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Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins—which are essential for peroxisome biogenesis, proliferation, and protein import—are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.

The leader of the capsid (LC) protein is exclusive to the Vesivirus genus, and it is needed for successful feline calicivirus (FCV) replication, as well as an efficient apoptosis induction through the mitochondrial pathway. In this work, we aimed to determine if the LC protein from the FCV is a viroporin. Although lacking in a transmembrane domain or an amphipathic helix, the LC protein from the FCV is toxic when expressed in bacteria and it oligomerizes through disulfide bonds, which are both key characteristics of viroporins. An electron microscopy analysis of LC-expressing E. coli cells suggest that the protein induces osmotic stress. Moreover, we found that the previously studied C40A LC mutant, that fails to induce apoptosis and that hinders the replication cycle, also oligomerizes but it has a reduced toxicity and fails to induce osmotic stress in bacteria. We propose that the LC protein is a viroporin that acts as a disulfide bond-dependent antimicrobial peptide, similar to the Ebola virus delta peptide.

Septins are a family of GTP-binding proteins identified in insects and mammals. Septins are components of the cytoskeleton and participate in cytokinesis, chromosomal segregation, intracellular vesicular traffic, and response to pathogens. Human septin 6 was identified as necessary for hepatitis C virus replication. Information about host factors necessary for flavivirus replication in mosquitoes is scarce. Thus, the role of septins in the replicative cycle of dengue virus in Aedes spp. derived cells was investigated. Through bioinformatic analysis, sequences of septin-like proteins were identified. Infected mosquito cells showed increased expression of Sep2. Colocalization analysis, proximity ligation and immunoprecipitation assays indicated that Sep2 interacts with proteins E, NS3 and NS5, but not NS1. Immunoelectron microscopy evidenced the presence of AalSep2 in replicative complexes. Finally, silencing of Sep2 expression resulted in a significant decrease in virus progeny, indicating that Sep2 is a host factor participating in dengue virus replication in mosquito cells.

Background Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available. Methods A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy. Results At L3–L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal–ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure. Conclusions A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages.

Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.

Cellular proteins have been identified to participate in calicivirus replication in association with viral proteins and/or viral RNAs. By mass spectrometry from pull-down assays, we identified several cellular proteins bound to the feline calicivirus (FCV) genomic RNA; among them the lipid raft-associated scaffold protein Annexin (Anx) A2. AnxA2 colocalizes with FCV NS6/7 protein and with the dsRNA in infected cells; moreover, it was found associated with the viral RNA in the membrane fraction corresponding to the replication complexes (RCs), suggesting its role during FCV replication. AnxA2-knockdown from CrFK cells prior to infection with FCV caused a delay in the cytopathic effect, a strong reduction of viral non-structural proteins and dsRNA production, and a decrease of FCV yield in both cell-associated and supernatant fractions. Taken together, these results indicate that AnxA2 associates to the genomic RNA of FCV and is required for an efficient FCV replication.

Highly dynamic ribosomes, glycogen granules, thinly fibrillar material, and multiple membrane-bound vesicles are embedded in the matrix-rich cytoplasm of Entamoeba spp. trophozoites. The absence of a Golgi apparatus in these amoebae has been commonly accepted. Here we challenge this observation by incubating Entamoeba histolytica and Entamoeba dispar with monensin, an ionophore that produces swelling of the Golgi apparatus. We observe changes in the trophozoites through standard transmission electron microscopy, cryofixation and cryosubstitution, and analyze the label and expression of known resident proteins of the cis-GM130 and trans-TGN38 Golgi network through confocal microscopy and Western blot assays. Cryosubstitution and standard methods using the treatment, preserved membranous lamellae resembling Golgi components. GM130 and TGN38 Golgi antigens were found by immunoelectron, immunoblot, and co-localization by confocal microscopy using the reagent NBD C6-ceramide. Our results indicate that previously undetected Golgi apparatus components are present in the cytoplasm of E. histolytica and E. dispar.

Acanthamoeba castellanii is the etiological agent of granulomatous amebic encephalitis, amebic keratitis, and skin lesions. In vitro and in vivo studies have demonstrated that Acanthamoeba trophozoites induce contact-dependent, and contact-independent pathogenic mechanisms. We have explored the potential role neuroactive substances may have in the migration of Acanthamoeba castellanii trophozoites using Transwell permeable supports in the presence of physiological concentrations of dopamine, glutamate, serotonin, or taurine diluted in PBS. Quantitation of migrated amoebae was carried out in scanning electron micrographs of the upper and under compartments sides of the chamber membranes. Our results showed that at 2 h of interaction, a statistically significant larger proportion of A. castellanii trophozoites migrated through the chamber membranes when neurotransmitters were placed in the lower compartments of the chambers compared to control. This migration effect was more evident under the presence of glutamate and taurine on the three surfaces (upper/lower membrane and bottom compartment) when the percentage of migrated trophozoites was analyzed. Scanning electron microscopy of trophozoites revealed that glutamate and taurine induced the formation of large adhesion lamellas and phagocytic stomas. These observations suggest that certain neuroactive substances could stimulate the migration of A. castellanii trophozoites in the central nervous system.

Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated by iron. An immunoproteomic analysis showed that trichomoniasis patient sera recognize various CPs, also some of them are present in vaginal washes (VWs). Thus, the goal of this work was to determine whether TvCP2 is expressed during infection and to assess the effect of iron on TvCP2 expression, localization and contribution to in vitro cellular damage. Western-blotting (WB) assays using TvCP2r and vaginitis patient serum samples showed that 6/9 Tv (+) but none of the Tv (−) patient sera recognized TvCP2r. WB using an anti-TvCP2r antibody and VWs from the same patients showed that in all of the Tv (+) but none of the Tv (−) VWs, the anti-TvCP2r antibody detected a 27 kDa protein band that corresponded to the mature TvCP2, which was confirmed by mass spectrometry analysis. Iron decreased the amount of TvCP2 mRNA and the protein localized on the parasite surface and cytoplasmic vesicles concomitant with the cytotoxic effect of TvCP2 on HeLa cells. Parasites pretreated with the anti-TvCP2r antibody also showed reduced levels of cytotoxicity and apoptosis induction in HeLa cell monolayers. In conclusion, these results show that TvCP2 is expressed during trichomonal infection and plays an important role in the in vitro HeLa cell cytotoxic damage under iron-restricted conditions.

Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbours three homologues of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.

Entamoeba histolytica, the causal agent of human amoebiasis, has two morphologically different phases: a resistant cyst and a trophozoite responsible for the invasion of the host tissues such as the colonic mucosa and the intestinal epithelium. During in vitro migration, trophozoites usually produce protuberances such as pseudopods and rarely filopodia, structures that have been observed in the interaction of trophozoites with human colonic epithelial tissue. To study the different membrane projections produced by the trophozoites, including pseudopods, filopodia, uropods, blebs, and others, we designed an induction system using erythrocyte extract or fibronectin (FN) in micropatterned grill lines (each micro-line containing multiple micro-portions of FN or erythrocyte extract) on which the trophozoites were placed in culture for migration assays. Using light, confocal, and scanning electron microscopy, we established that E. histolytica trophozoites frequently produce short and long filopodia, large retractile uropods in the rear, pseudopods, blebs, and others structures, also showing continuous migration periods. The present study provides a simple migration method to induce trophozoites to generate abundant membrane protrusion structures that are rarely obtained in normal or induced cultures, such as long filopodia; this method will allow a-better understanding of the interactions of trophozoites with FN and cell debris. E. histolytica trophozoites motility plays an important role in invasive amoebiasis. It has been proposed that both physical forces and chemical signals are involved in the trophozoite motility and migration. However, the in vivo molecules that drive the chemotactic migration remain to be determined. We propose the present assay to study host molecules that guide chemotactic behavior because the method is highly reproducible, and a live image of cell movement and migration can be quantified.

Transcription factor IID (TFIID) is a cornerstone in the transcription initiation in eukaryotes. It is composed of TBP and approximately 14 different subunits named TBP-associated factors (TAFs). TFIID has a key role in transcription of many genes involved in cell proliferation, cell growth, cell cycle, cell cycle checkpoint, and various other processes as well. Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, represents a major global health concern. Our research group has previously reported the genes coding the TATA box-binding protein (EhTBP) and TBP-related factor 1 (EhTRF1), which displayed different mRNA levels in trophozoites under different stress conditions. In this work, we identified the TBP-associated factor 1 (Ehtaf1) gene in the E. histolytica genome, which possess a well-conserved DUF domain and a Bromo domain located in the middle and C-terminus of the protein, respectively. The EhTAF1-DUF domain tertiary structure is similar to the corresponding HsTAF1 DUF domain. RT-qPCR experiments with RNA isolated from trophozoites harvested at different time points of the growth curve and under different stress conditions revealed that the Ehtaf1 gene was found slightly upregulated in the death phase of growth curve, but under heat shock stress, it was found upregulated 10 times, suggesting that Ehtaf1 might have an important role in the heat shock stress response. We also found that EhTAF1 is expressed in the nucleus and cytoplasm at 37 °C, but under heat shock stress, it is overexpressed in both the nucleus and cytoplasm, and partially colocalized with EhHSP70 in cytoplasm.

Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18 kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.

In the genusEntamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins duringEntamoeba invadensencystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment ofE. invadens.

Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins ofEntamoeba histolyticahas been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics andin vivovirulence ofE. histolytica. Using western blot, immunoprecipitation, microscopy assays, andin silicoanalysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

During Acanthamoeba castellanii trophozoite-cysts differentiation, four morphological stages were identified by scanning electron microscopy: trophozoite, precyst, immature cysts, and mature cysts. Fluorescence microscopy reveals the presence of small cumulus of actin in the cytoplasm of precysts after treatment with rhodamine phalloidin. By the contrary, in mature cysts, fluorescence was not observed. However, when excystation was induced, large fluorescent patches were present. By transmission electron microscopy, encysting amebas showed small cytoplasmic vesicles containing fibrillar material, surrounded by a narrow area of thin fibrils. Similar appearance was observed in pseudopods and phagocytic invaginations. In addition, large aggregates of rod-shape elements, similar to the chromatoid bodies, described in other amebas, were present in the cytoplasm. These cysts presented large areas with orange fluorescence after treatment with acridine orange.