Selective permeabilization of infected host cells with pore-forming proteins provides a novel tool to study protein synthesis and viability of the intracellular apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii

2001 ◽  
Vol 112 (1) ◽  
pp. 133-137 ◽  
Author(s):  
Stefan Baumeister ◽  
Kerstin Paprotka ◽  
Sucharit Bhakdi ◽  
Klaus Lingelbach
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Synthesis ◽  
Toxoplasma Gondii ◽  
Host Cells ◽  
Infected Host ◽  
Apicomplexan Parasites ◽  
Pore Forming Proteins

scholarly journals Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii

2004 ◽  
Vol 165 (3) ◽  
pp. 383-393 ◽  
Author(s):  
Elizabeth Gaskins ◽  
Stacey Gilk ◽  
Nicolette DeVore ◽  
Tara Mann ◽  
Gary Ward ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Complex ◽  
Integral Membrane Protein ◽  
Gliding Motility ◽  
Host Cells ◽  
Inner Membrane ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Two Stages

Apicomplexan parasites exhibit a unique form of substrate-dependent motility, gliding motility, which is essential during their invasion of host cells and during their spread between host cells. This process is dependent on actin filaments and myosin that are both located between the plasma membrane and two underlying membranes of the inner membrane complex. We have identified a protein complex in the apicomplexan parasite Toxoplasma gondii that contains the class XIV myosin required for gliding motility, TgMyoA, its associated light chain, TgMLC1, and two novel proteins, TgGAP45 and TgGAP50. We have localized this complex to the inner membrane complex of Toxoplasma, where it is anchored in the membrane by TgGAP50, an integral membrane glycoprotein. Assembly of the protein complex is spatially controlled and occurs in two stages. These results provide the first molecular description of an integral membrane protein as a specific receptor for a myosin motor, and further our understanding of the motile apparatus underlying gliding motility in apicomplexan parasites.

scholarly journals A Toxoplasma gondii Ortholog of Plasmodium GAMA Contributes to Parasite Attachment and Cell Invasion

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
My-Hang Huynh ◽  
Vern B. Carruthers
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Genetic Evidence ◽  
Specific Role ◽  
Host Cells ◽  
Human Pathogen ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Adhesive Proteins

ABSTRACT Toxoplasma gondii is a successful human pathogen in the same phylum as malaria-causing Plasmodium parasites. Invasion of a host cell is an essential process that begins with secretion of adhesive proteins onto the parasite surface for attachment and subsequent penetration of the host cell. Conserved invasion proteins likely play roles that were maintained through the divergence of these parasites. Here, we identify a new conserved invasion protein called glycosylphosphatidylinositol-anchored micronemal antigen (GAMA). Tachyzoites lacking TgGAMA were partially impaired in parasite attachment and invasion of host cells, yielding the first genetic evidence of a specific role in parasite entry into host cells. These findings widen our appreciation of the repertoire of conserved proteins that apicomplexan parasites employ for cell invasion. Toxoplasma gondii and its Plasmodium kin share a well-conserved invasion process, including sequential secretion of adhesive molecules for host cell attachment and invasion. However, only a few orthologs have been shown to be important for efficient invasion by both genera. Bioinformatic screening to uncover potential new players in invasion identified a previously unrecognized T. gondii ortholog of Plasmodium glycosylphosphatidylinositol-anchored micronemal antigen (TgGAMA). We show that TgGAMA localizes to the micronemes and is processed into several proteolytic products within the parasite prior to secretion onto the parasite surface during invasion. TgGAMA from parasite lysate bound to several different host cell types in vitro, suggesting a role in parasite attachment. Consistent with this function, tetracycline-regulatable TgGAMA and TgGAMA knockout strains showed significant reductions in host cell invasion at the attachment step, with no defects in any of the other stages of the parasite lytic cycle. Together, the results of this work reveal a new conserved component of the adhesive repertoire of apicomplexan parasites. IMPORTANCE Toxoplasma gondii is a successful human pathogen in the same phylum as malaria-causing Plasmodium parasites. Invasion of a host cell is an essential process that begins with secretion of adhesive proteins onto the parasite surface for attachment and subsequent penetration of the host cell. Conserved invasion proteins likely play roles that were maintained through the divergence of these parasites. Here, we identify a new conserved invasion protein called glycosylphosphatidylinositol-anchored micronemal antigen (GAMA). Tachyzoites lacking TgGAMA were partially impaired in parasite attachment and invasion of host cells, yielding the first genetic evidence of a specific role in parasite entry into host cells. These findings widen our appreciation of the repertoire of conserved proteins that apicomplexan parasites employ for cell invasion.

scholarly journals Serum Response Factor Regulates Immediate Early Host Gene Expression in Toxoplasma gondii-Infected Host Cells

PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e18335 ◽  
Author(s):  
Mandi Wiley ◽  
Crystal Teygong ◽  
Eric Phelps ◽  
Jay Radke ◽  
Ira J. Blader
Keyword(s):  
Gene Expression ◽  
Toxoplasma Gondii ◽  
Serum Response Factor ◽  
Host Gene ◽  
Host Cells ◽  
Immediate Early ◽  
Infected Host ◽  
Response Factor ◽  
Host Gene Expression ◽  
Serum Response

scholarly journals Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Shruthi Krishnamurthy ◽  
Bin Deng ◽  
Roxana del Rio ◽  
Kerry R. Buchholz ◽  
Moritz Treeck ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Host Cells ◽  
Receptor Protein ◽  
Host Cell Invasion ◽  
Cell Binding ◽  
Apicomplexan Parasites

ABSTRACT Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T . gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion. IMPORTANCE Nearly one-third of the world’s population is infected with the protozoan parasite Toxoplasma gondii , which causes life-threatening disease in neonates and immunocompromised individuals. T. gondii is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.

scholarly journals Structural role of essential light chains in the apicomplexan glideosome

2019 ◽  
Author(s):  
Samuel Pazicky ◽  
Karthikeyan Dhamotharan ◽  
Karol Kaszuba ◽  
Haydyn Mertens ◽  
Tim Gilberger ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Light Chain ◽  
Structural Information ◽  
Host Cells ◽  
Light Chains ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
C Terminus ◽  
Inner Membrane Complex

AbstractApicomplexan parasites, such as Plasmodium falciparum and Toxoplasma gondii, traverse the host tissues and invade the host cells exhibiting a specific type of motility called gliding. The molecular mechanism of gliding lies in the actin-myosin motor localized to the intermembrane space between the plasma membrane and inner membrane complex (IMC) of the parasites. Myosin A (MyoA) is a part of the glideosome, a large multi-protein complex, which is anchored in the outer membrane of the IMC. MyoA is bound to the proximal essential light chain (ELC) and distal myosin light chain (MLC1), which further interact with the glideosome associated proteins GAP40, GAP45 and GAP50. Whereas structures of several individual glideosome components and small dimeric complexes have been solved, structural information concerning the interaction of larger glideosome subunits and their role in glideosome function still remains to be elucidated. Here, we present structures of a T. gondii trimeric glideosome sub complex composed of a myosin A light chain domain with bound MLC1 and TgELC1 or TgELC2. Regardless of the differences between the secondary structure content observed for free P. falciparum PfELC and T. gondii TgELC1 or TgELC2, the proteins interact with a conserved region of TgMyoA to form structurally conserved complexes. Upon interaction, the essential light chains undergo contraction and induce α-helical structure in the myosin A C-terminus, stiffening the myosin lever arm. The complex formation is further stabilized through binding of a single calcium ion to T. gondii ELCs. Our work provides an important step towards the structural understanding of the entire glideosome and uncovering the role of its members in parasite motility and invasion.Author summaryApicomplexans, such as Toxoplasma gondii or the malaria agent Plasmodium falciparum, are small unicellular parasites that cause serious diseases in humans and other animals. These parasites move and infect the host cells by a unique type of motility called gliding. Gliding is empowered by an actin-myosin molecular motor located at the periphery of the parasites. Myosin interacts with additional proteins such as essential light chains to form the glideosome, a large protein assembly that anchors myosin in the inner membrane complex. Unfortunately, our understanding of the glideosome is insufficient because we lack the necessary structural information. Here we describe the first structures of trimeric glideosome sub complexes of T. gondii myosin A bound to two different light chain combinations, which show that T. gondii and P. falciparum form structurally conserved complexes. With an additional calcium-free complex structure, we demonstrate that calcium binding does not change the formation of the complexes, although it provides them with substantial stability. With additional data, we propose that the role of the essential light chains is to enhance myosin performance by inducing secondary structure in the C-terminus of myosin A. Our work represents an important step in unveiling the gliding mechanism of apicomplexan parasites.

scholarly journals Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure in Toxoplasma gondii

2020 ◽  
Author(s):  
William J. O’Shaughnessy ◽  
Xiaoyu Hu ◽  
Tsebaot Beraki ◽  
Matthew McDougal ◽  
Michael L. Reese
Keyword(s):  
Toxoplasma Gondii ◽  
Primary Cilia ◽  
Host Cells ◽  
Catastrophic Failure ◽  
Severe Phenotype ◽  
New Host ◽  
Apicomplexan Parasites ◽  
Cellular Processes

AbstractPrimary cilia are important organizing centers that control diverse cellular processes. Apicomplexan parasites like Toxoplasma gondii have a specialized cilium-like structure called the conoid that organizes the secretory and invasion machinery critical for the parasites’ lifestyle. The proteins that initiate the biogenesis of this structure are largely unknown. We identified the Toxoplasma ortholog of the conserved kinase ERK7 as essential to conoid assembly. Parasites in which ERK7 has been depleted lose their conoids late during maturation and are immotile and thus unable to invade new host cells. This is the most severe phenotype to conoid biogenesis yet reported, and is made more striking by the fact that ERK7 is not a conoid protein, as it localizes just basal to the structure. ERK7 has been recently implicated in ciliogenesis in metazoan cells, and our data suggest that this kinase has an ancient and central role in regulating ciliogenesis throughout Eukaryota.

scholarly journals Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

2021 ◽  
Vol 23 (1) ◽  
pp. 68
Author(s):  
Izra Abbaali ◽  
Danny A. Truong ◽  
Shania D. Day ◽  
Nancy Haro-Ramirez ◽  
Naomi S. Morrissette
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cells ◽  
Proof Of Concept ◽  
Systematic Analysis ◽  
Apicomplexan Parasites ◽  
Microtubule Disruption ◽  
Antiparasitic Drugs ◽  
General Response ◽  
Babesia Spp

Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.

scholarly journals Rhoptry and Dense Granule Secreted Effectors Regulate CD8+ T Cell Recognition of Toxoplasma gondii Infected Host Cells

2019 ◽  
Vol 10 ◽  
Author(s):  
Leah M. Rommereim ◽  
Barbara A. Fox ◽  
Kiah L. Butler ◽  
Viviana Cantillana ◽  
Gregory A. Taylor ◽  
...  
Keyword(s):  
T Cell ◽  
Toxoplasma Gondii ◽  
Cd8 T Cell ◽  
Dense Granule ◽  
Host Cells ◽  
Infected Host ◽  
Cell Recognition ◽  
T Cell Recognition

scholarly journals C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence

2019 ◽  
Vol 295 (4) ◽  
pp. 1066-1076 ◽  
Author(s):  
Andreia Albuquerque-Wendt ◽  
Damien Jacot ◽  
Nicolas Dos Santos Pacheco ◽  
Carla Seegers ◽  
Patricia Zarnovican ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protective Immunity ◽  
Host Cells ◽  
Apicomplexan Parasites ◽  
Tryptophan Residues ◽  
Parasite Survival ◽  
Parasite Motility ◽  
Parasite Egress ◽  
Thrombospondin Type 1 Repeats

C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase–deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

scholarly journals Plasma Membrane Association by N-Acylation Governs PKG Function in Toxoplasma gondii

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Kevin M. Brown ◽  
Shaojun Long ◽  
L. David Sibley
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Toxoplasma Gondii ◽  
Cyclic Gmp ◽  
Life Cycles ◽  
Host Cells ◽  
Protein Kinase G ◽  
Apicomplexan Parasites ◽  
Essential Proteins ◽  
Functional Differences

ABSTRACT Cyclic GMP (cGMP)-dependent protein kinase (protein kinase G [PKG]) is essential for microneme secretion, motility, invasion, and egress in apicomplexan parasites, However, the separate roles of two isoforms of the kinase that are expressed by some apicomplexans remain uncertain. Despite having identical regulatory and catalytic domains, PKGI is plasma membrane associated whereas PKGII is cytosolic in Toxoplasma gondii. To determine whether these isoforms are functionally distinct or redundant, we developed an auxin-inducible degron (AID) tagging system for conditional protein depletion in T. gondii. By combining AID regulation with genome editing strategies, we determined that PKGI is necessary and fully sufficient for PKG-dependent cellular processes. Conversely, PKGII is functionally insufficient and dispensable in the presence of PKGI. The difference in functionality mapped to the first 15 residues of PKGI, containing a myristoylated Gly residue at position 2 that is critical for membrane association and PKG function. Collectively, we have identified a novel requirement for cGMP signaling at the plasma membrane and developed a new system for examining essential proteins in T. gondii. IMPORTANCE Toxoplasma gondii is an obligate intracellular apicomplexan parasite and important clinical and veterinary pathogen that causes toxoplasmosis. Since apicomplexans can only propagate within host cells, efficient invasion is critically important for their life cycles. Previous studies using chemical genetics demonstrated that cyclic GMP signaling through protein kinase G (PKG)-controlled invasion by apicomplexan parasites. However, these studies did not resolve functional differences between two compartmentalized isoforms of the kinase. Here we developed a conditional protein regulation tool to interrogate PKG isoforms in T. gondii. We found that the cytosolic PKG isoform was largely insufficient and dispensable. In contrast, the plasma membrane-associated isoform was necessary and fully sufficient for PKG function. Our studies identify the plasma membrane as a key location for PKG activity and provide a broadly applicable system for examining essential proteins in T. gondii. Toxoplasma gondii is an obligate intracellular apicomplexan parasite and important clinical and veterinary pathogen that causes toxoplasmosis. Since apicomplexans can only propagate within host cells, efficient invasion is critically important for their life cycles. Previous studies using chemical genetics demonstrated that cyclic GMP signaling through protein kinase G (PKG)-controlled invasion by apicomplexan parasites. However, these studies did not resolve functional differences between two compartmentalized isoforms of the kinase. Here we developed a conditional protein regulation tool to interrogate PKG isoforms in T. gondii. We found that the cytosolic PKG isoform was largely insufficient and dispensable. In contrast, the plasma membrane-associated isoform was necessary and fully sufficient for PKG function. Our studies identify the plasma membrane as a key location for PKG activity and provide a broadly applicable system for examining essential proteins in T. gondii.

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