Marcel I. Ramirez

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles

The innate immune system is the first mechanism of vertebrate defense against pathogen infection. In this study, we present evidence for a novel immune evasion mechanism of Trypanosoma cruzi, mediated by host cell plasma membrane-derived vesicles. We found that T. cruzi metacyclic trypomastigotes induced microvesicle release from blood cells early in infection. Upon their release, microvesicles formed a complex on the T. cruzi surface with the complement C3 convertase, leading to its stabilization and inhibition, and ultimately resulting in increased parasite survival. Furthermore, we found that TGF-β–bearing microvesicles released from monocytes and lymphocytes promoted rapid cell invasion by T. cruzi, which also contributed to parasites escaping the complement attack. In addition, in vivo infection with T. cruzi showed a rapid increase of microvesicle levels in mouse plasma, and infection with exogenous microvesicles resulted in increased T. cruzi parasitemia. Altogether, these data support a role for microvesicles contributing to T. cruzi evasion of innate immunity.

Role of early lectin pathway activation in the complement-mediated killing of Trypanosoma cruzi.

The complement system is the first line of defence against pathogen infection and can be activated by the classic, alternative and lectin pathways. Trypanosoma cruzi, the causative agent of Chagas disease, has to evade complement system killing and invade the host cells to progress in infection. T. cruzi infectious stages resist complement-mediated killing by expressing surface receptors, which dissociate or prevent C3 convertase formation. Here, we present the first evidence that T. cruzi activates the complement lectin pathway. We detected rapid binding of mannan-binding lectin, H-ficolin, and L-ficolin to the surface of T. cruzi, and found that serum depleted of these molecules failed to kill parasites. Furthermore, lectin pathway activation by T. cruzi required the MBL-associated serine protease 2 (MASP2) activity resulting in C2 factor cleavage. In addition, we demonstrate that the infectious stage of T. cruzi inhibits the lectin pathway activation and complement killing expressing the complement C2 receptor inhibitor trispanning (CRIT) protein. Transgenic parasites overexpressing CRIT were highly resistant to complement-mediated killing. CRIT-derived peptides inhibited both C2 binding to the surface of T. cruzi and parasite killing. Biochemical studies revealed that the CRIT extracellular domain 1 inhibits MASP2 cleavage of C2 factor and thereby impairs C3 convertase formation. Our findings establish that the complement lectin pathway recognizes T. cruzi and provide molecular insights into how the infectious stage inhibits this activation to resist complement system killing.

Human Plasma Membrane-Derived Vesicles Halt Proliferation and Induce Differentiation of THP-1 Acute Monocytic Leukemia Cells

Plasma membrane-derived vesicles (PMVs) are small intact vesicles released from the cell surface that play a role in intercellular communication. We have examined the role of PMVs in the terminal differentiation of monocytes. The myeloid-differentiating agents all-trans retinoic acid/PMA and histamine, the inflammatory mediator that inhibits promonocyte proliferation, induced an intracellular Ca2+-mediated PMV (as opposed to exosome) release from THP-1 promonocytes. These PMVs cause THP-1 cells to enter G0–G1 cell cycle arrest and induce terminal monocyte-to-macrophage differentiation. Use of the TGF-β receptor antagonist SB-431542 and anti–TGF-β1 Ab showed that this was due to TGF-β1 carried on PMVs. Although TGF-β1 levels have been shown to increase in cell culture supernatants during macrophage differentiation and dendritic cell maturation, the presence of TGF-β1 in PMVs is yet to be reported. In this study, to our knowledge we show for the first time that TGF-β1 is carried on the surface of PMVs, and we confirm the presence within PMVs of certain leaderless proteins, with reported roles in myeloid cell differentiation. Our in vitro findings support a model in which TGF-β1–bearing PMVs, released from promonocytic leukemia cells (THP-1) or primary peripheral blood monocytes on exposure to sublytic complement or after treatment with a differentiation therapy agent, such as all-trans retinoic acid, significantly reduce proliferation of THP-1 cells. Such PMVs also induce the terminal differentiation of primary peripheral blood monocytes as well as THP-1 monocytes.

Complement C2 Receptor Inhibitor Trispanning: A Novel Human Complement Inhibitory Receptor

The complement system presents a powerful defense against infection and is tightly regulated to prevent damage to self by functionally equivalent soluble and membrane regulators. We describe complement C2 receptor inhibitor trispanning (CRIT), a novel human complement regulatory receptor, expressed on hemopoietic cells and a wide range of tissues throughout the body. CRIT is present in human parasites through horizontal transmission. Serum complement component C2 binds to the N-terminal extracellular domain 1 of CRIT, which, in peptide form, blocks C3 convertase formation and complement-mediated inflammation. Unlike C1 inhibitor, which inhibits the cleavage of C4 and C2, CRIT only blocks C2 cleavage but, in so doing, shares with C1 inhibitor the same functional effect, of preventing classical pathway C3 convertase formation. Ab blockage of cellular CRIT reduces inhibition of cytolysis, indicating that CRIT is a novel complement regulator protecting autologous cells.

Inefficient complement system clearance of Trypanosoma cruzi metacyclic trypomastigotes enables resistant strains to invade eukaryotic cells

The complement system is the main arm of the vertebrate innate immune system against pathogen infection. For the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, subverting the complement system and invading the host cells is crucial to succeed in infection. However, little attention has focused on whether the complement system can effectively control T. cruzi infection. To address this question, we decided to analyse: 1) which complement pathways are activated by T. cruzi using strains isolated from different hosts, 2) the capacity of these strains to resist the complement-mediated killing at nearly physiological conditions, and 3) whether the complement system could limit or control T. cruzi invasion of eukaryotic cells. The complement activating molecules C1q, C3, mannan-binding lectin and ficolins bound to all strains analysed; however, C3b and C4b deposition assays revealed that T. cruzi activates mainly the lectin and alternative complement pathways in non-immune human serum. Strikingly, we detected that metacyclic trypomastigotes of some T. cruzi strains were highly susceptible to complement-mediated killing in non-immune serum, while other strains were resistant. Furthermore, the rate of parasite invasion in eukaryotic cells was decreased by non-immune serum. Altogether, these results establish that the complement system recognizes T. cruzi metacyclic trypomastigotes, resulting in killing of susceptible strains. The complement system, therefore, acts as a physiological barrier which resistant strains have to evade for successful host infection.

Complement C2 Receptor Inhibitor Trispanning Confers an Increased Ability to Resist Complement-Mediated Lysis in Trypanosoma cruzi

The ability to resist complement differs between the Y and Colombiana Trypanosoma cruzi strains. We found that the Y strain of T. cruzi was more able to resist the classical and lectin pathways of complement activation than the Colombiana strain. The complement C2 receptor inhibitor trispanning gene (CRIT) is highly conserved in both strains. At the protein level, CRIT is expressed only in stationary-phase epimastigotes of the Y but not the Colombiana strain and is expressed in infectious metacyclic trypomastigotes of both strains. Y strain epimastigotes with an overexpressed CRIT gene (pTEX-CRIT) had higher survival in normal human serum (NHS). Overexpression of the Y strain CRIT gene in Colombiana epimastigote forms increased the parasites resistance to lysis mediated by the classical and lectin pathways but not to lysis mediated by alternative pathways. CRIT involvement on the parasite surface was confirmed by showing that the lytic activity of NHS against epimastigotes could be restored by adding excess C2.

Involvement of lectin pathway activation in the complement killing of Giardia intestinalis

Giardia intestinalis (syn. G. lamblia, G. duodenalis) is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. In humans, the clinical effects of Giardia infection range from the asymptomatic carrier state to a severe malabsorption syndrome possibly due to different virulence of the Giardia strain, the number of cysts ingested, the age of the host, and the state of the host immune system at the time of infection. The question about how G. intestinalis is controlled by the organism remains unanswered. Here, we investigated the role of the complement system and in particular, the lectin pathway during Giardia infections. We present the first evidence that G. intestinalis activate the complement lectin pathway and in doing so participate in eradication of the parasite. We detected rapid binding of mannan-binding lectin, H-ficolin and L-ficolin to the surface of G. intestinalis trophozoites and normal human serum depleted of these molecules failed to kill the parasites. Our finding provides insight into the role of lectin pathway in the control of G. intestinalis and about the nature of surface components of parasite.

Cell Adhesion and Ca2+ Signaling Activity in Stably Transfected Trypanosoma cruzi Epimastigotes Expressing the Metacyclic Stage-Specific Surface Molecule gp82

ABSTRACT Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82-kDa surface glycoprotein (gp82) that has been implicated in host cell invasion. gp82-mediated interaction of metacyclic forms with target cells induces in both cells activation of the signal transduction pathways, leading to intracellular Ca2+ mobilization, which is required for parasite internalization. Noninfective epimastigotes do not express detectable levels of gp82 and are unable to induce a Ca2+ response. We stably transfected epimastigotes with a T. cruzi expression vector carrying the metacyclic stage gp82 cDNA. These transfectants produced a functional gp82, which bound to and triggered a Ca2+ response in HeLa cells, in the same manner as the metacyclic trypomastigote gp82. Such properties were not found in epimastigotes transfected with the plasmid vector alone. Epimastigotes expressing gp82 on the surface adhered to HeLa cells but were not internalized. Treatment of gp82-expressing epimastigotes with forskolin, an activator of adenylyl cyclase that increases the metacyclic trypomastigote entry into target cells, did not promote parasite internalization. P175, an intracellular tyrosine phosphorylated protein, which appears to play a role in gp82-dependent signaling cascade in metacyclic forms, was undetectable in epimastigotes, either transfected or not with pTEX-gp82. Overall, our results indicate that gp82 is required but not sufficient for target cell invasion.

Mechanisms of complement lectin pathway activation and resistance by trypanosomatid parasites

Studies in the past decade have demonstrated a crucial role for the complement lectin pathway in host defence against protozoan microbes. Recognition of pathogen surface molecules by mannan-binding lectin and ficolins revealed new mechanisms of innate immune defence and a diversity of parasite strategies of immune evasion. In the present review, we will discuss the current knowledge of: (1) the molecular mechanism of lectin pathway activation by trypanosomes; (2) the mechanisms of complement evasion by trypanosomes; and (3) host genetic deficiencies of complement lectin pathway factors that contribute to infection susceptibility and disease progression. This review will focus on trypanosomatids, the parasites that cause Chagas disease, leishmaniasis and sleeping sickness (African trypanosomiasis).