Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by <Emphasis Type="Italic">Neospora caninum</Emphasis> tachyzoites

Neospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4^c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4^c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.     

Changes in β-giardin sequence of <Emphasis Type="Italic">Giardia intestinalis</Emphasis> sensitive and resistant to albendazole strains

Giardia intestinalis can develop resistance to albendazole, although the molecular mechanism is not understood. The aim of this study was to investigate the differences and permanent mutation in the β-giardin gene of G. intestinalis strains: sensitive, resistant, or recovered-resistance to albendazole. The β-giardin gene was amplified by nested polymerase chain reaction. The IC₅₀ values varied from 0.29 to 0.38 μg/mL for strains sensitive to albendazole. For resistant strains, the IC₅₀ range was 1.31–2.12 μg/mL. Recovered-sensitivity albendazole strains’ IC₅₀ values were 0.33–0.49 μg/mL, and for strains with recovered-resistance, the IC₅₀ was 1.42–2.74 μg/mL. β-giardin amplicon (720 bp) was sequenced and analysis sequence revealed several amino acid mutations from resistant and recovered-sensitive strains of G. intestinalis. Most of the mutations were located in the ROD domain of β-giardin with a change from the sequence “TIARERA” in sensitive strains instead “IDRPRE” in resistant strains. A comparative sequence analysis in resistant, recovered-sensitive, and resistant-recovered strains revealed permanent mutation. This is the first report of combinatorial serine–proline–arginine repeats in the ROD domain of β-giardin, whereas such repeats have been reported previously in the HEAD domain of SF-assemblin proteins. This is the first time that the resistance to albendazole correlates with genetics but it is not necessarily caused by mutations in the β-giardin gene of G. intestinalis.     

Allopurinol is effective to modify the evolution of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> infection in mice

There is a real need for new and less toxic drugs for the treatment of Chagas disease, as nifurtimox and benznidazole are effective but toxic and provoke unpleasant side effects, especially in adult patients. Allopurinol, commonly used to treat the hiperuricemia, is also used by the Trypanosoma cruzi’s hypoxantine guanine fosforyltransferase as an alternative substrate incorporating it into the parasite’s ribonucleic acid, provoking the death of the parasite. However, the results of using allopurinol as chemotherapy for Chagas disease are not clear. For that, we investigated the evolution of the T. cruzi infection in mice treated with allopurinol (5, 10 or 15 mg/kg for 90 days) obtaining a reduction in the parasitaemia (p < 0.05), no electrocardiographic alterations (p < 0.05) and a conserved myocardial and cardiac β-receptors’ affinity values with the highest dose of the drug, compared to those of the uninfected mice. Cruzipain immunoglobulin G levels remained high in all the groups as well as the survival (70%, 90 days post-infection). Allopurinol prevented the acute phase evolving into the chronic cardiac disease.     

Excreted/secreted glycoproteins of <Emphasis Type="Italic">G. intestinalis</Emphasis> play an essential role in the antibody response

In the present work, glycoproteins in the excretory/secretory products of G. intestinalis were identified and the reactivity in serum of immunized mice with these molecules was evaluated by western blotting before and after chemical treatment or enzymatic deglycosylation. Glycoproteins of 58 and 63 kDa were revealed in E/S products after periodic acid-Schiff (PAS) stain. Studies of carbohydrate specificity using digoxigenin-labeled lectins, revealed the presence of O-glycans and N-glycans. Chemical treatment of excretory/secretory products with sodium meta-periodate or enzymatic deglycosylation with N-glycosidase F reduced the reactivity in serum for proteins of 36, 58 and 63 kDa, respectively. These results show the presence of glycoproteins in E/S products of G. intestinalis and suggest that the antibody response is directed against glycoepitopes. The expression of carbohydrate moieties in the E/S-G. intestinalis may play an essential role in the antibody response and may be a target for serodiagnosis or immune intervention in human giardiasis.     

Identification of differentially expressed proteins in a <Emphasis Type="Italic">mat1-2-</Emphasis>deleted strain of <Emphasis Type="Italic">Gibberella zeae</Emphasis>, using a comparative proteomics analysis

Gibberella zeae is a self-fertile ascomycetous fungus that causes important diseases of cereal crops. A comprehensive understanding of sexual reproduction in G. zeae is needed for disease control. To identify fungal proteins involved in this process, we compared the protein profiles of a wild-type strain and its self-sterile strain deleted for MAT1-2, a master regulator of sexual reproduction in G. zeae. Using 2-DE and either MALDI-TOF or ESI-Q-TOF MS, we identified 13 protein spots that showed statistically significant differences in expression levels between the two strains; 11 were reduced and two were increased in abundance in the ΔMAT1-2 strain. Six of the 13 proteins were similar to those related to cell wall structure and the others were orthologs of proteins involved in metabolism or environmental stress responses. We confirmed that all the genes of the proteins examined were down-regulated during the sexual development stage in the ΔMAT1-2, ΔMAT1-1, and other strains deleted for a MAP kinase or a G-protein gene. These data suggest that differences in the protein expression levels are mostly affected by down-regulation of the corresponding genes in the ΔMAT1-2 strain. To date, this is the first proteomics approach successfully identifying proteins differentially regulated by MAT1-2 in G. zeae. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Subversion of innate and adaptive immune responses by <Emphasis Type="Italic">Toxoplasma Gondii</Emphasis>

The intracellular apicomplexan parasite Toxoplasma gondii is able to survive and persist in immunocompetent intermediate hosts for the host’s life span. This is despite the induction of a vigorous humoral and—more importantly—cell-mediated immune response during infection. In order to establish and maintain such chronic infections, however, T. gondii has evolved multiple strategies to avoid or to interfere with potentially efficient anti-parasitic immune responses of the host. Such immune evasion includes (1) indirect mechanisms by altering the expression and secretion of immunomodulatory cytokines or by altering the viability of immune cells and (2) direct mechanisms by establishing a lifestyle within a suitable intracellular niche and by interference with intracellular signaling cascades, thereby abolishing a number of antimicrobial effector mechanisms of the host. Despite the parasite’s ability to interfere successfully with the host’s efforts to eradicate the infection, the immune response is, however, not completely abrogated but is rather partially diminished after infection. T. gondii thus keeps a delicate balance between induction and suppression of the host’s immune response in order to guarantee the survival of the host as a safe harbor for parasite development and to allow its transmission to the definitive host.     

Possible role of calmodulin in excystation of Giardia lamblia

The protozoan Giardia lamblia initiates infection when trophozoites emerge from a cyst in the hosts by the excystation process. Although this process is crucial to the initiation of infection by G. lamblia, little is known about its regulation. To study the possible involvement of calmodulin (CaM) in excystation we tested the effect of several CaM antagonists (TFP, W-7, and W-5) on this cellular function. Except for W-5 the rest of these compounds inhibited excystation. The protein kinase C inhibitor H-7 had no effect on excystation, suggesting that CaM antagonists acted by selectively inhibiting CaM. Furthermore, CaM was redistributed after the induction of excystation and there was an increase in its fluorescence and activity. These results suggest that a CaM-dependent process is involved in G. lamblia excystation. Received: 10 March 1998 / Accepted: 22 April 1998     

Return to sender: use of Plasmodium ER retrieval sequences to study protein transport in the infected erythrocyte and predict putative ER protein families

We have investigated how knowledge of endoplasmic reticulum (ER) retrieval signals can be used to study specific trafficking pathways in the malaria-infected erythrocyte. We show that addition of various lumenal ER retrieval signals to soluble green fluorescent protein (GFP) chimaera causes retrieval of the fusion protein in the parasite’s ER. In contrast, adding these signals to the C-terminus of a membrane bound protein does not affect its eventual sub-cellular localization. This demonstrates proof of principle that ER retrieval signals can be used to study the solubility state of Plasmodium falciparum proteins during their transport to the host erythrocyte. Furthermore, using our knowledge of ER retrieval signals, we identify Plasmodium ER protein families and assign putative functions to them.     

Effect of proteins from the spirochete <Emphasis Type="Italic">Borrelia burgdorferi sensu lato</Emphasis> on myelinated nerve excitability

We studied the effect of spirochete Borrelia burgdorferi sensu lato cell membrane proteins on excitability of myelinated nerve fiber. It was found that cell surface proteins of spirochetes B. burgdorferi s. s. bind to Ranvier nodes of the axon and to Schwann cells. Binding of B. burgdorferi s. s. and B. garinii to the nerve fiber modulates the amplitude and conduction velocity of the action potential, while B. afzelii had no effect on these parameters. The decrease in the spike amplitude and conduction velocity during sorption of B. burgdorferi s. s. or cell wall proteins was accompanied by desorption of membrane-bound calcium. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 1, pp. 42–45, January, 2007     

Synchronization of occurrence of the ovarian philometrid, <Emphasis Type="Italic">Philometra carolinensis,</Emphasis> with the spawning season of its fish host,the spotted seatrout, <Emphasis Type="Italic">Cynoscion nebulosus</Emphasis>

The philometrid Philometra carolinensis inhabits the ovaries of the spotted seatrout, Cynoscion nebulosus. A 2-year study in estuaries of South Carolina showed that each year adult female worms were present only during the spawning season of the host and that only sexually mature fish were infected. Overall prevalence was 13.1%. Young-of-the-year fish were uninfected and mature 1-year-old fish were less frequently infected than older fish. Abundance of the philometrid was significantly different in age-1 and -2 spotted seatrout. Prevalence, mean abundance, and intensity peaked during the first 2 months of the host’s 4-month spawning season, which then declined abruptly. Occurrence of the philometrid in the fish host was unaffected by water temperature, salinity, and dissolved oxygen. Histological studies revealed that the worms were hematophagous. Worms induced disruption of the ovarian lamellar walls resulting in the interruption of development and the loss of host eggs into the ovarian lumen prior to their maturation. The data show that development of this parasite is linked to the host’s reproductive status and suggest that paratenesis plays an important role in the maintenance of the parasite’s life cycle.     

Multi-membrane-bound structures of Apicomplexa: II. the ovoid mitochondrial cytoplasmic (OMC) complex of Toxoplasma gondii tachyzoites

Apicomplexa including the causative agents of toxoplasmosis and malaria reportedly possess one or few tubular-shaped mitochondria that permeate, more or less branched, throughout these unicellular parasites. Electron micrographs generated herein from serial-sectioned Toxoplasma gondii tachyzoites demonstrated, however, a greater diversity regarding both the shape of the cultured parasite’s single mitochondrion and its sub-structural organization. Moreover, a unique subcellular construction was detected that basically comprised a pouch-shaped subdivision of the tachyzoite mitochondrion plus a fraction of parasitic cytoplasm enclosed therein. This composite assembling, termed ovoid mitochondrial cytoplasmic (OMC) complex, characteristically displayed a highly reduced matrix lumen of its mitochondrial border construction, which furthermore often failed to possess any cristae or contained tightly pleated cristae, thus creating a pouch-shaped multi-laminar wall of four or more membranous layers, respectively. Given this architecture, cross-sectioned OMC complexes of T. gondii tachyzoites frequently mimicked in size and shape the parasites’ plastid-like organelle (apicoplast). Moreover, like the apicoplast, the OMC complex was often found adjacent to the tachyzoite’s single Golgi complex and constantly located in close proximity to the outer membrane of the parasite’s nuclear envelope. The T. gondii OMC complex differed, however, from the apicoplast in its exact fine structural organization and a stage-restricted presence that was apparently linked to mitochondrial growth and/or division. Any special function(s) possibly performed by the T. gondii OMC complex remains, nevertheless, to be elucidated.     

The Differential Effect of Endothelial Cell Factors on <Emphasis Type="Italic">In Vitro</Emphasis> Motility of Malignant and Non-malignant Cells

Motility of cancer cells plays a critical role in tumor metastasis, and as such is a target for intervention. The motility of malignant Calu-1 human lung epithelial carcinoma cells is upregulated when placed on a human umbilical vein endothelial cell monolayer, while that of non-malignant L132 human lung epithelial cells is not. To dissect the factor(s) causing such differential behaviors, the motile responses of both cell lines to endothelial cell factors—secreted to the media, on the endothelial cell surface, and secreted to the extracellular matrix—and to individual extracellular matrix proteins were compared. Cell motility was quantified by tracking the cell movement on a surface with time-lapse video microscopy, which was analyzed with the persistent random walk model of motility. None of the factors tested had a remarkable effect on L132 cell motility, but the Calu-1 cell motility was significantly upregulated by endothelial cell extracellular matrix and by laminin, fibronectin, collagen I and collagen VI individually. Flow cytometry analysis revealed significantly higher expression levels of integrin subunits β1, α2, α3, and α6, which are known receptors for these extracellular matrix proteins, on the Calu-1 than L132 cells, implicating a role of these integrins in the observed motile behaviors of these cell lines.     

Proteins of the Plasmodium falciparum two transmembrane Maurer’s cleft protein family, PfMC-2TM, and the 130?kDa Maurer’s cleft protein define different domains of the infected erythrocyte intramembranous network

Plasmodium falciparum Maurer’s clefts participate in the transport of macromolecules within the cytoplasm, including the transport of virulence proteins to the erythrocyte membrane surface. We identified a family of genes PfMC-2TM encoding transmembrane proteins located within the intramembranous network of the infected erythrocyte using monoclonal antibody SP1C1. The distribution of the PfMC-2TM protein family within domains of the network was investigated by colocalization and confocal microscopy studies using monoclonal antibody SP1C1 specific for PFMC-2TM and monoclonal antibody SP1A6 specific for the130 kDa Maurer’s cleft protein. Peptide-specific antibodies were prepared against six peptides from different domains of PfMC-2TM and used with the Mabs, as well as known antibodies specific to Maurer’s clefts proteins (ring-expressed protein and membrane-associated histidine-rich protein 1), the erythrocyte membrane protein 1 (PfEMP-1), and serine-rich antigen in colocalization studies. We show that PfMC-2TM is located in the Maurer’s clefts throughout the intracellular blood stage, and immunoelectron microscopy shows domains of PfMC-2TM localized in the parasitophorous vacuole and parasitophorous vacuole membrane. The distribution of the 130 kDa Maurer’s cleft protein changes from within the parasite to the clefts during intracellular development as the parasite matures from young trophozoite to segmented schizont.     

<Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Antigen 85A Induces Th-1 Immune Responses in Systemic Sarcoidosis

Sarcoidosis is a granulomatous disease of unknown etiology, characterized by a Th-1 immunophenotype. Although humoral immune responses by sarcoidosis subjects to mycobacterial proteins have been detected, mycobacterial antigens capable of inducing cellular immune responses in sarcoidosis subjects have not been reported. We used the enzyme-linked immunospot assay to assess for recognition of the Mycobacterium tuberculosis mycolyl transferase, Antigen 85A, by peripheral blood mononuclear cells from 25 sarcoidosis subjects, 22 PPD− (purified protein derivative) healthy volunteers, and 16 PPD+ healthy subjects. Reactivity to Ag85A whole protein was observed in 15 of 25 sarcoidosis subjects compared to 2 of 22 PPD− subjects (p=0.0006, Fisher’s exact test) and to 14 of 16 PPD+ subjects (p=0.084, Fisher’s exact test). Monoclonal antibody against HLA-DR inhibited recognition. In addition to immune recognition of Ag85A whole protein, peptide-mapping studies identified four immunogenic Ag85A peptides, which induced Th-1 immune responses in individual sarcoidosis subjects, suggesting that multiple epitopes from a mycobacterial protein may have a role in sarcoidosis immunopathogenesis.     

Interaction of β-Giardin with the Bop1 Protein in Giardia lamblia

A cDNA clone encoding a putative Bop1 homologous protein was identified in Giardia lamblia. Since Bop1 is a nucleolar protein involved in rRNA processing, thereby controlling the cell cycle, we investigated components of cell cycle control in G. lamblia by identifying the protein(s) that interact with Bop 1. Through an immunoaffinity column made with polyclonal antibodies specific to the recombinant Bop1 of G. lamblia, a pool of proteins was obtained from the crude extracts of Giardia and then used as antigens to immunize rats. By employing the resultant sera for cDNA library immunoscreening, we isolated cDNA clones encoding an immunopurified protein, which turned out to contain the gene for β-giardin, a Giardia-specific cytoskeletal protein. The interaction between Bop1 and β-giardin was confirmed via two different methods, yeast two-hybrid assay and coimmunoprecipitation.     

Biochemical characterization of mitogen-activated protein (MAP) kinase activity in Toxoplasma gondii

Mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) are activated by many extracellular stimuli. In this study, we investigated whether MAP kinase and tyrosine kinases were involved in transducing signals in Toxoplasma gondii. Using anti-phosphotyrosine and anti-active ERK antibodies, we identified several phosphorylated proteins in Toxoplasma. In particular, phosphorylation of a 47 kDa and a 43 kDa protein increased strongly after calcium influx. MAP kinase activity, caused by calcium influx, was determined using either a specific synthetic peptide, or an in gel kinase assay. Conversely, calcium chelators (BAPTA and EGTA) and a calcium channel blocker (nifedipine) inhibited this activation. Also, a specific inhibitor of MAP kinase kinase (PD 098059) blocked MAP kinase activity. Three specific anti-MAP kinase antibodies recognized the 47 kDa and 43 kDa proteins, which were putatively identified as ERK1- and ERK2-homologs, respectively. These findings provide early evidence of signal transduction involving members of the MAP kinase family in T. gondii. Received: 28 September 1999 / Accepted: 12 October 1999     

Characterization of axenic isolates of Giardia intestinalis established from humans and animals in Germany

A total of 15 isolates of Giardia intestinalis, the first axenic cultures of this organism to be described from Germany, were established in Bonn from faecal cysts obtained from human and animal stool specimens. Measurement of in vitro growth kinetics for 12 of the isolates revealed 3 phenotypes (`rapid', `medium-rate' and `slow' growers) characterized by generation times of 9–11 h (5 isolates), 12–15 h (5 isolates) and ≥18–20 h (2 isolates), respectively. Cloned sublines exhibited growth rates similar to those of the parent isolates. Genetic analyses involving use of the polymerase chain reaction to amplify segments of genes encoding variant-specific surface proteins or the enzyme glutamate dehydrogenase, coupled with the detection of restriction-fragment-length polymorphisms, identified genotypes belonging to three previously described genetic groups. Seven isolates (from humans, a calf and a chinchilla) were typed to genetic group I – a potentially zoonotic genotype belonging to assemblage A, one of two major genetic lineages defined by analysis of G. intestinalis from humans and animals. Six isolates (all from humans) showed identity with the group II genotype – recovered thus far only from humans and also belonging to assemblage A. Two isolates (one from a human, the other from a monkey housed at the Cologne zoo) were classified as assemblage B genotypes. The in vitro growth rates correlated strongly with genotype, group I or group II (assemblage A) genotypes accounting for all of the `rapid' and `medium-rate' cultures and both assemblage B isolates being `slow growers'. The data indicate that genetically based metabolic differences may determine how rapidly G. intestinalis isolates can grow in axenic culture. Received: 28 August 1997 / Accepted: 26 October 1997