bims-toxgon 2023-09-03 papers

Issue of 2023–09–03
seven papers selected by
Lakesh Kumar, BITS Pilani

Parasites Hosts Dis. 2023 Aug;61(3): 231-239

Recent progress in vaccine development targeting pre-clinical human toxoplasmosis.

Toxoplasma gondii is an intracellular parasitic organism affecting all warm-blooded vertebrates. Due to the unavailability of commercialized human T. gondii vaccine, many studies have been reported investigating the protective efficacy of pre-clinical T. gondii vaccines expressing diverse antigens. Careful antigen selection and implementing multifarious immunization strategies could enhance protection against toxoplasmosis in animal models. Although none of the available vaccines could remove the tissue-dwelling parasites from the host organism, findings from these pre-clinical toxoplasmosis vaccine studies highlighted their developmental potential and provided insights into rational vaccine design. We herein explored the progress of T. gondii vaccine development using DNA, protein subunit, and virus-like particle vaccine platforms. Specifically, we summarized the findings from the pre-clinical toxoplasmosis vaccine studies involving T. gondii challenge infection in mice published in the past 5 years.

Keywords: DNA; Toxoplasma gondii; protein subunit; vaccine; virus-like particle

DOI: https://doi.org/10.3347/PHD.22097

Vet Parasitol. 2023 Aug 10. pii: S0304-4017(23)00137-1. [Epub ahead of print]322 110006

New morphological observations on the initial events of Toxoplasma gondii entry into host cells.

Everson Reili de Souza Teles, Juliana de Araujo Portes, Wanderley de Souza.

Toxoplasma gondii is an obligate intracellular protozoan of worldwide distribution. It is effective in the infection of various homoeothermic animals of economic importance. The process of T. gondii invasion of host cells occurs in less than 20 s by the active mechanism of penetration. First, a mobile junction is formed due to the association between the apical end of the parasite and the host cell surface. Then, the secretion of invasive and docking proteins allows the formation of the mobile junction before the complete internalization of the parasite. Here, using high-resolution microscopy, it was described new morphological observations of the early events of host cell invasion by tachyzoites of T. gondii. Attempts were made to synchronize the interaction process using low temperatures and treatment of the host cells with cytochalasin D, a drug that interferes with the actin dynamics. Images were obtained showing that the parasite and the host cells seem to release small vesicles with diameters varying from 25 to 100 nm. Furthermore, tunneling nanotubes emerge from the host cell surface and interact with the parasite even at long distance. These observations add new details of adhesion and entry events, such as surface projections of the host cell plasma membrane, pseudopods, and nanotubes radiating from the host cell toward the parasite. In addition, scanning microscopy revealed intense vesiculation, with a morphological characteristic of extracellular microvesicles, during the entry of the tachyzoite into the host cell.

Keywords: Electron Microscopy; Parasite-host cell interaction; Toxoplasma gondii

DOI: https://doi.org/10.1016/j.vetpar.2023.110006

PLoS Pathog. 2023 Aug 31. 19(8): e1011566

Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii.

Beejan Asady, Vera Samples, Julia D Romano, Jelena Levitskaya, Bao Lige, Pratik Khare, Anne Le, Isabelle Coppens.

As an obligate intracellular parasite, Toxoplasma gondii must import essential nutrients from the host cell into the parasitophorous vacuole. We previously reported that the parasite scavenges cholesterol from host endocytic organelles for incorporation into membranes and storage as cholesteryl esters in lipid droplets. In this study, we have investigated whether Toxoplasma utilizes cholesterol as a precursor for the synthesis of metabolites, such as steroids. In mammalian cells, steroidogenesis occurs in mitochondria and involves membrane-bound type I cytochrome P450 oxidases that are activated through interaction with heme-binding proteins containing a cytochrome b5 domain, such as members of the membrane-associated progesterone receptor (MAPR) family. Our LC-MS targeted lipidomics detect selective classes of hormone steroids in Toxoplasma, with a predominance for anti-inflammatory hydroxypregnenolone species, deoxycorticosterone and dehydroepiandrosterone. The genome of Toxoplasma contains homologs encoding a single type I CYP450 enzyme (we named TgCYP450mt) and a single MAPR (we named TgMAPR). We showed that TgMAPR is a hemoprotein with conserved residues in a heme-binding cytochrome b5 domain. Both TgCYP450 and TgMAPR localize to the mitochondrion and show interactions in in situ proximity ligation assays. Genetic ablation of cyp450mt is not tolerated by Toxoplasma; we therefore engineered a conditional knockout strain and showed that iΔTgCYP450mt parasites exhibit growth impairment in cultured cells. Parasite strains deficient for mapr could be generated; however, ΔTgMAPR parasites suffer from poor global fitness, loss of plasma membrane integrity, aberrant mitochondrial cristae, and an abnormally long S-phase in their cell cycle. Compared to wild-type parasites, iΔTgCYP450mt and ΔTgMAPR lost virulence in mice and metabolomics studies reveal that both mutants have reduced levels of steroids. These observations point to a steroidogenic pathway operational in the mitochondrion of a protozoan that involves an evolutionary conserved TgCYP450mt enzyme and its binding partner TgMAPR.

DOI: https://doi.org/10.1371/journal.ppat.1011566

mBio. 2023 Aug 30. e0127923

A Toxoplasma gondii lipoxygenase-like enzyme is necessary for virulence and changes localization associated with the host immune response.

Carlos J Ramírez-Flores, Billy Joel Erazo Flores, Andrés M Tibabuzo Perdomo, Katie L Barnes, Sarah K Wilson, Carolina Mendoza Cavazos, Laura J Knoll.

While the asexual cycle of Toxoplasma gondii can occur in any warm-blooded animal, the sexual cycle is restricted to the feline intestine. We previously determined that because cats lack delta-6-desaturase activity in their intestines, they build up excess linoleic acid, which signals T. gondii to undergo sexual development. We hypothesized that T. gondii oxygenates linoleic acid to signal sexual development, so we examined the T. gondii genome for lipoxygenases-like enzymes (TgLOXL) enzymes. We identified seven TgLOXLs that were at least 100-fold more abundant in the cat intestinal versus the tissue culture tachyzoite stage. Parasites deleted in TgLOXL1 (TgΔLOXL1) had no significant growth differences in tissue culture fibroblast cells. Because the sexual development assay begins with brain cysts, we infected mice with TgΔLOXL1 and were surprised to find that TgΔLOXL1 had reduced virulence. The TgΔLOXL1 parasitemia was reduced by 3 d post-infection and largely cleared by 7 d post-infection. At 3 d post-infection, the cytokines interferon gamma (IFN-γ), IL-6, MCP-1, and TNF-α were significantly reduced in TgΔLOXL1-infected mice, which prompted us to examine TgΔLOXL1 in IFN-γ KO mice. We found that IFN-γ KO mice infected with TgΔLOXL1 succumbed to acute infection with the same kinetics as the parental and complemented strains, suggesting that TgLOXL1 plays a role in the IFN-γ signaling cascade. In tissue culture fibroblasts, TgLOXL1 was localized within the parasite, but in leukocytes from infected mice and activated macrophages, TgLOXL1 was localized within the host cytoplasm. These results suggest that TgLOXL1 changes localization in response to host immune activation. IMPORTANCE Lipoxygenases (LOXs) are enzymes that catalyze the deoxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid. These modifications create signaling molecules that are best characterized for modulating the immune response. Deletion of the first lipoxygenase-like enzyme characterized for Toxoplasma gondii (TgLOXL1) generated a less virulent strain, and infected mice showed a decreased immune response. This virulence defect was dependent on the mouse cytokine interferon gamma IFNγ. TgLOXL1 changes location from inside the parasite in tissue culture conditions to vesicular structures within the host immune cells during mouse infection. These results suggest that TgLOXL1 plays a role in the modification of the host immune response in mice.

Keywords: Toxoplasma gondii; cytokines; immune response; leukocytes; lipoxygenase

DOI: https://doi.org/10.1128/mbio.01279-23

Microbiology (Reading). 2023 Aug;169(8):

Timing of dense granule biogenesis in asexual malaria parasites.

Tansy Vallintine, Christiaan van Ooij.

Malaria is an important infectious disease that continues to claim hundreds of thousands of lives annually. The disease is caused by infection of host erythrocytes by apicomplexan parasites of the genus Plasmodium. The parasite contains three different apical organelles - micronemes, rhoptries and dense granules (DGs) - whose contents are secreted to mediate binding to and invasion of the host cell and the extensive remodelling of the host cell that occurs following invasion. Whereas the roles of micronemes and rhoptries in binding and invasion of the host erythrocyte have been studied in detail, the roles of DGs in Plasmodium parasites are poorly understood. They have been proposed to control host cell remodelling through regulated protein secretion after invasion, but many basic aspects of the biology of DGs remain unknown. Here we describe DG biogenesis timing for the first time, using RESA localization as a proxy for the timing of DG formation. We show that DG formation commences approximately 37 min prior to schizont egress, as measured by the recruitment of the DG marker RESA. Furthermore, using a bioinformatics approach, we aimed to predict additional cargo of the DGs and identified the J-dot protein HSP40 as a DG protein, further supporting the very early role of these organelles in the interaction of the parasite with the host cell.

Keywords: Plasmodium; apical organelles; apicomplexa; dense granules; malaria

DOI: https://doi.org/10.1099/mic.0.001389

Commun Biol. 2023 Aug 31. 6(1): 895

Structural insights into MIC2 recognition by MIC2-associated protein in Toxoplasma gondii.

Su Zhang, Fangfang Wang, Dujuan Zhang, Dongsheng Liu, Wei Ding, Timothy A Springer, Gaojie Song.

Microneme protein 2 (MIC2) and MIC2-associated protein (M2AP) play crucial roles in the gliding motility and host cell invasion of Toxoplasma gondii. Complex formation between MIC2 and M2AP is required for maturation and transport from the microneme to the parasite surface. Previous studies showed that M2AP associates with the 6th TSR domain of MIC2 (TSR6), but the detailed interaction remains unclear. In this study, we report crystal structures of M2AP alone and in complex with TSR6. TSR domains have an unusually thin, long structure with a layer of intercalated residues on one side. The non-layered side of TSR6 with hotspot residue His-620 at the center binds to M2AP. Remarkably, we show that TSR6 residue Y602 is dynamic; it equilibrates between being part of the layer (the layered state) and in a flipped-out state in the absence of M2AP. However, when bound to M2AP, Y602 shifts to the flipped-out state. Our findings provide insights into the association and stabilization of MIC2-M2AP complex, and may be used to develop new therapies to prevent infections caused by this parasite.

DOI: https://doi.org/10.1038/s42003-023-05277-0

bioRxiv. 2023 Aug 14. pii: 2023.08.10.552871. [Epub ahead of print]

Bioluminescence Assay of Lysine Deacylase Sirtuin Activity.

Alexandria N Van Scoyk, Orlando Antelope, Anca Franzini, Donald E Ayer, Randall T Peterson, Anthony D Pomicter, Shawn C Owen, Michael W Deininger.

Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine towards maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple pathological conditions, including cancer. Measuring sirtuins' activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed 'SIRT ify ', a high-sensitivity assay for measuring sirtuin activity in vitro and in vivo . SIRT ify is based on a split-version of the NanoLuc® luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine acylations and confirm the effects of sirtuin modulators. SIRT ify effectively quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.

DOI: https://doi.org/10.1101/2023.08.10.552871