Targeted Demethylation of FOXP3-TSDR Enhances the Suppressive Capacity of STAT6-deficient Inducible T Regulatory Cells.

In vitro induced T regulatory cells (iTregs) are promising for addressing inflammation-driven diseases. However, current protocols for the generation and expansion of iTregs fail to induce extensive demethylation of the Treg-specific demethylated region (TSDR) within the FOXP3 gene, recognized as the master regulator for regulatory T cells (Tregs). This deficiency results in the rapid loss of Foxp3 expression and an unstable regulatory phenotype. Nevertheless, inhibition of STAT6 signaling effectively stabilizes Foxp3 expression in iTregs. Thus, this study aimed to develop a protocol combining epigenetic editing with STAT6 deficiency to improve iTregs' ability to maintain stable suppressive function and a functional phenotype. Our findings demonstrate that the combination of STAT6 deficiency (STAT6-/-) with targeted demethylation of the TSDR using a CRISPR-TET1 tool leads to extensive demethylation of FOXP3-TSDR. Demethylation in STAT6-/- iTregs was associated with enhanced expression of Foxp3 and suppressive markers such as CTLA-4, PD-1, IL-10, and TGF-ß. Furthermore, the edited STAT6-/- iTregs exhibited an increased capacity to suppress CD8+ and CD4+ lymphocytes and could more efficiently impair Th1-signature gene expression compared to conventional iTregs. In conclusion, the deactivation of STAT6 and TSDR-targeted demethylation via CRISPR-TET1 is sufficient to induce iTregs with heightened stability and increased suppressive capacity, offering potential applications against inflammatory and autoimmune diseases.

Phosphatidylserine synthase in the endoplasmic reticulum of Toxoplasma is essential for its lytic cycle in human cells.

Glycerophospholipids have emerged as a significant contributor to the intracellular growth of pathogenic protist Toxoplasma gondii. Phosphatidylserine (PtdSer) is one such lipid, attributed to the locomotion and motility-dependent invasion and egress events in its acutely infectious tachyzoite stage. However, the de novo synthesis of PtdSer and the importance of the pathway in tachyzoites remain poorly understood. We show that a base-exchange-type PtdSer synthase (PSS) located in the parasite's endoplasmic reticulum produces PtdSer, which is rapidly converted to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase (PSD) activity. The PSS-PSD pathway enables the synthesis of several lipid species, including PtdSer (16:0/18:1) and PtdEtn (18:2/20:4, 18:1/18:2 and 18:2/22:5). The PSS-depleted strain exhibited a lower abundance of the major ester-linked PtdEtn species and concurrent accrual of host-derived ether-PtdEtn species. Most phosphatidylthreonine (PtdThr) species-an exclusive natural analog of PtdSer, also made in the endoplasmic reticulum-were repressed. PtdSer species, however, remained largely unaltered, likely due to the serine-exchange reaction of PtdThr synthase in favor of PtdSer upon PSS depletion. Not least, the loss of PSS abrogated the lytic cycle of tachyzoites, impairing the cell division, motility, and egress. In a nutshell, our data demonstrate a critical role of PSS in the biogenesis of PtdSer and PtdEtn species and its physiologically essential repurposing for the asexual reproduction of a clinically relevant intracellular pathogen.

STAT6 controls the stability and suppressive function of regulatory T cells.

Signal transducer and activator of transcription 6 (STAT6) promotes tumorigenesis by decreasing the Forkhead box P3+ (Foxp3+) cell frequency allowing for the infiltration of inflammatory cells during the early stages of colitis-associated cancer (CAC). In this study, we dissected the role of STAT6 in the generation of inducible in vitro regulatory T cells (iTregs) and peripheral in vivo Tregs (pTregs) under inflammatory conditions. In in vitro assays, when STAT6 was lacking, iTregs preserved a stable phenotype and expressed high levels of Foxp3 and CD25 during long expansion periods, even in the presence of IL-6. This effect was associated with increased in vitro suppressive ability, over-expression of programmed death-1 (PD-1), CTLA-4, and Foxp3, and decreased IFN-γ expression. Furthermore, iTregs developed during STAT6 deficiency showed a higher demethylation status for the FOXP3 Treg-specific demethylated region (TSDR), coupled with lower DNA methyltransferase 1 (DNMT1) mRNA expression, suggesting that STAT6 may lead to Foxp3 silencing. Using a mouse model of CAC, the STAT6-/- pTregs expressed a more activated phenotype at the intestine, had higher suppressive capacity, and expressed more significant levels of PD-1 and latency-associated peptide of TGF-ß (LAP) associated with their ability to attenuate tumor development. These data suggest that STAT6 signaling impairs the induction, stability, and suppressive capacity of Tregs developed in vitro or in vivo during gut inflammation.

Genome Editing in Bacteria: CRISPR-Cas and Beyond.

Genome editing in bacteria encompasses a wide array of laborious and multi-step methods such as suicide plasmids. The discovery and applications of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas based technologies have revolutionized genome editing in eukaryotic organisms due to its simplicity and programmability. Nevertheless, this system has not been as widely favored for bacterial genome editing. In this review, we summarize the main approaches and difficulties associated with CRISPR-Cas-mediated genome editing in bacteria and present some alternatives to circumvent these issues, including CRISPR nickases, Cas12a, base editors, CRISPR-associated transposases, prime-editing, endogenous CRISPR systems, and the use of pre-made ribonucleoprotein complexes of Cas proteins and guide RNAs. Finally, we also address fluorescent-protein-based methods to evaluate the efficacy of CRISPR-based systems for genome editing in bacteria. CRISPR-Cas still holds promise as a generalized genome-editing tool in bacteria and is developing further optimization for an expanded application in these organisms. This review provides a rarely offered comprehensive view of genome editing. It also aims to familiarize the microbiology community with an ever-growing genome-editing toolbox for bacteria.

Differential gene expression of virulence factors modulates infectivity of TcI Trypanosoma cruzi strains.

Trypanosoma cruzi is the etiological agent of Chagas disease, whose clinical outcome ranges from asymptomatic individuals to chronic fatal megasyndromes. Despite being central to pathogenesis, the regulation of parasite virulence factors' expression remains largely unknown. In this work, the relative expression of several parasite virulence factors between two TcI strains (Ninoa, low virulence and Qro, high virulence) was assessed by qRT-PCR of total and of polysome-associated mRNA, as well as by western blots. Trypomastigotes were also incubated with specific anti-sense morpholino oligonucleotides to block the translation of a selected virulence factor, calreticulin, in both strains. Ninoa trypomastigotes showed significantly lower levels of trypomastigote-decay acceleration factor, complement regulatory protein, complement C2 receptor inhibitor trispanning, and glycoproteins 82 and 90 mRNAs compared with Qro. There was a significantly lower recruitment of complement regulatory protein and complement C2 receptor inhibitor trispanning mRNAs to polysomes and higher recruitment of MASP mRNA to monosomes in Ninoa strain. Calreticulin mRNA displayed both a higher total mRNA level and recruitment to translationally active polysomes in the Ninoa strain (low virulence) than in the Qro strain (high virulence). When calreticulin was downregulated by ≈ 50% by anti-sense morpholino oligonucleotides, a significant decrease of parasite invasion in mammalian cells was found in both strains. Calreticulin downregulation, however, only increased significantly the activation of the complement system by Ninoa trypomastigotes. These results suggest a role for the regulation of virulence factors' gene expression in the differential virulence among T. cruzi strains. Furthermore, a possible function of calreticulin in parasite invasion not related to its binding to complement factors is shown.

Complement system contributes to modulate the infectivity of susceptible TcI strains of Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi is a protozoan parasite and an etiological agent of Chagas disease. There is a wide variability in the clinical outcome of its infection, ranging from asymptomatic individuals to those with chronic fatal mega syndromes. Both parasite and host factors, as well as their interplay, are thought to be involved in the process. OBJECTIVES: To evaluate the resistance to complement-mediated killing in two T. cruzi TcI strains with differential virulence and the subsequent effect on their infectivity in mammalian cells. METHODS: Tissue-culture derived trypomastigotes of both strains were incubated in guinea pig serum and subjected to flow cytometry in order to determine their viability and complement activations. Trypomastigotes were also incubated on host cells monolayers in the presence of serum, and infectivity was evaluated under different conditions of complement pathway inhibition. Relative expression of the main parasite-specific complement receptors between the two strains was assessed by quantitative real-time polymerase chain reaction. FINDINGS: In this work, we showed that two TcI strains, one with lower virulence (Ninoa) compared to the other (Qro), differ in their resistance to the lytic activity of complement system, hence causing a compromised ability of Ninoa strain to invade mammalian cells. These results correlate with the three-fold lower messenger RNA (mRNA) levels of complement regulatory protein (CRP), trypomastigote-decay acceleration factor (T-DAF), and complement C2 receptor inhibitor trispanning (CRIT) in Ninoa compared to those in Qro. On the other hand, calreticulin (CRT) mRNA and surface protein levels were higher in Ninoa strain and promoted its infectivity when the lectin pathway of the complement system was inhibited. MAIN CONCLUSIONS: This work suggests the complex interplay of CRP, T-DAF, CRIT, and CRT, and the diagnostic value of mRNA levels in the assessment of virulence potential of T. cruzi strains, particularly when dealing with isolates with similar genetic background.

Complement system contributes to modulate the infectivity of susceptible TcI strains of Trypanosoma cruzi

BACKGROUND Trypanosoma cruzi is a protozoan parasite and an etiological agent of Chagas disease. There is a wide variability in the clinical outcome of its infection, ranging from asymptomatic individuals to those with chronic fatal mega syndromes. Both parasite and host factors, as well as their interplay, are thought to be involved in the process. OBJECTIVES To evaluate the resistance to complement-mediated killing in two T. cruzi TcI strains with differential virulence and the subsequent effect on their infectivity in mammalian cells. METHODS Tissue-culture derived trypomastigotes of both strains were incubated in guinea pig serum and subjected to flow cytometry in order to determine their viability and complement activations. Trypomastigotes were also incubated on host cells monolayers in the presence of serum, and infectivity was evaluated under different conditions of complement pathway inhibition. Relative expression of the main parasite-specific complement receptors between the two strains was assessed by quantitative real-time polymerase chain reaction. FINDINGS In this work, we showed that two TcI strains, one with lower virulence (Ninoa) compared to the other (Qro), differ in their resistance to the lytic activity of complement system, hence causing a compromised ability of Ninoa strain to invade mammalian cells. These results correlate with the three-fold lower messenger RNA (mRNA) levels of complement regulatory protein (CRP), trypomastigote-decay acceleration factor (T-DAF), and complement C2 receptor inhibitor trispanning (CRIT) in Ninoa compared to those in Qro. On the other hand, calreticulin (CRT) mRNA and surface protein levels were higher in Ninoa strain and promoted its infectivity when the lectin pathway of the complement system was inhibited. MAIN CONCLUSIONS This work suggests the complex interplay of CRP, T-DAF, CRIT, and CRT, and the diagnostic value of mRNA levels in the assessment of virulence potential of T. cruzi strains, particularly when dealing with isolates with similar genetic background.

Optogenetic monitoring identifies phosphatidylthreonine-regulated calcium homeostasis in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite, which inflicts acute as well as chronic infections in a wide range of warm-blooded vertebrates. Our recent work has demonstrated the natural occurrence and autonomous synthesis of an exclusive lipid phosphatidylthreonine in T. gondii. Targeted gene disruption of phosphatidylthreonine synthase impairs the parasite virulence due to unforeseen attenuation of the consecutive events of motility, egress and invasion. However, the underlying basis of such an intriguing phenotype in the parasite mutant remains unknown. Using an optogenetic sensor (gene-encoded calcium indicator, GCaMP6s), we show that loss of phosphatidylthreonine depletes calcium stores in intracellular tachyzoites, which leads to dysregulation of calcium release into the cytosol during the egress phase of the mutant. Consistently, the parasite motility and egress phenotypes in the mutant can be entirely restored by ionophore-induced mobilization of calcium. Collectively, our results suggest a novel regulatory function of phosphatidylthreonine in calcium signaling of a prevalent parasitic protist. Moreover, our application of an optogenetic sensor to monitor subcellular calcium in a model intracellular pathogen exemplifies its wider utility to other entwined systems.

Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence.

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.

Dynein light chain 8a of Toxoplasma gondii, a unique conoid-localized ß-strand-swapped homodimer, is required for an efficient parasite growth.

Dynein light chain 8 (DLC8) is a ubiquitous eukaryotic protein regulating diverse cellular functions. We show that the obligate intracellular parasite Toxoplasma gondii harbors 4 DLC8 proteins (TgDLC8a-d), of which only TgDLC8a clusters in the mainstream LC8 class. TgDLC8b-d proteins form a divergent and alveolate-specific clade. TgDLC8b-d proteins are largely cytosolic, whereas TgDLC8a resides in the conoid at the apical end of T. gondii. The apical location of TgDLC8a is also not shared by its nearly identical Eimeria (EtDLC8a), Plasmodium (PfDLC8), or human (HsDLC8) orthologs. Notwithstanding an exclusive conoid targeting, TgDLC8a exhibits a classical LC8 structure. It forms a homodimer by swapping of the ß strands that interact with the antiparallel ß' strands of the opposing monomers. The TgDLC8a dimer contains two identical binding grooves and appears to be adapted for multitarget recognition. By contrast, the previously reported PfDLC8 homodimer is shaped by binding of the ß strand with the parallel ß' strand and lacks such a distinct binding interface. Our comparisons suggest an unexpected structural and functional divergence of the two otherwise conserved proteins from apicomplexan parasites. Finally, we demonstrate that a phosphomimetic S88E mutation renders the TgDLC8a-S88E mutant monomeric and cytosolic in T. gondii, and its overexpression inhibits the parasite growth in human fibroblasts.