent an alternative to improve plant growth and yield as well as to act as agents of biocontrol. This study characterized isolates of Streptomyces spp. (Stm) as PGPR, determined the antagonism of these isolates against Pectobacterium carotovorum subsp. brasiliensis (Pcb), evaluated the ability of Stm on promoting growth and modulating the defense-related metabolism of tomato plants, and the potential of Stm isolates on reducing soft rot disease in this species. The VOC profile of Stm was also verified. Promotion of plant growth was assessed indirectly through VOC emission and by direct interaction with Stm isolates in the roots. Evaluation of soft rot disease was performed in vitro on plants treated with Stm and challenged with Pcb. Enzymes related to plant defense were then analyzed in plants treated with three selected isolates of Stm, and PM1 was chosen for further Pcb-challenging experiment. Streptomyces spp. isolates displayed characteristics of PGPR. PM3 was the isolate with efficient antagonism against Pcb by dual-culture. Most of the isolates promoted growth of root and shoot of tomato plants by VOC, and PM5 was the isolate that most promoted growth by direct interaction with Stm. Soft rot disease and mortality of plants were significantly reduced when plants were treated with StmPM1. Modulation of secondary metabolism was observed with Stm treatment, and fast response of polyphenoloxidases was detected in plants pretreated with StmPM1 and challenged with Pcb. Peroxidase was significantly activated three days after infection with Pcb in plants pretreated with StmPM1. Results suggest that Streptomyces sp. PM1 and PM5 have the potential to act as PGPR.

Influence of Ecto-nucleoside triphosphate diphosphohydrolase activity on Trypanosoma cruzi infectivity and virulence.

BACKGROUND: The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. METHODOLOGY/PRINCIPAL FINDINGS: We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites. CONCLUSIONS/SIGNIFICANCE: Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.