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]

Abstract

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.
PMID:
 
28430827
 
DOI:
 
10.1371/journal.ppat.1006331

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.

KEYWORDS:

CD103; CD8+ T cell memory; Toxoplasma gondii; chronic infection; neuroimmunology; tissue-resident memory cells
PMID:
28424687
PMCID:
PMC5372813
DOI:
10.3389/fimmu.2017.00335

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.

KEYWORDS:

Apicomplexa; Toxoplasma; apicoplast; lipids; patatin; phospholipase
PMID:
28419631
DOI:
10.1111/mmi.13694

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.


BACKGROUND:

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.

METHODOLOGY/PRINCIPAL FINDINGS:

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.

CONCLUSION/SIGNIFICANCE:

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.
PMID:
28414794
DOI:
10.1371/journal.pone.0176144

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.

KEYWORDS:

Autophagy; Cell signalling; Host cell; Plasmodium; Theileria; Toxoplasma
PMID:
28411879
DOI:
10.1016/j.bj.2017.01.001

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.
PMID:
28406951
DOI:
10.1371/journal.pone.0175644

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.
PMID:
28406972
DOI:
10.1371/journal.pone.0175159

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.

KEYWORDS:

chromatin remodeling; epigenetics; immune evasion; innate immunity; intracellular pathogen; serine/threonine kinases; signal transduction; transcription factors
PMID:
28404792
DOI:
10.1128/CMR.00005-17

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.

KEYWORDS:

Felines; PCR; Sexual transmission; Tachyzoites; Toxoplasmosis
PMID:
28404209
DOI:
10.1016/j.vetpar.2017.04.003

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.

KEYWORDS:

Apicomplexan parasites; Arginine vasopressin; Behavioral manipulation; Estrogen; Gender; Medial amygdala; Parasites; Progesterone; Testosterone
PMID:
28400143
DOI:
10.1016/j.bbi.2017.04.005

Monday, April 10, 2017

Non-coding RNAs in Host-Pathogen Interactions: Subversion of Mammalian Cell Functions by Protozoan Parasites

 2017 Mar 21;8:474. doi: 10.3389/fmicb.2017.00474. eCollection 2017.

Abstract

Pathogens have evolved mechanisms to modulate host cell functions and avoid recognition and destruction by the host damage response. For many years, researchers have focused on proteins as the main effectors used by pathogens to hijack host cell pathways, but only recently with the development of deep RNA sequencing these molecules were brought to light as key players in infectious diseases. Protozoan parasites such as those from the genera PlasmodiumToxoplasmaLeishmania, and Trypanosomacause life-threatening diseases and are responsible for 1000s of deaths worldwide every year. Some of these parasites replicate intracellularly when infecting mammalian hosts, whereas others can survive and replicate extracellularly in the bloodstream. Each of these parasites uses specific evasion mechanisms to avoid being killed by the host defense system. An increasing number of studies have shown that these pathogens can transfer non-coding RNA molecules to the host cells to modulate their functions. This transference usually happens via extracellular vesicles, which are small membrane vesicles secreted by the microorganism. In this mini-review we will combine published work regarding several protozoan parasites that were shown to use non-coding RNAs in inter-kingdom communication and briefly discuss future perspectives in the field.

KEYWORDS: 

extracellular vesicles; infection; miRNA; non-coding RNA; parasitic diseases; protozoan parasites
PMID:
 
28377760
 
PMCID:
 
PMC5359270
 
DOI:
 
10.3389/fmicb.2017.00474

From Toxoplasmosis to Schizophrenia via NMDA Dysfunction: Peptide Overlap between Toxoplasma gondii and N-Methyl-d-Aspartate Receptors As a Potential Mechanistic Link

 2017 Mar 15;8:37. doi: 10.3389/fpsyt.2017.00037. eCollection 2017.

Abstract

The present work aims at investigating how Toxoplasma gondii (T. gondii) infection may be linked to N-methyl-d-aspartate receptor (NMDAR) dysfunction in schizophrenia and related disorders and puts forward the hypothesis that immune responses against T. gondii may involve NMDARs. Indeed, the analysis of the protozoan proteome and NMDAR subunits for peptide commonalities shows a massive peptide overlap and supports the possibility that anti-T. gondii immune responses raised during active protozoan infection may cross-react with host NMDARs, determining disruption of neural circuits and cognitive deficits. In particular, the NMDA 2D subunit, which is mainly expressed in parvalbumin-positive interneurons, appears to be a hotspot for potential T. gondii-induced cross-reactive immune attacks.

KEYWORDS: 

N-methyl-d-aspartate receptors; NMDA 2D; Toxoplasma gondii; gamma oscillations; immune cross-reactivity; parvalbumin-positive interneurons; peptide commonality; schizophrenia
PMID:
 
28360866
 
PMCID:
 
PMC5350139
 
DOI:
 
10.3389/fpsyt.2017.00037

An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells

 2017 Mar 31;12(3):e0173018. doi: 10.1371/journal.pone.0173018. eCollection 2017.

Abstract

Toxoplasmosis is a zoonotic infection affecting approximately 30% of the world's human population. After sexual reproduction in the definitive feline host, Toxoplasma oocysts, each containing 8 sporozoites, are shed into the environment where they can go on to infect humans and other warm-blooded intermediate hosts. Here, we use an in vitro model to assess host transcriptomic changes that occur in the earliest stages of such infections. We show that infection of rat intestinal epithelial cells with mature sporozoites primarily results in higher expression of genes associated with Tumor Necrosis Factor alpha (TNFα) signaling via NF-κB. Furthermore, we find that, consistent with their biology, these mature, invaded sporozoites display a transcriptome intermediate between the previously reported day 10 oocysts and that of their tachyzoite counterparts. Thus, this study uncovers novel host and pathogen factors that may be critical for the establishment of a successful intracellular niche following sporozoite-initiated infection.
PMID:
 
28362800
 
PMCID:
 
PMC5376300
 
DOI:
 
10.1371/journal.pone.0173018

Widespread 5-methylcytosine in the genomes of avian Coccidia and other apicomplexan parasites

 2017 Mar 30. doi: 10.1007/s00436-017-5434-x. [Epub ahead of print]

Gong Z1,2Yin H1,2Ma X1,2Liu B1,2Han Z1,2Gou L1,2Cai J3,4.

Abstract

To date, little is known about cytosine methylation in the genomic DNA of apicomplexan parasites, although it has been confirmed that this important epigenetic modification exists in many lower eukaryotes, plants, and animals. In the present study, ELISA-based detection demonstrated that low levels of 5-methylcytosine (5-mC) are present in Eimeria spp., Toxoplasma gondii, Cryptosporidium spp., and Neospora caninum. The proportions of 5-mC in genomic DNA were 0.18 ± 0.02% in E tenella sporulated oocysts, 0.19 ± 0.01% in E. tenella second-generation merozoites, 0.22 ± 0.04% in T. gondii tachyzoites, 0.28 ± 0.03% in N. caninum tachyzoites, and 0.06 ± 0.01, 0.11 ± 0.01, and 0.09 ± 0.01% in C. andersoni, C. baileyi, and C. parvum sporulated oocysts, respectively. In addition, we found that the percentages of 5-mC in E. tenella varied considerably at different life stages, with sporozoites having the highest percentage of 5-mC (0.78 ± 0.10%). Similar stage differences in 5-mC were also found in E. maxima, E. necatrix, and E. acervulina, the levels of 5-mC in their sporozoites being 4.3-, 1.8-, 2.5-, and 2.0-fold higher than that of sporulated oocysts, respectively (p < 0.01). Furthermore, a total DNA methyltransferase-like activity was detected in whole cell extracts prepared from E. tenella sporozoites. In conclusion, genomic DNA methylation is present in these apicomplexan parasites and may play a role in the stage conversion of Eimeria.

KEYWORDS: 

5-Methylcytosine; Apicomplexan parasites; DNA methyltransferase-like activity; Eimeria spp.; Genomic DNA
PMID:
 
28361273
 
DOI:
 
10.1007/s00436-017-5434-x

Optogenetic monitoring identifies phosphatidylthreonine-regulated calcium homeostasis in Toxoplasma gondii

 2016 May 2;3(5):215-223. doi: 10.15698/mic2016.05.500.

Abstract

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.

KEYWORDS: 

Toxoplasma gondii; calcium homeostasis; gene-encoded calcium indicator; intracellular parasite; lytic cycle; optogenetics; phosphatidylthreonine
PMID:
 
28357357
 
PMCID:
 
PMC5349149
 
DOI:
 
10.15698/mic2016.05.500