scholarly journals The apical annuli of Toxoplasma gondii are composed of coiled-coil and signaling proteins embedded in the IMC sutures

2019 ◽  
Author(s):  
Klemens Engelberg ◽  
Chun-Ti Chen ◽  
Tyler Bechtel ◽  
Victoria Sánchez Guzmán ◽  
Allison A. Drozda ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Coiled Coil ◽  
Super Resolution ◽  
Building Blocks ◽  
Signaling Proteins ◽  
Waste Products ◽  
Membrane Complex ◽  
Inner Membrane Complex

AbstractThe apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2 and a methyltransferase (AAMT). Moreover, Inner Membrane Complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy (SR-SIM) identified a concentric organization comprising four rings with diameters ranging from 200-400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggest that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed the AAPs are conserved narrowly in Coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signaling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signaling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.

scholarly journals Requirement of Toxoplasma gondii metacaspases for IMC1 maturation, endodyogeny and virulence in mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Muzi Li ◽  
Jing Liu ◽  
Yayun Wu ◽  
Yihan Wu ◽  
Xiaodong Sun ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Normal Growth ◽  
Double Knockout ◽  
Protein Aggregate ◽  
Knockout Strain ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Single Knockout

Abstract Background Metacaspases are multifunctional proteins found in plants, fungi and protozoa, and are involved in processes such as insoluble protein aggregate clearance and cell proliferation. Our previous study demonstrated that metacaspase-1 (MCA1) contributes to parasite apoptosis in Toxoplasma gondii. Deletion of MCA1 from T. gondii has no effect on the growth and virulence of the parasites. Three metacaspases were identified in the ToxoDB Toxoplasma Informatics Resource, and the function of metacaspase-2 (MCA2) and metacaspase-3 (MCA3) has not been demonstrated. Methods In this study, we constructed MCA1, MCA2 and MCA1/MCA2 transgenic strains from RHΔku80 (Δku80), including overexpressing strains and knockout strains, to clarify the function of MCA1 and MCA2 of T. gondii. Results MCA1 and MCA2 were distributed in the cytoplasm with punctuated aggregation, and part of the punctuated aggregation of MCA1 and MCA2 was localized on the inner membrane complex of T. gondii. The proliferation of the MCA1/MCA2 double-knockout strain was significantly reduced; however, the two single knockout strains (MCA1 knockout strain and MCA2 knockout strain) exhibited normal growth rates as compared to the parental strain, Δku80. In addition, endodyogeny was impaired in the tachyzoites whose MCA1 and MCA2 were both deleted due to multiple nuclei and abnormal expression of IMC1. We further found that IMC1 of the double-knockout strain was detergent-soluble, indicating that MCA1 and MCA2 are associated with IMC1 maturation. Compared to the parental Δku80 strain, the double-knockout strain was more readily induced from tachyzoites to bradyzoites in vitro. Furthermore, the double-knockout strain was less pathogenic in mice and was able to develop bradyzoites in the brain, which formed cysts and established chronic infection. Conclusion MCA1 and MCA2 are important factors which participate in IMC1 maturation and endodyogeny of T. gondii. The double-knockout strain has slower proliferation and was able to develop bradyzoites both in vitro and in vivo. Graphic abstract

scholarly journals Two alveolin network proteins are essential for the subpellicular microtubules assembly and conoid anchoring to the apical pole of mature Toxoplasma gondii

2020 ◽  
Author(s):  
Nicolò Tosetti ◽  
Nicolas Dos Santos Pacheco ◽  
Eloïse Bertiaux ◽  
Bohumil Maco ◽  
Lorène Bournonville ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Microtubule Organizing Center ◽  
Membrane Complex ◽  
Catastrophic Loss ◽  
Organizing Center ◽  
Apical Complex ◽  
Critical Components ◽  
Inner Membrane Complex

AbstractToxoplasma gondii belongs to the coccidian sub-group of Apicomplexa that possess an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic and dynamic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR), a microtubule-organizing center for the 22 subpellicular microtubules (SPMTs). The SPMTs are linked to the Inner Membrane Complex (IMC), a patchwork of flattened vesicles, via an intricate network of small filaments composed of alveolins proteins. Here, we capitalize on super-resolution techniques including stimulated emission depletion (STED) microscopy and ultrastructure expansion microscopy (U-ExM) to localize the Apical Cap protein 9 (AC9) and its close partner AC10, identified by BioID, to the alveolin network and intercalated between the SPMTs. Conditional depletion of AC9 or AC10 using the Auxin-induced Degron (AiD) system uncovered a severe loss of fitness. Parasites lacking AC9 or AC10 replicate normally but are defective in microneme secretion and hence fail to invade and egress from infected cells. Remarkably, a series of crucial apical complex proteins (MyoH, AKMT, FRM1, CPH1, ICMAP1 and RNG2) are lost in the mature parasites although they are still present in the forming daughter cells. Electron microscopy on intracellular or deoxycholate-extracted parasites revealed that the mature parasite mutants are conoidless. Closer examination of the SPMTs by U-ExM highlighted the disassembly of the SPMTs in the apical cap region that is presumably at the origin of the catastrophic loss of APR and conoid. AC9 and AC10 are two critical components of the alveolin network that ensure the integrity of the whole apical complex in T. gondii and likely other coccidians.

scholarly journals A De novo Peptide from a High Throughput Peptide Library Blocks Myosin A -MTIP Complex Formation in Plasmodium falciparum

2020 ◽  
Vol 21 (17) ◽  
pp. 6158
Author(s):  
Zill e Anam ◽  
Nishant Joshi ◽  
Sakshi Gupta ◽  
Preeti Yadav ◽  
Ayushi Chaurasiya ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Peptide Library ◽  
Tail Domain ◽  
Interacting Protein ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
De Novo Peptide

Apicomplexan parasites, through their motor machinery, produce the required propulsive force critical for host cell-entry. The conserved components of this so-called glideosome machinery are myosin A and myosin A Tail Interacting Protein (MTIP). MTIP tethers myosin A to the inner membrane complex of the parasite through 20 amino acid-long C-terminal end of myosin A that makes direct contacts with MTIP, allowing the invasion of Plasmodium falciparum in erythrocytes. Here, we discovered through screening a peptide library, a de-novo peptide ZA1 that binds the myosin A tail domain. We demonstrated that ZA1 bound strongly to myosin A tail and was able to disrupt the native myosin A tail MTIP complex both in vitro and in vivo. We then showed that a shortened peptide derived from ZA1, named ZA1S, was able to bind myosin A and block parasite invasion. Overall, our study identified a novel anti-malarial peptide that could be used in combination with other antimalarials for blocking the invasion of Plasmodium falciparum.

scholarly journals Novel Approaches Reveal that Toxoplasma gondii Bradyzoites within Tissue Cysts Are Dynamic and Replicating Entities In Vivo

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Elizabeth Watts ◽  
Yihua Zhao ◽  
Animesh Dhara ◽  
Becca Eller ◽  
Abhijit Patwardhan ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Packing Density ◽  
Critical Role ◽  
Chronic Phase ◽  
Life Cycle Stage ◽  
Content Type ◽  
Symptomatic Disease ◽  
Membrane Complex ◽  
Inner Membrane Complex

ABSTRACTDespite their critical role in chronic toxoplasmosis, the biology ofToxoplasma gondiibradyzoites is poorly understood. In an attempt to address this gap, we optimized approaches to purify tissue cysts and analyzed the replicative potential of bradyzoites within these cysts. In order to quantify individual bradyzoites within tissue cysts, we have developed imaging software, BradyCount 1.0, that allows the rapid establishment of bradyzoite burdens within imaged optical sections of purified tissue cysts. While in general larger tissue cysts contain more bradyzoites, their relative “occupancy” was typically lower than that of smaller cysts, resulting in a lower packing density. The packing density permits a direct measure of how bradyzoites develop within cysts, allowing for comparisons across progression of the chronic phase. In order to capture bradyzoite endodyogeny, we exploited the differential intensity of TgIMC3, an inner membrane complex protein that intensely labels newly formed/forming daughters within bradyzoites and decays over time in the absence of further division. To our surprise, we were able to capture not only sporadic and asynchronous division but also synchronous replication of all bradyzoites within mature tissue cysts. Furthermore, the time-dependent decay of TgIMC3 intensity was exploited to gain insights into the temporal patterns of bradyzoite replicationin vivo. Despite the fact that bradyzoites are considered replicatively dormant, we find evidence for cyclical, episodic bradyzoite growth within tissue cystsin vivo. These findings directly challenge the prevailing notion of bradyzoites as dormant nonreplicative entities in chronic toxoplasmosis and have implications on our understanding of this enigmatic and clinically important life cycle stage.IMPORTANCEThe protozoanToxoplasma gondiiestablishes a lifelong chronic infection mediated by the bradyzoite form of the parasite within tissue cysts. Technical challenges have limited even the most basic studies on bradyzoites and the tissue cystsin vivo. Bradyzoites, which are viewed as dormant, poorly replicating or nonreplicating entities, were found to be surprisingly active, exhibiting not only the capacity for growth but also previously unrecognized patterns of replication that point to their being considerably more dynamic than previously imagined. These newly revealed properties force us to reexamine the most basic questions regarding bradyzoite biology and the progression of the chronic phase of toxoplasmosis. By developing new tools and approaches to study the chronic phase at the level of bradyzoites, we expose new avenues to tackle both drug development and a better understanding of events that may lead to reactivated symptomatic disease.

scholarly journals The apical annuli of Toxoplasma gondii are composed of coiled‐coil and signalling proteins embedded in the inner membrane complex sutures

2019 ◽  
Vol 22 (1) ◽  
Author(s):  
Klemens Engelberg ◽  
Chun‐Ti Chen ◽  
Tyler Bechtel ◽  
Victoria Sánchez Guzmán ◽  
Allison A. Drozda ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Coiled Coil ◽  
Inner Membrane ◽  
Membrane Complex ◽  
Inner Membrane Complex

scholarly journals Identification and Molecular Dissection of IMC32, a Conserved Toxoplasma Inner Membrane Complex Protein That Is Essential for Parasite Replication

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Juan A. Torres ◽  
Rebecca R. Pasquarelli ◽  
Peter S. Back ◽  
Andy S. Moon ◽  
Peter J. Bradley
Keyword(s):  
Toxoplasma Gondii ◽  
Coiled Coil ◽  
Inner Membrane ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Conserved Regions ◽  
Membrane Complex ◽  
Parasite Survival ◽  
Parasite Replication ◽  
Inner Membrane Complex

ABSTRACT The inner membrane complex (IMC) is a unique organelle of apicomplexan parasites that plays critical roles in parasite motility, host cell invasion, and replication. Despite the common functions of the organelle, relatively few IMC proteins are conserved across the phylum and the precise roles of many IMC components remain to be characterized. Here, we identify a novel component of the Toxoplasma gondii IMC (IMC32) that localizes to the body portion of the IMC and is recruited to developing daughter buds early during endodyogeny. IMC32 is essential for parasite survival, as its conditional depletion results in a complete collapse of the IMC that is lethal to the parasite. We demonstrate that localization of IMC32 is dependent on both an N-terminal palmitoylation site and a series of C-terminal coiled-coil domains. Using deletion analyses and functional complementation, we show that two conserved regions within the C-terminal coiled-coil domains play critical roles in protein function during replication. Together, this work reveals an essential component of parasite replication that provides a novel target for therapeutic intervention of T. gondii and related apicomplexan parasites. IMPORTANCE The IMC is an important organelle that apicomplexan parasites use to maintain their intracellular lifestyle. While many IMC proteins have been identified, only a few central players that are essential for internal budding have been described and even fewer are conserved across the phylum. Here, we identify IMC32, a novel component of the Toxoplasma gondii IMC that localizes to very early daughter buds, indicating a role in the early stages of parasite replication. We then demonstrate that IMC32 is essential for parasite survival and pinpoint conserved regions within the protein that are important for membrane association and daughter cell formation. As IMC32 is unique to these parasites and not present in their mammalian hosts, it serves as a new target for the development of drugs that exclusively affect these important intracellular pathogens.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

scholarly journals A molecular architectural design that promises potent antimicrobial activity against multidrug-resistant pathogens

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Bing Yuan ◽  
Jiaojiao Liu ◽  
Zhixiong Deng ◽  
Lin Wei ◽  
Wenwen Li ◽  
...  
Keyword(s):  
Architectural Design ◽  
Building Blocks ◽  
Multidrug Resistant ◽  
In Vitro Cytotoxicity ◽  
Antimicrobial Drugs ◽  
Minimal Inhibitory Concentrations ◽  
Antimicrobial Efficiency ◽  
Multidrug Resistant Pathogens

AbstractAddressing the devastating threat of drug-resistant pathogens requires the discovery of new antibiotics with advanced action mechanisms and/or novel strategies for drug design. Herein, from a biophysical perspective, we design a class of synthetic antibacterial complexes with specialized architectures based on melittin (Mel), a natural antimicrobial peptide, and poly(ethylene glycol) (PEG), a clinically available agent, as building blocks that show potent and architecture-modulated antibacterial activity. Among the complexes, the flexibly linear complex consisting of one Mel terminally connected with a long-chained PEG (e.g., PEG12k–1*Mel) shows the most pronounced improvement in performance compared with pristine Mel, with up to 500% improvement in antimicrobial efficiency, excellent in vitro activity against multidrug-resistant pathogens (over a range of minimal inhibitory concentrations of 2–32 µg mL−1), a 68% decrease in in vitro cytotoxicity, and a 57% decrease in in vivo acute toxicity. A lipid-specific mode of action in membrane recognition and an accelerated “channel” effect in perforating the bacterial membrane of the complex are described. Our results introduce a new way to design highly efficient and low-toxicity antimicrobial drugs based on architectural modulations with clinically available agents.

scholarly journals Synthesis, Characterization and Evaluation of Peptide Nanostructures for Biomedical Applications

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4587
Author(s):  
Fanny d’Orlyé ◽  
Laura Trapiella-Alfonso ◽  
Camille Lescot ◽  
Marie Pinvidic ◽  
Bich-Thuy Doan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biomedical Applications ◽  
Chemical Reactivity ◽  
Physical Stability ◽  
Chemical Properties ◽  
Building Blocks ◽  
Imaging Probes ◽  
Therapeutic Molecules

There is a challenging need for the development of new alternative nanostructures that can allow the coupling and/or encapsulation of therapeutic/diagnostic molecules while reducing their toxicity and improving their circulation and in-vivo targeting. Among the new materials using natural building blocks, peptides have attracted significant interest because of their simple structure, relative chemical and physical stability, diversity of sequences and forms, their easy functionalization with (bio)molecules and the possibility of synthesizing them in large quantities. A number of them have the ability to self-assemble into nanotubes, -spheres, -vesicles or -rods under mild conditions, which opens up new applications in biology and nanomedicine due to their intrinsic biocompatibility and biodegradability as well as their surface chemical reactivity via amino- and carboxyl groups. In order to obtain nanostructures suitable for biomedical applications, the structure, size, shape and surface chemistry of these nanoplatforms must be optimized. These properties depend directly on the nature and sequence of the amino acids that constitute them. It is therefore essential to control the order in which the amino acids are introduced during the synthesis of short peptide chains and to evaluate their in-vitro and in-vivo physico-chemical properties before testing them for biomedical applications. This review therefore focuses on the synthesis, functionalization and characterization of peptide sequences that can self-assemble to form nanostructures. The synthesis in batch or with new continuous flow and microflow techniques will be described and compared in terms of amino acids sequence, purification processes, functionalization or encapsulation of targeting ligands, imaging probes as well as therapeutic molecules. Their chemical and biological characterization will be presented to evaluate their purity, toxicity, biocompatibility and biodistribution, and some therapeutic properties in vitro and in vivo. Finally, their main applications in the biomedical field will be presented so as to highlight their importance and advantages over classical nanostructures.

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