A bacterial-like mechanosensitive channel controls osmoregulation and infectivity in Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease undergoes drastic cellular morphological and biochemical changes as it passes from extracellular epimastigote and trypomastigote forms, to intracellular/tissue non-motile stage. Here we describe and characterize a mechanosensitive channel in T. cruzi (TcMscS), which is homologous to bacterial MscS. TcMscS is present in the contractile vacuole of extracellular stages but redistributes to the plasma membrane in intracellular amastigotes. The heterologously expressed TcMscS is activated by membrane tension (12 mN/m) forming a large 0.4 nS pore permeable to both anions and cations and possibly small osmolytes. The TcMscS knockdown and knockout T. cruzi strains, generated by CRISPR-Cas9 gene targeting, show slower growth in intracellular and extracellular stages, inability to robustly regulate volume and a dramatically decreased infectivity. Our study shows that the tension-driven mechanosensitive channel fulfills multiple roles in different stages of T. cruzi life cycle and is essential for infectivity.

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Lysosomal Fusion Is Essential for the Retention of Trypanosoma cruzi Inside Host Cells

Journal of Experimental Medicine ◽

10.1084/jem.20041408 ◽

2004 ◽

Vol 200 (9) ◽

pp. 1135-1143 ◽

Cited By ~ 94

Author(s):

Luciana O. Andrade ◽

Norma W. Andrews

Keyword(s):

Plasma Membrane ◽

Life Cycle ◽

Trypanosoma Cruzi ◽

Parasitophorous Vacuole ◽

Cell Types ◽

Significant Fraction ◽

Host Cells ◽

Productive Infection ◽

Kinase Inhibition

Trypomastigotes, the highly motile infective forms of Trypanosoma cruzi, are capable of infecting several cell types. Invasion occurs either by direct recruitment and fusion of lysosomes at the plasma membrane, or through invagination of the plasma membrane followed by intracellular fusion with lysosomes. The lysosome-like parasitophorous vacuole is subsequently disrupted, releasing the parasites for replication in the cytosol. The role of this early residence within lysosomes in the intracellular cycle of T. cruzi has remained unclear. For several other cytosolic pathogens, survival inside host cells depends on an early escape from phagosomes before lysosomal fusion. Here, we show that when lysosome-mediated T. cruzi invasion is blocked through phosophoinositide 3-kinase inhibition, a significant fraction of the internalized parasites are not subsequently retained inside host cells for a productive infection. A direct correlation was observed between the lysosomal fusion rates after invasion and the intracellular retention of trypomastigotes. Thus, formation of a parasitophorous vacuole with lysosomal properties is essential for preventing these highly motile parasites from exiting host cells and for allowing completion of the intracellular life cycle.

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Multiple roles for the ESCRT machinery in maintaining plasma membrane homeostasis

10.1101/2020.02.25.964452 ◽

2020 ◽

Author(s):

Oliver Schmidt ◽

Yannick Weyer ◽

Simon Sprenger ◽

Michael A. Widerin ◽

Sebastian Eising ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Integrity ◽

Multiple Roles ◽

Membrane Tension ◽

Membrane Remodeling ◽

Membrane Stress ◽

Endosomal Sorting ◽

Protein Levels ◽

Escrt Machinery ◽

Calcineurin Phosphatase

ABSTRACTThe endosomal sorting complexes required for transport (ESCRT) execute evolutionary conserved membrane remodeling processes. Here we used budding yeast to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. In response to reduced membrane tension and inhibition of the target of rapamycin complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help to maintain membrane integrity. Conversely, the growth of ESCRT mutants strongly depends on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This is caused by calcineurin phosphatase activity which causes Orm2 to accumulate at the endoplasmic reticulum (ER) in ESCRT mutants. Orm2 is a repressor of SL biosynthesis and its accumulation provokes increased membrane stress. This necessitates TORC2 signaling through its downstream kinase Ypk1 to control Orm2 protein levels and prevent a detrimental imbalance of SL metabolism. Our findings reveal new aspects of antagonistic calcineurin/TORC2 signaling for the regulation of SL biosynthesis and the maintenance of PM homeostasis, and suggest that the ESCRT machinery contributes directly and indirectly to these processes.

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Major Surface Protease of Trypanosomatids: One Size Fits All?

Infection and Immunity ◽

10.1128/iai.00776-09 ◽

2009 ◽

Vol 78 (1) ◽

pp. 22-31 ◽

Cited By ~ 58

Author(s):

Chaoqun Yao

Keyword(s):

Extracellular Matrix ◽

Plasma Membrane ◽

Trypanosoma Cruzi ◽

Trypanosoma Brucei ◽

Life Cycles ◽

Cellular Functions ◽

Time Points ◽

Intracellular Amastigotes ◽

Extracellular Release ◽

Major Surface

ABSTRACT Major surface protease (MSP or GP63) is the most abundant glycoprotein localized to the plasma membrane of Leishmania promastigotes. MSP plays several important roles in the pathogenesis of leishmaniasis, including but not limited to (i) evasion of complement-mediated lysis, (ii) facilitation of macrophage (Mø) phagocytosis of promastigotes, (iii) interaction with the extracellular matrix, (iv) inhibition of natural killer cellular functions, (v) resistance to antimicrobial peptide killing, (vi) degradation of Mø and fibroblast cytosolic proteins, and (vii) promotion of survival of intracellular amastigotes. MSP homologues have been found in all other trypanosomatids studied to date including heteroxenous members of Trypanosoma cruzi, the extracellular Trypanosoma brucei, unusual intraerythrocytic Endotrypanum spp., phytoparasitic Phytomonas spp., and numerous monoxenous species. These proteins are likely to perform roles different from those described for Leishmania spp. Multiple MSPs in individual cells may play distinct roles at some time points in trypanosomatid life cycles and collaborative or redundant roles at others. The cellular locations and the extracellular release of MSPs are also discussed in connection with MSP functions in leishmanial promastigotes.

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A freeze-fracture-etched study of the physarum polycephalum plasma membrane during early formation of the macroplasmodial stage

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147570 ◽

1993 ◽

Vol 51 ◽

pp. 346-347

Author(s):

Randolph W. Taylor ◽

Henrie Treadwell

Keyword(s):

Plasma Membrane ◽

Life Cycle ◽

Growth Phase ◽

Filter Paper ◽

Physarum Polycephalum ◽

Freeze Fracture ◽

Petri Dish ◽

Wire Screen ◽

Wide Mouth ◽

Sterile Filter

The plasma membrane of the Slime Mold, Physarum polycephalum, process unique morphological distinctions at different stages of the life cycle. Investigations of the plasma membrane of P. polycephalum, particularly, the arrangements of the intramembranous particles has provided useful information concerning possible changes occurring in higher organisms. In this report Freeze-fracture-etched techniques were used to investigate 3 hours post-fusion of the macroplasmodia stage of the P. polycephalum plasma membrane.Microplasmodia of Physarum polycephalum (M3C), axenically maintained, were collected in mid-expotential growth phase by centrifugation. Aliquots of microplasmodia were spread in 3 cm circles with a wide mouth pipette onto sterile filter paper which was supported on a wire screen contained in a petri dish. The cells were starved for 2 hrs at 24°C. After starvation, the cells were feed semidefined medium supplemented with hemin and incubated at 24°C. Three hours after incubation, samples were collected randomly from the petri plates, placed in plancettes and frozen with a propane-nitrogen jet freezer.

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Flagellar Motility ofTrypanosoma cruziEpimastigotes

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/520380 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

G. Ballesteros-Rodea ◽

M. Santillán ◽

S. Martínez-Calvillo ◽

R. Manning-Cela

Keyword(s):

Life Cycle ◽

Trypanosoma Cruzi ◽

Quantitative Description ◽

Flagellar Motility ◽

Parasite Life Cycle ◽

Large Variability ◽

Rotational Angle ◽

Ciliary Beating ◽

Parasite Motility ◽

Parasite Behavior

The hemoflagellateTrypanosoma cruziis the causative agent of American trypanosomiasis. Despite the importance of motility in the parasite life cycle, little is known aboutT. cruzimotility, and there is no quantitative description of its flagellar beating. Using video microscopy and quantitative vectorial analysis of epimastigote trajectories, we find a forward parasite motility defined by tip-to-base symmetrical flagellar beats. This motion is occasionally interrupted by base-to-tip highly asymmetric beats, which represent the ciliary beat of trypanosomatid flagella. The switch between flagellar and ciliary beating facilitates the parasite's reorientation, which produces a large variability of movement and trajectories that results in different distance ranges traveled by the cells. An analysis of the distance, speed, and rotational angle indicates that epimastigote movement is not completely random, and the phenomenon is highly dependent on the parasite behavior and is characterized by directed and tumbling parasite motion as well as their combination, resulting in the alternation of rectilinear and intricate motility paths.

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Plasmodium Kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission

10.1101/665836 ◽

2019 ◽

Cited By ~ 3

Author(s):

Mohammad Zeeshan ◽

Fiona Shilliday ◽

Tianyang Liu ◽

Steven Abel ◽

Tobias Mourier ◽

...

Keyword(s):

Life Cycle ◽

Cell Division ◽

Gene Targeting ◽

Intracellular Transport ◽

Regulation Of Gene Expression ◽

Microtubule Depolymerization ◽

Expression Of Genes ◽

Genome Wide ◽

Spindle Dynamics ◽

Chromosome Alignment

AbstractKinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics are not well understood in the different proliferative stages. Genome-wide homology analysis of Plasmodium spp. revealed the presence of two Kinesin-8 motor proteins (Kinesin-8X and Kinesin-8B). Here we have studied the biochemical properties of Kinesin-8X and its role in parasite proliferation. In vitro, Kinesin-8X showed motile and depolymerization activities like other Kinesin-8 motors. To understand its role in cell division, we have used protein tagging and live cell imaging to define the location of Plasmodium Kinesin-8X during all proliferative stages of the P berghei life cycle. Furthermore, we have used gene targeting to analyse the function of Kinesin-8X. The results reveal a spatio-temporal involvement of Kinesin-8X in spindle dynamics and its association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the Kinesin-8X gene showed that this protein is required for endomitotic division during oocyst development and is therefore necessary for parasite replication within the mosquito gut, and for transmission to the vertebrate host. Consistently, transcriptome analysis of Δkinesin-8X parasites reveals modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression supporting a role in cell division.Author SummaryKinesins are microtubule-based motors that play key roles in intracellular transport, cell division and motility. Members of the Kinesin-8 family contribute to chromosome alignment during cell division in many eukaryotes. However, the roles of kinesins in the atypical cell division in Plasmodium, the causative agent of malaria, is not known. In contrast to many other eukaryotes, Plasmodium proliferates by endomitosis, in which genome replication and division occur within a nucleus bounded by a persistent nuclear envelope. We show that the Plasmodium genome encodes only nine kinesins and we further investigate the role of Kinesin-8X throughout the Plasmodium life cycle using biochemical and gene targeting approaches. We show that Plasmodium Kinesin-8X has microtubule-based motility and depolymerization activity. We also show that Kinesin-8X is probably localized on putative MTOCs and spindles during cell division in most of the stages of P. berghei life cycle. By gene deletion we demonstrate that Kinesin-8X is essential for normal oocyst development and sporozoite formation. Genome-wide RNA analysis of Δkinesin-8X parasites reveals modulated expression of genes involved in microtubule-based processes. Overall, the data suggest that Kinesin-8X is a molecular motor that plays essential roles during endomitosis in oocyst development in the mosquito, contributing to parasite transmission.

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An Intracellular Ammonium Transporter Is Necessary for Replication, Differentiation, and Resistance to Starvation and Osmotic Stress in Trypanosoma cruzi

mSphere ◽

10.1128/msphere.00377-17 ◽

2018 ◽

Vol 3 (1) ◽

Cited By ~ 10

Author(s):

Teresa Cruz-Bustos ◽

Evgeniy Potapenko ◽

Melissa Storey ◽

Roberto Docampo

Keyword(s):

Life Cycle ◽

Amino Acid ◽

Osmotic Stress ◽

Trypanosoma Cruzi ◽

Etiologic Agent ◽

Ammonium Transporter ◽

Amino Acid Consumption ◽

Acid Consumption ◽

Acidic Compartments ◽

Production Of Ammonia

Trypanosoma cruzi is an important human and animal pathogen and the etiologic agent of Chagas disease. The parasite undergoes drastic changes in its metabolism during its life cycle. Amino acid consumption becomes important in the infective stages and leads to the production of ammonia (NH3), which needs to be detoxified. We report here the identification of an ammonium (NH4 +) transporter that localizes to acidic compartments and is important for replication, differentiation, and resistance to starvation and osmotic stress.

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Acetylenic fatty acids from Porcelia macrocarpa (Annonaceae) against trypomastigotes of Trypanosoma cruzi: Effect of octadec-9-ynoic acid in plasma membrane electric potential

Bioorganic Chemistry ◽

10.1016/j.bioorg.2018.03.025 ◽

2018 ◽

Vol 78 ◽

pp. 307-311 ◽

Cited By ~ 9

Author(s):

Vinicius S. Londero ◽

Thais A. da Costa-Silva ◽

Kaio S. Gomes ◽

Daiane D. Ferreira ◽

Juliana T. Mesquita ◽

...

Keyword(s):

Fatty Acids ◽

Plasma Membrane ◽

Trypanosoma Cruzi ◽

Electric Potential ◽

Acetylenic Fatty Acids

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Gene deletion suggests a role for Trypanosoma cruzi surface glycoprotein GP72 in the insect and mammalian stages of the life cycle

Journal of Cell Science ◽

10.1242/jcs.106.4.1023 ◽

1993 ◽

Vol 106 (4) ◽

pp. 1023-1033 ◽

Cited By ~ 1

Author(s):

A.R. de Jesus ◽

R. Cooper ◽

M. Espinosa ◽

J.E. Gomes ◽

E.S. Garcia ◽

...

Keyword(s):

Life Cycle ◽

Trypanosoma Cruzi ◽

Gene Deletion ◽

Surface Glycoprotein ◽

Triatoma Infestans ◽

Insect Vector ◽

Null Mutant ◽

Apparent Lack ◽

Targeted Gene Deletion ◽

Mouse Macrophages

We have explored the biological function of a surface glycoprotein (GP72) of Trypanosoma cruzi by studying a null mutant parasite, generated by targeted gene deletion. GP72 deletion affected parasite morphology in several stages of the life cycle. Insect midgut (epimastigote) forms had a detached flagellum (apomastigote) in the null mutant. The abnormal flagellar phenotype persisted during development of the infective (metacyclic) forms but there was no impairment in the acquisition of complement resistance, sialidase expression or cell infectivity. The GP72 null mutant could efficiently infect and proliferate in mouse macrophages and non-phagocytic L6E9 cells. The mammalian stages of the life cycle also showed major morphological abnormalities. During early subcultures in L6E9 cells, few extracellular fully flagellated forms, expressing markers characteristic of trypomastigotes, were seen. The extracellular population consisted almost exclusively of rounded forms with short flagella (micromastigote), which expressed an amastigote-specific surface marker and no sialidase. The propagation of the parasite was not affected, despite the apparent lack of the trypomastigote forms, which are thought to be primarily responsible for cell invasion. After some subcultures, the extracellular population changed to about equal numbers of micromastigotes and a range of flagellated forms that still did not include true trypomastigotes. Instead, the kinetoplast remained close to the nucleus and the flagellum emerged from the middle of the cell (mesomastigote). Half of the flagellum adhered to the cell body and the remainder was free at the anterior end. In Triatoma infestans, the survival of the mutant was dramatically reduced, suggesting that either GP72 itself, or the altered properties of the flagellum, were critical for establishment in the insect vector.

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Participation of concanavalin A binding sites in the interaction between Trypanosoma cruzi and macrophages

Journal of Cell Science ◽

10.1242/jcs.62.1.287 ◽

1983 ◽

Vol 62 (1) ◽

pp. 287-299

Author(s):

M.N. Meirelles ◽

A. Martinez-Palomo ◽

T. Souto-Padron ◽

W. De Souza

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Trypanosoma Cruzi ◽

Uniform Distribution ◽

Concanavalin A ◽

Binding Sites ◽

Peritoneal Macrophages ◽

Untreated Mouse ◽

Mouse Peritoneal Macrophages

Untreated mouse peritoneal macrophages as well as macrophages treated with concanavalin A (ConA) were incubated in the presence of untreated or ConA-treated epimastigotes and trypomastigotes of Trypanosoma cruzi. Treatment of epimastigotes or trypomastigotes with ConA increased or decreased their uptake by macrophages, respectively. Treatment of their macrophages with ConA reduced by 70% and increased by five times the ingestion of epimastigotes and trypomastigotes, respectively. These results are discussed in relation to previous studies on the mobility of ConA receptors in the membrane of the parasite. Using fluorescein- or ferritin-labelled ConA we observed that ConA binding sites located on the plasma membrane of macrophages are internalized during endocytosis of T. cruzi, and observed in association with the membrane of the endocytic vacuole. Vacuoles without parasites showed a uniform distribution of ConA binding sites, while these sites were distributed in patches in vacuoles containing parasites. These results, in association with others previously reported, suggest the involvement of glycoproteins and/or glycolipids localized on the cell surface of T. cruzi and macrophages during the T. cruzi-macrophage interaction.

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