Tuesday, April 25, 2017

A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity in Toxoplasma gondii

2017 Apr 24. doi: 10.1111/cmi.12749. [Epub ahead of print]

Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, while many of the calcium-dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation we have determined that a nonsense mutation in a previously uncharacterized protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localizes to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network (TVN). Our findings support a connection between the TVN and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.


GRA41; Toxoplasma gondii; calcium; dense granule; egress; tubulovesicular network

Saturday, April 22, 2017

Dual role of the Toxoplasma gondii clathrin adaptor AP1 in the sorting of rhoptry and microneme proteins and in parasite division

 2017 Apr 21;13(4):e1006331. doi: 10.1371/journal.ppat.1006331. [Epub ahead of print]


Toxoplasma gondii possesses a highly polarized secretory system, which efficiently assembles de novo micronemes and rhoptries during parasite replication. These apical secretory organelles release their contents into host cells promoting parasite invasion and survival. Using a CreLox-based inducible knock-out strategy and the ddFKBP over-expression system, we unraveled novel functions of the clathrin adaptor complex TgAP1. First, our data indicate that AP1 in T. gondii likely functions as a conserved heterotetrameric complex composed of the four subunits γ, β, μ1, σ1 and interacts with known regulators of clathrin-mediated vesicular budding such as the unique ENTH-domain containing protein, which we named Epsin-like protein (TgEpsL). Disruption of the μ1 subunit resulted in the mis-sorting of microneme proteins at the level of the Trans-Golgi-Network (TGN). Furthermore, we demonstrated that TgAP1 regulates rhoptry biogenesis by activating rhoptry protein exit from the TGN, but also participates in the post-Golgi maturation process of preROP compartments into apically anchored club-shaped mature organelles. For this latter activity, our data indicate a specific functional relationship between TgAP1 and the Rab5A-positive endosome-like compartment. In addition, we unraveled an original role for TgAP1 in the regulation of parasite division. APμ1-depleted parasites undergo normal daughter cell budding and basal complex assembly but fail to segregate at the end of cytokinesis.

Friday, April 21, 2017

CD103+ CD8 T Cells in the Toxoplasma-Infected Brain Exhibit a Tissue-Resident Memory Transcriptional Profile

2017 Mar 29;8:335. doi: 10.3389/fimmu.2017.00335. eCollection 2017.

During chronic infection, memory T cells acquire a unique phenotype and become dependent on different survival signals than those needed for memory T cells generated during an acute infection. The distinction between the role of effector and memory T cells in an environment of persistent antigen remains unclear. Here, in the context of chronic Toxoplasma gondii infection, we demonstrate that a population of CD8 T cells exhibiting a tissue-resident memory (TRM) phenotype accumulates within the brain. We show that this population is distributed throughout the brain in both parenchymal and extraparenchymal spaces. Furthermore, this population is transcriptionally distinct and exhibits a transcriptional signature consistent with the TRM observed in acute viral infections. Finally, we establish that the CD103+ TRM population has an intrinsic capacity to produce both IFN-γ and TNF-α, cytokines critical for parasite control within the central nervous system (CNS). The contribution of this population to pro-inflammatory cytokine production suggests an important role for TRM in protective and ongoing immune responses in the infected CNS. Accession number: GSE95105.


CD103; CD8+ T cell memory; Toxoplasma gondii; chronic infection; neuroimmunology; tissue-resident memory cells

Wednesday, April 19, 2017

TgPL2, a patatin-like phospholipase domain-containing protein, is involved in the maintenance of apicoplast lipids homeostasis in Toxoplasma

2017 Apr 17. doi: 10.1111/mmi.13694. [Epub ahead of print]

Patatin-like phospholipases are involved in numerous cellular functions, including lipid metabolism and membranes remodeling. The patatin-like catalytic domain, whose phospholipase activity relies on a serine-aspartate dyad and an anion binding box, is widely spread among prokaryotes and eukaryotes. We describe TgPL2, a novel patatin-like phospholipase domain-containing protein from the parasitic protist Toxoplasma gondii. TgPL2 is a large protein, in which the key motifs for enzymatic activity are conserved in the patatin-like domain. Using immunofluorescent assays and immunoelectron microscopy analysis, we have shown that TgPL2 localizes to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. This plastid hosts several important biosynthetic pathways, which makes it an attractive organelle for identifying new potential drug targets. We thus addressed TgPL2 function by generating a conditional knockdown mutant and demonstrated it has an essential contribution for maintaining the integrity of the plastid. In absence of TgPL2, the organelle is rapidly lost and remaining apicoplasts appear enlarged, with an abnormal accumulation of membranous structures, suggesting a defect in lipids homeostasis. More precisely, analyses of lipid content upon TgPL2 depletion suggest this protein is important for maintaining levels of apicoplast-generated fatty acids, and also regulating phosphatidylcholine and lysophosphatidylcholine levels in the parasite. This article is protected by copyright. All rights reserved.


Apicomplexa; Toxoplasma; apicoplast; lipids; patatin; phospholipase

Tuesday, April 18, 2017

Intestinal, extra-intestinal and systemic sequelae of Toxoplasma gondii induced acute ileitis in mice harboring a human gut microbiota

2017 Apr 17;12(4):e0176144. doi: 10.1371/journal.pone.0176144. eCollection 2017.


Within seven days following peroral high dose infection with Toxoplasma gondii susceptible conventionally colonized mice develop acute ileitis due to an underlying T helper cell (Th) -1 type immunopathology. We here addressed whether mice harboring a human intestinal microbiota developed intestinal, extra-intestinal and systemic sequelae upon ileitis induction.


Secondary abiotic mice were generated by broad-spectrum antibiotic treatment and associated with a complex human intestinal microbiota following peroral fecal microbiota transplantation. Within three weeks the human microbiota had stably established in the murine intestinal tract as assessed by quantitative cultural and culture-independent (i.e. molecular 16S rRNA based) methods. At day 7 post infection (p.i.) with 50 cysts of T. gondii strain ME49 by gavage human microbiota associated (hma) mice displayed severe clinical, macroscopic and microscopic sequelae indicating acute ileitis. In diseased hma mice increased numbers of innate and adaptive immune cells within the ileal mucosa and lamina propria and elevated intestinal secretion of pro-inflammatory mediators including IFN-γ, IL-12 and nitric oxide could be observed at day 7 p.i. Ileitis development was accompanied by substantial shifts in intestinal microbiota composition of hma mice characterized by elevated total bacterial loads and increased numbers of intestinal Gram-negative commensals such as enterobacteria and Bacteroides / Prevotella species overgrowing the small and large intestinal lumen. Furthermore, viable bacteria translocated from the inflamed ileum to extra-intestinal including systemic compartments. Notably, pro-inflammatory immune responses were not restricted to the intestinal tract as indicated by increased pro-inflammatory cytokine secretion in extra-intestinal (i.e. liver and kidney) and systemic compartments including spleen and serum.


With respect to the intestinal microbiota composition "humanized" mice display acute ileitis following peroral high dose T. gondii infection. Thus, hma mice constitute a suitable model to further dissect the interactions between pathogens, human microbiota and vertebrate host immunity during acute intestinal inflammation.

Monday, April 17, 2017

Apicomplexan autophagy and modulation of autophagy in parasite-infected host cells

2017 Feb;40(1):23-30. doi: 10.1016/j.bj.2017.01.001. Epub 2017 Mar 23.

Apicomplexan parasites are responsible for a number of important human pathologies. Obviously, as Eukaryotes they share a number of cellular features and pathways with their respective host cells. One of them is autophagy, a process involved in the degradation of the cell's own components. These intracellular parasites nonetheless seem to present a number of original features compared to their very evolutionarily distant host cells. In mammals and other metazoans, autophagy has been identified as an important contributor to the defence against microbial pathogens. Thus, host autophagy also likely plays a key role in the control of apicomplexan parasites, although its potential manipulation and subversion by intracellular parasites creates a complex interplay in the regulation of host and parasite autophagy. In this mini-review, we summarise current knowledge on autophagy in both parasites and their host cells, in the context of infection by three Apicomplexa: Plasmodium, Toxoplasma, and Theileria.


Autophagy; Cell signalling; Host cell; Plasmodium; Theileria; Toxoplasma

Friday, April 14, 2017

Protection induced by virus-like particles containing Toxoplasma gondii microneme protein 8 against highly virulent RH strain of Toxoplasma gondii infection

2017 Apr 13;12(4):e0175644. doi: 10.1371/journal.pone.0175644. eCollection 2017.

Toxoplasma gondii (T. gondii) microneme protein 8 (MIC8) represents a novel, functional distinct invasion factor. In this study, we generated virus-like particles (VLPs) targeting Toxoplasma gondii MIC8 for the first time, and investigated the protection against highly virulent RH strain of T. gondii in a mouse model. We found that VLP vaccination induced Toxoplasma gondii-specific IgG and IgG1 antibody responses in the sera. Upon challenge infection with RH strain of T. gondii tachyzoites, vaccinated mice showed a significant increase of both IgG antibodies in sera and IgA antibodies in feces compared to those before challenge, and a rapid expansion of both germinal center B cell (B220+, GL7+) and T cell (CD4+, CD8+) populations. Importantly, intranasally immunized mice showed higher neutralizing antibodies and displayed no proinflammatory cytokine IFN-γ in the spleen. Mice were completely protected from a lethal challenge infection with the highly virulent T. gondii (RH) showing no body weight loss (100% survival). Our study shows the effective protection against T. gondii infection provided by VLPs containing microneme protein 8 of T. gondii, thus indicating a potential T. gondii vaccine candidate.

Disruption of outer blood-retinal barrier by Toxoplasma gondii-infected monocytes is mediated by paracrinely activated FAK signaling

2017 Apr 13;12(4):e0175159. doi: 10.1371/journal.pone.0175159. eCollection 2017.

Song HB1,2,3, Jun HO1, Kim JH1, Lee YH4, Choi MH3, Kim JH1,2,5.

Ocular toxoplasmosis is mediated by monocytes infected with Toxoplasma gondii that are disseminated to target organs. Although infected monocytes can easily access to outer blood-retinal barrier due to leaky choroidal vasculatures, not much is known about the effect of T. gondii-infected monocytes on outer blood-retinal barrier. We prepared human monocytes, THP-1, infected with T. gondii and human retinal pigment epithelial cells, ARPE-19, grown on transwells as an in vitro model of outer blood-retinal barrier. Exposure to infected monocytes resulted in disruption of tight junction protein, ZO-1, and decrease in transepithelial electrical resistance of retinal pigment epithelium. Supernatants alone separated from infected monocytes also decreased transepithelial electrical resistance and disrupted tight junction protein. Further investigation revealed that the supernatants could activate focal adhesion kinase (FAK) signaling in retinal pigment epithelium and the disruption was attenuated by FAK inhibitor. The disrupted barrier was partly restored by blocking CXCL8, a FAK activating factor secreted by infected monocytes. In this study, we demonstrated that monocytes infected with T. gondii can disrupt outer blood-retinal barrier, which is mediated by paracrinely activated FAK signaling. FAK signaling can be a target of therapeutic approach to prevent negative influence of infected monocytes on outer blood-retinal barrier.

Toxoplasma Effectors Targeting Host Signaling and Transcription

2017 Jul;30(3):615-645. doi: 10.1128/CMR.00005-17.

Early electron microscopy studies revealed the elaborate cellular features that define the unique adaptations of apicomplexan parasites. Among these were bulbous rhoptry (ROP) organelles and small, dense granules (GRAs), both of which are secreted during invasion of host cells. These early morphological studies were followed by the exploration of the cellular contents of these secretory organelles, revealing them to be comprised of highly divergent protein families with few conserved domains or predicted functions. In parallel, studies on host-pathogen interactions identified many host signaling pathways that were mysteriously altered by infection. It was only with the advent of forward and reverse genetic strategies that the connections between individual parasite effectors and the specific host pathways that they targeted finally became clear. The current repertoire of parasite effectors includes ROP kinases and pseudokinases that are secreted during invasion and that block host immune pathways. Similarly, many secretory GRA proteins alter host gene expression by activating host transcription factors, through modification of chromatin, or by inducing small noncoding RNAs. These effectors highlight novel mechanisms by which T. gondii has learned to harness host signaling to favor intracellular survival and will guide future studies designed to uncover the additional complexity of this intricate host-pathogen interaction.


chromatin remodeling; epigenetics; immune evasion; innate immunity; intracellular pathogen; serine/threonine kinases; signal transduction; transcription factors

Investigation of Toxoplasma gondii in semen, testicle and epididymis tissues of primo-infected cats

2017 Apr 3. pii: S0304-4017(17)30136-X. doi: 10.1016/j.vetpar.2017.04.003. [Epub ahead of print]

This study aimed to investigate the presence of Toxoplasma gondii in semen, testicle and epididymis tissues of cats experimentally infected by this coccidium. A total of 12 male felines without a definite breed that were of reproductive age and serologically negative for T. gondii were selected and distributed to the following three experimental groups: GI, inoculated with 600 tissue cysts of the P strain of T. gondii (isolate III); GII, inoculated with 2×105 tachyzoites of the RH strain (isolate I); and GIII, not inoculated (control group). Prior to inoculation (day -7 and 0) and on post inoculation days (PIDs) 7, 14, 21, 28, 42, 56, and 70, all felines were subjected to assessments of anti-T. gondii IgG by indirect immunofluorescence (IIF) and assessments of parasitemia. Collection of semen (electroejaculation) was performed on the specified dates, followed by nested PCR and bioassays in mice to detect T. gondii. On PID 70, all 12 felines were orchiectomized, and the presence of the parasite in the testicles and epididymides was evaluated by nested PCR, murine bioassay, and histopathological and immunohistochemical analyses. All felines inoculated with T. gondii (GI and GII) seroconverted to the toxoplasmic infection after PID 14; on PID 7, seroconversion of three felines (P4, RH2 and RH4) could observed, and all exhibited detectable titers by PID 64. The GII felines exhibited greater serological titers compared with GI felines. The maximum serological titer (IgG) was observed in feline RH3 (titer 1024), while in other experimental felines, a maximum titer of 256 was detected. Parasitemic peaks were diagnosed in all felines of groups I and II from PIDs 7-42. A total of five parasitemic peaks were diagnosed in GI and nine in GII. In none of the experimental time points was the presence of T. gondii diagnosed in seminal samples collected from the felines or in the testicle or epididymis tissues collected from these animals. Thus, sexual transmission in domestic cats does not appear to be a major route of T. gondii infection, possibly demonstrating the tendency of this protozoan to develop a response directed to the formation and excretion of oocysts in the feces of these definite hosts, which act as its main route of perpetuation in the environment.


Felines; PCR; Sexual transmission; Tachyzoites; Toxoplasmosis

Loss of predator aversion in female rats after Toxoplasma gondii infection is not dependent on ovarian steroids

2017 Apr 8. pii: S0889-1591(17)30107-1. doi: 10.1016/j.bbi.2017.04.005. [Epub ahead of print]

Toxoplasma gondii infection reduces aversion to cat odors in male rats. Relevant proximate mechanisms include interaction of gonadal testosterone and brain nonapeptide arginine-vasopressin. Both of these substrates are sexually dimorphic with preferential expression in males; suggesting either absence of behavioral change in females or mediation by analogous neuroendocrine substrates. Here we demonstrate that Toxoplasma gondii infection reduces aversion to cat odor in female rats. This change is not accompanied by altered steroid hormones; cannot be rescued by gonadal removal; and, does not depend on arginine-vasopressin. Thus behavioral change in males and female occur through non-analogous mechanisms that remain hitherto unknown.


Apicomplexan parasites; Arginine vasopressin; Behavioral manipulation; Estrogen; Gender; Medial amygdala; Parasites; Progesterone; Testosterone