An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and γ irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

A novel clade of protistan parasites near the animal-fungal divergence.

Sequences of nuclear-encoded small-subunit rRNA genes have been determined for representatives of the enigmatic genera Dermocystidium, Ichthyophonus, and Psorospermium, protistan parasites of fish and crustaceans. The small-subunit rRNA genes from these parasites and from the "rosette agent" (also a parasite of fish) together form a novel, statistically supported clade. Phylogenetic analyses demonstrate this clade to diverge near the animal-fungal dichotomy, although more precise resolution is problematic. In the most parsimonious and maximally likely phylogenetic frameworks inferred from the most stably aligned sequence regions, the clade constitutes the most basal branch of the metazoa; but within a limited range of model parameters, and in some analyses that incorporate less well-aligned sequence regions, an alternative topology in which it diverges immediately before the animal-fungal dichotomy was recovered. Mitochondrial cristae of Dermocystidium spp. are flat, whereas those of Ichthyophonus hoferi appear tubulovesiculate. These results extend our understanding of the types of organisms from which metazoa and fungi may have evolved.

Antigenic variation and the within-host dynamics of parasites.

Many parasites exhibit antigenic variation within their hosts. We use mathematical models to investigate the dynamical interaction between an antigenically varying parasite and the host's immune system. The models incorporate antigenic variation in the parasite population and the generation of immune responses directed against (i) antigens specific to individual parasite variants and (ii) antigens common to all the parasite variants. Analysis of the models allows us to evaluate the relative importance of variant-specific and cross-reactive immune responses in controlling the parasite. Early in the course of infection within the host, when parasite diversity is below a defined threshold value (the value is determined by the biological properties of the parasite and of the host's immune response), the variant-specific immune responses are predominant. Later, when the parasite diversity is high, the cross-reactive immune response is largely responsible for controlling the parasitemia. It is argued that increasing antigenic diversity leads to a switch from variant-specific to cross-reactive immune responses. These simple models mimic various features of observed infections recorded in the experimental literature, including an initial peak in parasitemia, a long and variable duration of infection with fluctuating parasitemia that ends with either the clearance of the parasite or persistent infection.

Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine.

Plasmodium falciparum malaria parasites were transformed with plasmids containing P. falciparum or Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (dhfr-ts) coding sequences that confer resistance to pyrimethamine. Under pyrimethamine pressure, transformed parasites were obtained that maintained the transfected plasmids as unrearranged episomes for several weeks. These parasite populations were replaced after 2 to 3 months by parasites that had incorporated the transfected DNA into nuclear chromosomes. Depending upon the particular construct used for transformation, homologous integration was detected in the P. falciparum dhfr-ts locus (chromosome 4) or in hrp3 and hrp2 sequences that were used in the plasmid constructs as gene control regions (chromosomes 13 and 8, respectively). Transformation by homologous integration sets the stage for targeted gene alterations and knock-outs that will advance understanding of P. falciparum.

Restriction-modification systems as genomic parasites in competition for specific sequences.

Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virus-infected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.

Sphingolipid synthesis as a target for chemotherapy against malaria parasites.

The human malaria parasite Plasmodium falciparum contains sphingomyelin synthase in its Golgi apparatus and in a network of tubovesicular membranes in the cytoplasm of the infected erythrocyte. Palmitoyl and decanoyl analogues of 1-phenyl-2-acylamino-3-morpholino-1-propanol inhibit the enzyme activity in infected erythrocytes. An average of 35% of the activity is extremely sensitive to these drugs and undergoes a rapid, linear decrease at drug concentrations of 0.05-1 microM. The remaining 65% suffers a slower linear inhibition at drug concentrations ranging from 25 to 500 microM. Evidence is presented that inhibition of the sensitive fraction alone selectively disrupts the appearance of the interconnected tubular network in the host cell cytoplasm, without blocking secretory development at the parasite plasma membrane or in organelles within the parasite, such as the Golgi and the digestive food vacuole. This inhibition also blocks parasite proliferation in culture, indicating that the sensitive sphingomyelin synthase activity as well as the tubovesicular network may provide rational targets for drugs against malaria.

Sex proportions of Haemoproteus blood parasites and local mate competition.

Recent genetic evidence suggests that parasitic protozoa often reproduce by "selfing," defined as sexual stages from a single, clonal lineage fertilizing each other. Selfing favors production of an excess of female over male progeny. We tested whether the proportion of male gametocytes of blood parasites of the genus Haemoproteus was affected by variables that could influence the probability of selfing. Proportions of male Haemoproteus gametocytes from 11 passerine host populations were not affected by the age of the parasites' avian hosts, date in season, sex of host, intensity of host's infection, or prevalence of parasites within host populations.

Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites.

Immunization of rodents and humans with irradiation-attenuated malaria sporozoites confers preerythrocytic stage-specific protective immunity to challenge infection. This immunity is directed against intrahepatic parasites and involves T cells and interferon gamma, which prevent development of exoerythrocytic stages and subsequent blood infection. The present study was undertaken to determine how protective immunity is achieved after immunization of rodent hosts with irradiated Plasmodium berghei sporozoites. We present evidence that irradiated parasites persist in hepatocytes of rats and mice for up to 6 months after immunization. A relationship between the persistence of parasites and the maintenance of protective immunity was observed. Protective immunity was abrogated in irradiated-sporozoite-immunized rats following the application of chemotherapy to remove preexisting liver parasites. Additionally, protective immunity against sporozoite challenge was established in rats vaccinated with early and late hepatic stages of irradiated parasites. These results show that irradiation-attenuated sporozoites produce persistent intrahepatic stages in vivo necessary for the induction and maintenance of protective immunity.

Parkinson's disease and gastrointestinal dysfunctions: morphological, neurochemical and functional modifications of the enteric nervous system associated with central dopaminergic impairment

Parkinson’s disease (PD) is frequently associated with gastrointestinal (GI) symptoms, mostly represented by abdominal distension, constipation and defecatory dysfunctions. Despite GI dysfunctions have a major impact on the clinical picture of PD, there is currently a lack of information on the neurochemical, pathological and functional correlates of GI dysmotility associated with PD. Moreover, there is a need of effective and safe pharmacological therapies for managing GI disturbances in PD patients. The present research project has been undertaken to investigate the relationships between PD and related GI dysfunctions by means of investigations in an animal model of PD induced by intranigral injection of 6-hydroxydopamine (6-OHDA). The use of the 6-OHDA experimental model of PD in the present program has allowed to pursue the following goals: 1) to examine the impact of central dopaminergic denervation on colonic excitatory cholinergic and tachykininergic neuromotility by means of molecular, histomorphologic and functional approaches; 2) to elucidate the role of gut inflammation in the onset and progression of colonic dysmotility associated with PD, characterizing the degree of inflammation and oxidative damage in colonic tissues, as well as identifying the immune cells involved in the production of pro-inflammatory cytokines in the gut; 3) to evaluate the impact of chronic treatment with L-DOPA plus benserazide on colonic neuromuscular activity both in control and PD animals. The results suggest that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory cholinergic neurotransmission and an enhanced tachykininergic control, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate. These motor alterations might result from the occurrence of a condition of gut inflammation associated with central intranigral denervation. The treatment with L-DOPA/BE following central dopaminergic neurodegeneration can restore colonic motility, likely through a normalization of the cholinergic enteric neurotransmission, and it can also improve the colonic inflammation associated with central dopaminergic denervation.

Symbionts in Mesozooplankton Communities from NE Atlantic Ocean: Ecology and Recruitment of Parasites to the Marine Trophic Web

Beca JAE-Predoctoral CISC; Proyecto LARECO CTM2011-25929