bims-toxgon 2023-06-18 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2023–06–18
eight papers selected by
Lakesh Kumar, BITS Pilani

A cathepsin C-like protease mediates the post-translation modification of Toxoplasma gondii secretory proteins for optimal invasion and egress.
Exploring Toxoplasma gondii´s Biology within the Intestinal Epithelium: intestinal-derived models to unravel sexual differentiation.
A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats.
Evaluation of the protective immune response of an attenuated strain of Toxoplasma gondii with long-term passages on the Gecko cell line.
Microfluidic Biosensor Based on Molybdenum Disulfide (MoS2) Modified Thin-Core Microfiber for Immune Detection of Toxoplasma gondii.
A Plasmodium membrane receptor platform integrates cues for egress and invasion in blood forms and activation of transmission stages.
Plasmodium GPI-anchored micronemal antigen is essential for parasite transmission through the mosquito host.
The AMPK-dependent inhibition of autophagy plays a crucial role in protecting photoreceptor from photooxidative injury.

mBio. 2023 Jun 16. e0017423

A cathepsin C-like protease mediates the post-translation modification of Toxoplasma gondii secretory proteins for optimal invasion and egress.

L Brock Thornton, Melanie Key, Chiara Micchelli, Andrew J Stasic, Samuel Kwain, Katherine Floyd, Silvia N J Moreno, Brian N Dominy, Daniel C Whitehead, Zhicheng Dou.

  Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, Toxoplasma gondii must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans. Previous work has shown that some micronemal invasion effectors experience a series of proteolytic cleavages within the parasite's secretion pathway for maturation, such as the aspartyl protease (TgASP3) and the cathepsin L-like protease (TgCPL), localized within the post-Golgi compartment and the endolysosomal system, respectively. Furthermore, it has been shown that the precise maturation of micronemal effectors is critical for Toxoplasma invasion and egress. Here, we show that an endosome-like compartment (ELC)-residing cathepsin C-like protease (TgCPC1) mediates the final trimming of some micronemal effectors, and its loss further results in defects in the steps of invasion, egress, and migration throughout the parasite's lytic cycle. Notably, the deletion of TgCPC1 completely blocks the activation of subtilisin-like protease 1 (TgSUB1) in the parasites, which globally impairs the surface-trimming of many key micronemal invasion and egress effectors. Additionally, we found that Toxoplasma is not efficiently inhibited by the chemical inhibitor targeting the malarial CPC ortholog, suggesting that these cathepsin C-like orthologs are structurally different within the apicomplexan phylum. Collectively, our findings identify a novel function of TgCPC1 in processing micronemal proteins within the Toxoplasma parasite's secretory pathway and expand the understanding of the roles of cathepsin C protease.Toxoplasma gondii is a microbial pathogen that is well adapted for disseminating infections. It can infect virtually all warm-blooded animals. Approximately one-third of the human population carries toxoplasmosis. During infection, the parasites sequentially secrete protein effectors from the microneme, rhoptry, and dense granule, three organelles exclusively found in apicomplexan parasites, to help establish their lytic cycle. Proteolytic cleavage of these secretory proteins is required for the parasite's optimal function. Previous work has revealed that two proteases residing within the parasite's secretory pathway cleave micronemal and rhoptry proteins, which mediate parasite invasion and egress. Here, we demonstrate that a cathepsin C-like protease (TgCPC1) is involved in processing several invasion and egress effectors. The genetic deletion of TgCPC1 prevented the complete maturation of some effectors in the parasites. Strikingly, the deletion led to a full inactivation of one surface-anchored protease, which globally impaired the trimming of some key micronemal proteins before secretion. Therefore, this finding represents a novel post-translational mechanism for the processing of virulence factors within microbial pathogens.

Keywords:  Toxoplasma gondii; aminopeptidase; apicomplexan; cathepsin C; digestive vacuole; egress; invasion; lysosome; protease; protein trafficking

DOI:  https://doi.org/10.1128/mbio.00174-23
Front Cell Infect Microbiol. 2023 ;13 1134471

Exploring Toxoplasma gondii´s Biology within the Intestinal Epithelium: intestinal-derived models to unravel sexual differentiation.

Florencia Sena, Saira Cancela, Mariela Bollati-Fogolín, Romina Pagotto, María E Francia.

  A variety of intestinal-derived culture systems have been developed to mimic in vivo cell behavior and organization, incorporating different tissue and microenvironmental elements. Great insight into the biology of the causative agent of toxoplasmosis, Toxoplasma gondii, has been attained by using diverse in vitro cellular models. Nonetheless, there are still processes key to its transmission and persistence which remain to be elucidated, such as the mechanisms underlying its systemic dissemination and sexual differentiation both of which occur at the intestinal level. Because this event occurs in a complex and specific cellular environment (the intestine upon ingestion of infective forms, and the feline intestine, respectively), traditional reductionist in vitro cellular models fail to recreate conditions resembling in vivo physiology. The development of new biomaterials and the advances in cell culture knowledge have opened the door to a next generation of more physiologically relevant cellular models. Among them, organoids have become a valuable tool for unmasking the underlying mechanism involved in T. gondii sexual differentiation. Murine-derived intestinal organoids mimicking the biochemistry of the feline intestine have allowed the generation of pre-sexual and sexual stages of T. gondii for the first time in vitro, opening a window of opportunity to tackling these stages by "felinizing" a wide variety of animal cell cultures. Here, we reviewed intestinal in vitro and ex vivo models and discussed their strengths and limitations in the context of a quest for faithful models to in vitro emulate the biology of the enteric stages of T. gondii.

Keywords:  Toxoplasma gondii; ex vivo models; felinization; in vitro models; intestine; sexual differentiation

DOI:  https://doi.org/10.3389/fcimb.2023.1134471
Infect Dis Poverty. 2023 Jun 15. 12(1): 60

A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats.

Yu Shen, Bin Zheng, Hao Sun, Songrui Wu, Jiyuan Fan, Jianzu Ding, Meng Gao, Qingming Kong, Di Lou, Haojie Ding, Xunhui Zhuo, Shaohong Lu.

   BACKGROUND: Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondii vaccine for the control of toxoplasmosis, and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats.
METHODS: First, the ompdc and uprt genes of T. gondii were deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and subsets of T lymphocytes. Finally, the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out. GraphPad Prism software was used to conduct the log-rank (Mantel-Cox) test, Student's t test and one-way ANOVA.
RESULTS: The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system. Compared with the wild-type strain, the mutant notably reduced proliferation (P < 0.05). In addition, the mutant exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. Notably, limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, compared with nonimmunized group, high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) in mice were detected by the mutant (P < 0.05). Remarkably, all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains. The immunized sera and splenocytes, especially CD8+ T cells, could significantly extend (P < 0.05) the survival time of mice challenged with the RHΔku80 strain compared with naïve mice. In addition, compared with nonimmunized cats, cats immunized with the mutant produced high levels of antibodies and cytokines (P < 0.05), and notably decreased the shedding numbers of oocysts in feces (95.3%).
CONCLUSIONS: The avirulent RHΔompdcΔuprt strain can provide strong anti-T. gondii immune responses, and is a promising candidate for developing a safe and effective live attenuated vaccine.

Keywords:  Cat; Immunization; Live attenuated vaccine; Mouse; Orotidine-5'-monophosphate decarboxylase; Phosphoribosyltransferase; Toxoplasma gondii

DOI:  https://doi.org/10.1186/s40249-023-01109-9
Vet Parasitol. 2023 Jun 02. pii: S0304-4017(23)00100-0. [Epub ahead of print]320 109969

Evaluation of the protective immune response of an attenuated strain of Toxoplasma gondii with long-term passages on the Gecko cell line.

Roghayeh Ramezanpoor Ronizi, Mehdi Namavari, Elham Moazamian.

  Toxoplasma gondii is an obligate intracellular parasite that causes the zoonoses disease, named toxoplasmosis, with global prevalence. Until now, no cost-effective treatment method has been found to deal with toxoplasma, and vaccination is the best way to deal with the infection. In the case of pathogenic protozoa, mainly live vaccines have had successful results compared to other vaccine platforms. This study evaluated the efficacy of a live experimental vaccine through long-term passages on the Gecko cell line (Z1) in inducing a protective immune response in BALB/c mice. Thirty mice were divided into three equal groups; G1: the immunized/challenged group (injection of attenuated strain), G2: the immunized/unchallenged group (injection of attenuated strain), and G3: the control group (injection of culture medium).One month after immunization, the studied mice were challenged with 1ₓ103 live tachyzoites of Toxoplasma acute RH strain. We performed Serological investigations, including evaluating antibodies, interferon-gamma (IFN-γ), and interleukins 2, 4, 10, and 12 (IL-2,4,10,12). At the study's end, a molecular test was performed on brain and liver tissues in the immunized groups to check the presence of parasites. The results from the serological tests for the evaluation of antibodies, interferon-gamma (IFN-γ), and interleukins 10 and 12 (IL-10, 12) show a significant difference (p < 0.05) between the vaccinated group and the control group, which are essential indicators of protective immunity against toxoplasma infection. Thus, in the vaccinated group, the survival rate of mice against the challenge was 70%. Also, in group two (G2), the attenuated strain of Toxoplasma gondii had no pathogenicity, and all mice survived until the end of the study period. Molecular results also showed the absence of parasites in the brain and liver tissues in this immunized group and the parasite was found in only one case of liver tissue in G1. Therefore, the attenuated strain has caused significant and protective humoral and cellular immune responses in vaccinated groups. This study showed that with the long-term passage of the acute strain on the Gecko cell line, it is possible to quickly obtain a non-diseased attenuated strain with the ability to induce protective immunity. This successful finding can introduce further research to achieve a promising vaccine in the target animals.

Keywords:  Attenuated strain; Gecko cell line; Immunogenicity; Toxoplasma gondii; Vaccine

DOI:  https://doi.org/10.1016/j.vetpar.2023.109969
Sensors (Basel). 2023 May 31. pii: 5218. [Epub ahead of print]23(11):

Microfluidic Biosensor Based on Molybdenum Disulfide (MoS2) Modified Thin-Core Microfiber for Immune Detection of Toxoplasma gondii.

Huiji Chen, Binbin Luo, Shengxi Wu, Shenghui Shi, Qin Dai, Zehua Peng, Mingfu Zhao.

  Toxoplasma gondii (T. gondii) is a zoonotic parasite that is widely distributed and seriously endangers public health and human health. Therefore, accurate and effective detection of T. gondii is crucial. This study proposes a microfluidic biosensor using a thin-core microfiber (TCMF) coated with molybdenum disulfide (MoS2) for immune detection of T. gondii. The single-mode fiber was fused with the thin-core fiber, and the TCMF was obtained by arc discharging and flame heating. In order to avoid interference and protect the sensing structure, the TCMF was encapsulated in the microfluidic chip. MoS2 and T. gondii antigen were modified on the surface of TCMF for the immune detection of T. gondii. Experimental results showed that the detection range of the proposed biosensor for T. gondii monoclonal antibody solutions was 1 pg/mL to 10 ng/mL with sensitivity of 3.358 nm/log(mg/mL); the detection of limit was calculated to be 87 fg/mL through the Langmuir model; the dissociation constant and the affinity constant were calculated to be about 5.79 × 10-13 M and 1.727 × 1014 M-1, respectively. The specificity and clinical characteristics of the biosensor was explored. The rabies virus, pseudorabies virus, and T. gondii serum were used to confirm the excellent specificity and clinical characteristics of the biosensor, indicating that the proposed biosensor has great application potential in the biomedical field.

Keywords:  microfluidic chip; molybdenum disulfide; optical fiber biosensor; thin-core microfiber; toxoplasma gondii

DOI:  https://doi.org/10.3390/s23115218
Sci Adv. 2023 Jun 16. 9(24): eadf2161

A Plasmodium membrane receptor platform integrates cues for egress and invasion in blood forms and activation of transmission stages.

Ronja Marie Kuehnel, Emma Ganga, Aurélia C Balestra, Catherine Suarez, Matthias Wyss, Natacha Klages, Lorenzo Brusini, Bohumil Maco, Nicolas Brancucci, Till S Voss, Dominique Soldati, Mathieu Brochet.

Critical events in the life cycle of malaria-causing parasites depend on cyclic guanosine monophosphate homeostasis by guanylyl cyclases (GCs) and phosphodiesterases, including merozoite egress or invasion of erythrocytes and gametocyte activation. These processes rely on a single GCα, but in the absence of known signaling receptors, how this pathway integrates distinct triggers is unknown. We show that temperature-dependent epistatic interactions between phosphodiesterases counterbalance GCα basal activity preventing gametocyte activation before mosquito blood feed. GCα interacts with two multipass membrane cofactors in schizonts and gametocytes: UGO (unique GC organizer) and SLF (signaling linking factor). While SLF regulates GCα basal activity, UGO is essential for GCα up-regulation in response to natural signals inducing merozoite egress and gametocyte activation. This work identifies a GC membrane receptor platform that senses signals triggering processes specific to an intracellular parasitic lifestyle, including host cell egress and invasion to ensure intraerythrocytic amplification and transmission to mosquitoes.

DOI: https://doi.org/10.1126/sciadv.adf2161
Mol Microbiol. 2023 Jun 14.

Plasmodium GPI-anchored micronemal antigen is essential for parasite transmission through the mosquito host.

Charlie Jennison, Janna M Armstrong, Dorender A Dankwa, Nina Hertoghs, Sudhir Kumar, Biley A Abatiyow, Myo Naung, Nana K Minkah, Kristian E Swearingen, Robert Moritz, Alyssa E Barry, Stefan H I Kappe, Ashley M Vaughan.

  Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.

Keywords:   Plasmodium berghei ; malaria; microneme; mosquito stage; sporozoite

DOI:  https://doi.org/10.1111/mmi.15078
J Photochem Photobiol B. 2023 Jun 05. pii: S1011-1344(23)00089-1. [Epub ahead of print]245 112735

The AMPK-dependent inhibition of autophagy plays a crucial role in protecting photoreceptor from photooxidative injury.

Yu-Lin Li, Tian-Zi Zhang, Li-Kun Han, Chang He, Yi-Ran Pan, Bin Fan, Guang-Yu Li.

  Excessive light exposure can potentially cause irreversible damage to the various photoreceptor cells, and this aspect has been considered as an important factor leading to the progression of the different retinal diseases. AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) are crucial intracellular signaling hubs involved in the regulation of cellular metabolism, energy homeostasis, cellular growth and autophagy. A number of previous studies have indicated that either AMPK activation or mTOR inhibition can promote autophagy in most cases. In the current study, we have established an in vitro as well as in vivo photooxidation-damaged photoreceptor model and investigated the possible influence of visible light exposure in the AMPK/mTOR/autophagy signaling pathway. We have also explored the potential regulatory effects of AMPK/mTOR on light-induced autophagy and protection achieved by suppressing autophagy in photooxidation-damaged photoreceptors. We observed that light exposure led to a significant activation of mTOR and autophagy in the photoreceptor cells. However, intriguingly, AMPK activation or mTOR inhibition significantly inhibited rather than promoting autophagy, which was termed as AMPK-dependent inhibition of autophagy. In addition, either indirectly suppressing autophagy by AMPK activation/ mTOR inhibition or directly blocking autophagy with an inhibitor exerted a significant protective effect on the photoreceptor cells against the photooxidative damage. Neuroprotective effects caused by the AMPK-dependent inhibition of autophagy were also verified with a retinal light injured mouse model in vivo. Overall, our findings demonstrated that AMPK / mTOR pathway could inhibit autophagy through AMPK-dependent inhibition of autophagy to significantly protect the photoreceptors from photooxidative injury, which may aid to further develop novel targeted retinal neuroprotective drugs.

Keywords:  AMPK; Autophagy; Photooxidation; Photoreceptor; Retinal light injury; Retinal neuroprotection

DOI:  https://doi.org/10.1016/j.jphotobiol.2023.112735