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

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.

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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 ◽

10.1128/mbio.00754-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 13

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.

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A Member of the Ferlin Calcium Sensor Family Is Essential for Toxoplasma gondii Rhoptry Secretion

mBio ◽

10.1128/mbio.01510-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 12

Author(s):

Bradley I. Coleman ◽

Sudeshna Saha ◽

Seiko Sato ◽

Klemens Engelberg ◽

David J. P. Ferguson ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Host Cells ◽

Model Organisms ◽

Host Cell Invasion ◽

Content Type ◽

Calcium Dependent ◽

Calcium Sensing ◽

Intracellular Ca

ABSTRACT Invasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility, and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+. Later stages of invasion are considered Ca2+ independent, including the secretion of proteins required for host cell entry and remodeling from the parasite’s rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms, such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. Here we focus on one ferlin that is conserved across the Apicomplexa, T. gondii FER2 (TgFER2). Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Knockdown of TgFER2 prevented rhoptry secretion, and these parasites failed to form the moving junction at the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate the requirement of TgFER2 for rhoptry secretion in Toxoplasma tachyzoites and suggest a possible Ca2+ dependence of rhoptry secretion. These findings provide the first mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion. IMPORTANCE Apicomplexan protozoan parasites, such as those causing malaria and toxoplasmosis, must invade the cells of their hosts in order to establish a pathogenic infection. Timely release of proteins from a series of apical organelles is required for invasion. Neither the vesicular fusion events that underlie secretion nor the observed reliance of the various processes on changes in intracellular calcium concentrations is completely understood. We identified a group of three proteins with strong homology to the calcium-sensing ferlin family, which are known to be involved in protein secretion in other organisms. Surprisingly, decreasing the amounts of one of these proteins (TgFER2) did not have any effect on the typically calcium-dependent steps in invasion. Instead, TgFER2 was essential for the release of proteins from organelles called rhoptries. These data provide a tantalizing first look at the mechanisms controlling the very poorly understood process of rhoptry secretion, which is essential for the parasite’s infection cycle.

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High Resolution Low Voltage Field Emission Scanning Electron Microscopy on De-Embedded Thick Sections Reveals Structural Interactions of the Surface of the Apicomplexan Parasite Toxoplasma Gondii with the Host Cell in the Parasitophorous Vacuole

Microscopy and Microanalysis ◽

10.1017/s143192760003573x ◽

2000 ◽

Vol 6 (S2) ◽

pp. 648-649

Author(s):

Heide Schatten ◽

David Sibley ◽

Hans Ris

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Biochemical Characterization ◽

Low Voltage ◽

Cytoskeletal Proteins ◽

Host Cells ◽

Farm Animals ◽

Host Cell Invasion ◽

Apicomplexan Parasites

The protozoan parasite Toxoplasma gondii represents a large group of Apicomplexan parasites with a highly unusual motility system that is crucial for cell locomotion and host cell invasion. Studying the motility system and interactions with host cells will increase our knowledge on how to prevent infection. Apicomplexan parasites can cause considerable health problems to animals including sheep, goats, pigs, and chicken. Eimeria is known as a pathogen of coccidiosis in chicken, and Cryptosporidium causes cryptospiridiosis in cattle and other farm animals. Toxoplasma gondii can cause infection that results in abortion, central nervous disorders and death of stressed and immunocompromised farm animals. Common to all Apicomplexan parasites is an unconventional motility system that is thought to be actively involved in host-cell invasion. However, the structural and biochemical characterization of Apicomplexan parasites has proven more difficult than previously thought because of genetic and behavioral differences of the parasites’ cytoskeletal proteins that differ from those in well studied mammalian systems.

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Functional Analysis of Rhomboid Proteases during Toxoplasma Invasion

mBio ◽

10.1128/mbio.01795-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 39

Author(s):

Bang Shen ◽

Jeffrey S. Buguliskis ◽

Tobie D. Lee ◽

L. David Sibley

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Gliding Motility ◽

Surface Proteins ◽

Host Cell Invasion ◽

Content Type ◽

Apicomplexan Parasites ◽

Transmembrane Segments ◽

Rhomboid Proteases

ABSTRACT Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii. IMPORTANCE Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T. gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.

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A member of the ferlin calcium sensor family is essential for Toxoplasma gondii rhoptry secretion

10.1101/304048 ◽

2018 ◽

Author(s):

Bradley I. Coleman ◽

Sudeshna Saha ◽

Seiko Sato ◽

Klemens Engelberg ◽

David J. P. Ferguson ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Membrane Skeleton ◽

Host Cells ◽

Model Organisms ◽

Host Cell Invasion ◽

Apicomplexan Parasites ◽

Intracellular Ca ◽

Related Proteins

AbstractInvasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+. Later stages of invasion are considered Ca2+-independent, including the secretion of proteins required for host cell entry and remodeling from the parasite’s rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. One ferlin that is conserved across the Apicomplexa, TgFER2, localizes to the parasite’s cortical membrane skeleton, apical end, and rhoptries. Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Specifically, knockdown of TgFER2 prevented rhoptry secretion and these parasites failed to form the moving junction on the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate that the putative Ca2+ sensor TgFER2 is required for the secretion of rhoptries. These findings provide the first regulatory and mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion.Graphical abstract

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1,3,4-Thiadiazoles Effectively Inhibit Proliferation of Toxoplasma gondii

Cells ◽

10.3390/cells10051053 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1053

Author(s):

Lidia Węglińska ◽

Adrian Bekier ◽

Katarzyna Dzitko ◽

Barbara Pacholczyk-Sienicka ◽

Łukasz Albrecht ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Direct Action ◽

Human Fibroblasts ◽

Imidazole Ring ◽

Host Cells ◽

In Vitro Assays ◽

Host Cell Invasion

Congenital and acquired toxoplasmosis caused by the food- and water-born parasite Toxoplasma gondii (T. gondii) is one of the most prevalent zoonotic infection of global importance. T. gondii is an obligate intracellular parasite with limited capacity for extracellular survival, thus a successful, efficient and robust host cell invasion process is crucial for its survival, proliferation and transmission. In this study, we screened a series of novel 1,3,4-thiadiazole-2-halophenylamines functionalized at the C5 position with the imidazole ring (1b–12b) for their effects on T. gondii host cell invasion and proliferation. To achieve this goal, these compounds were initially subjected to in vitro assays to assess their cytotoxicity on human fibroblasts and then antiparasitic efficacy. Results showed that all of them compare favorably to control drugs sulfadiazine and trimethoprim in terms of T. gondii growth inhibition (IC50) and selectivity toward the parasite, expressed as selectivity index (SI). Subsequently, the most potent of them with meta-fluoro 2b, meta-chloro 5b, meta-bromo 8b, meta-iodo 11b and para-iodo 12b substitution were tested for their efficacy in inhibition of tachyzoites invasion and subsequent proliferation by direct action on established intracellular infection. All the compounds significantly inhibited the parasite invasion and intracellular proliferation via direct action on both tachyzoites and parasitophorous vacuoles formation. The most effective was para-iodo derivative 12b that caused reduction in the percentage of infected host cells by 44% and number of tachyzoites per vacuole by 93% compared to non-treated host cells. Collectively, these studies indicate that 1,3,4-thiadiazoles 1b–12b, especially 12b with IC50 of 4.70 µg/mL and SI of 20.89, could be considered as early hit compounds for future design and synthesis of anti-Toxoplasma agents that effectively and selectively block the invasion and subsequent proliferation of T. gondii into host cells.

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Toxoplasma gondii Development of Its Replicative Niche: in Its Host Cell and Beyond

Eukaryotic Cell ◽

10.1128/ec.00081-14 ◽

2014 ◽

Vol 13 (8) ◽

pp. 965-976 ◽

Cited By ~ 32

Author(s):

Ira J. Blader ◽

Anita A. Koshy

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Host Cells ◽

Content Type ◽

Obligate Intracellular ◽

Cellular Processes ◽

High Prevalence ◽

Life Threatening ◽

Cell Processes ◽

Cellular Pathways

ABSTRACTIntracellular pathogens can replicate efficiently only after they manipulate and modify their host cells to create an environment conducive to replication. While diverse cellular pathways are targeted by different pathogens, metabolism, membrane and cytoskeletal architecture formation, and cell death are the three primary cellular processes that are modified by infections.Toxoplasma gondiiis an obligate intracellular protozoan that infects ∼30% of the world's population and causes severe and life-threatening disease in developing fetuses, in immune-comprised patients, and in certain otherwise healthy individuals who are primarily found in South America. The high prevalence ofToxoplasmain humans is in large part a result of its ability to modulate these three host cell processes. Here, we highlight recent work defining the mechanisms by whichToxoplasmainteracts with these processes. In addition, we hypothesize why some processes are modified not only in the infected host cell but also in neighboring uninfected cells.

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Two Phosphoglucomutase Paralogs Facilitate Ionophore-Triggered Secretion of the Toxoplasma Micronemes

mSphere ◽

10.1128/msphere.00521-17 ◽

2017 ◽

Vol 2 (6) ◽

Cited By ~ 7

Author(s):

Sudeshna Saha ◽

Bradley I. Coleman ◽

Rashmi Dubey ◽

Ira J. Blader ◽

Marc-Jan Gubbels

Keyword(s):

Toxoplasma Gondii ◽

Phosphatidic Acid ◽

Host Cell ◽

Lytic Cycle ◽

Ionophore A23187 ◽

Host Cell Invasion ◽

Content Type ◽

Apicomplexan Parasites ◽

Genetic Deletion

ABSTRACT Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice. Paralogs of the widely prevalent phosphoglucomutase (PGM) protein called parafusin function in calcium (Ca2+)-mediated exocytosis across eukaryotes. In Toxoplasma gondii, the parafusin-related protein 1 (PRP1) has been associated with Ca2+-dependent microneme organelle secretion required for essential processes like host cell invasion and egress. Using reverse genetics, we observed PRP1 to be dispensable for completion of the lytic cycle, including host cell invasion and egress by the parasite. However, the absence of the gene affected increased microneme release triggered by A23187, a Ca2+ ionophore used to raise the cytoplasmic Ca2+ concentration mimicking the physiological role of Ca2+ during invasion and egress. The basal levels of constitutive microneme release in extracellular parasites and phosphatidic acid-triggered microneme secretion were unaffected in the mutant. The phenotype of the deletion mutant of the second PGM-encoding gene in Toxoplasma, PGM2, was similar to the phenotype of the PRP1 deletion mutant. Furthermore, the ability of the tachyzoites to induce acute infection in the mice remained normal in the absence of both PGM paralogs. Our data thus reveal that the microneme secretion upon high Ca2+ flux is facilitated by the Toxoplasma PGM paralogs, PRP1 and PGM2. However, this protein-mediated release is neither essential for lytic cycle completion nor for acute virulence of the parasite. IMPORTANCE Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice.

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Cysteine Protease Inhibitors Block Toxoplasma gondii Microneme Secretion and Cell Invasion

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01059-06 ◽

2007 ◽

Vol 51 (2) ◽

pp. 679-688 ◽

Cited By ~ 37

Author(s):

Chin Fen Teo ◽

Xing Wang Zhou ◽

Matthew Bogyo ◽

Vern B. Carruthers

Keyword(s):

Toxoplasma Gondii ◽

Protease Inhibitors ◽

Cell Invasion ◽

Cysteine Protease ◽

Gliding Motility ◽

Host Cells ◽

Vinyl Sulfone ◽

Cysteine Protease Inhibitors ◽

Adhesive Proteins ◽

Phenyl Vinyl

ABSTRACT Toxoplasma gondii enters host cells via an active, self-driven process to fulfill its need for intracellular replication and survival. Successful host cell invasion is governed by sequential release of secretory proteins from three specialized organelles, including the micronemes, which contribute adhesive proteins necessary for parasite attachment and penetration. Cumulative evidence from studies of Trypanosoma species and malaria parasites has shown that cysteine protease inhibitors represent potent anti-parasitic agents capable of curing infections in vivo. In this study, we screened a series of selective cysteine protease inhibitors for their effects on T. gondii cell invasion. Two of these compounds, morpholinourea-leucyl-homophenolalaninyl-phenyl-vinyl-sulfone and N-benzoxycarbonyl-(leucyl)3-phenyl-vinyl-sulfone, impaired T. gondii invasion and gliding motility at low-micromolar concentrations. Unexpectedly, these inhibitors did not affect surface proteolysis of microneme products but instead impaired an earlier step by precluding the secretion of microneme-derived adhesins to the parasite surface. Our findings suggest that cysteine protease activity is required for microneme secretion and cell invasion by T. gondii.

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Host Cell Invasion by Toxoplasma gondii Is Temporally Regulated by the Host Microtubule Cytoskeleton

Eukaryotic Cell ◽

10.1128/ec.00079-10 ◽

2010 ◽

Vol 9 (11) ◽

pp. 1680-1689 ◽

Cited By ~ 30

Author(s):

Kristin R. Sweeney ◽

Naomi S. Morrissette ◽

Stephanie LaChapelle ◽

Ira J. Blader

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Viral Infections ◽

Protozoan Parasite ◽

Host Cells ◽

Cell Tropism ◽

Microtubule Cytoskeleton ◽

Parasite Invasion ◽

Content Type ◽

Microtubule Disruption

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we found that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather, host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections, where they function to traffic the pathogen or pathogen-derived material from the host cell's periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.

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