Proteomic analysis of plasma exosomes from Cystic Echinococcosis patients provides in vivo support for distinct immune response profiles in active vs inactive infection and suggests potential biomarkers.

The reference diagnostic method of human abdominal Cystic Echinococcosis (CE) is imaging, particularly ultrasound, supported by serology when imaging is inconclusive. However, current diagnostic tools are neither optimal nor widely available. The availability of a test detecting circulating biomarkers would considerably improve CE diagnosis and cyst staging (active vs inactive), as well as treatments and follow-up of patients. Exosomes are extracellular vesicles involved in intercellular communication, including immune system responses, and are a recognized source of biomarkers. With the aim of identifying potential biomarkers, plasma pools from patients infected by active or inactive CE, as well as from control subjects, were processed to isolate exosomes for proteomic label-free quantitative analysis. Results were statistically processed and subjected to bioinformatics analysis to define distinct features associated with parasite viability. First, a few parasite proteins were identified that were specifically associated with either active or inactive CE, which represent potential biomarkers to be validated in further studies. Second, numerous identified proteins of human origin were common to active and inactive CE, confirming an overlap of several immune response pathways. However, a subset of human proteins specific to either active or inactive CE, and central in the respective protein-protein interaction networks, were identified. These include the Src family kinases Src and Lyn, and the immune-suppressive cytokine TGF-ß in active CE, and Cdc42 in inactive CE. The Src and Lyn Kinases were confirmed as potential markers of active CE in totally independent plasma pools. In addition, insights were obtained on immune response profiles: largely consistent with previous evidence, our observations hint to a Th1/Th2/regulatory immune environment in patients with active CE and a Th1/inflammatory environment with a component of the wound healing response in the presence of inactive CE. Of note, our results were obtained for the first time from the analysis of samples obtained in vivo from a well-characterized, large cohort of human subjects.

Interspecies conservation of gene order and intron-exon structure in a genomic locus of high gene density and complexity in Plasmodium.

A 13.6 kb contig of chromosome 5 of Plasmodium berghei, a rodent malaria parasite, has been sequenced and analysed for its coding potential. Assembly and comparison of this genomic locus with the orthologous locus on chromosome 10 of the human malaria Plasmodium falciparum revealed an unexpectedly high level of conservation of the gene organisation and complexity, only partially predicted by current gene-finder algorithms. Adjacent putative genes, transcribed from complementary strands, overlap in their untranslated regions, introns and exons, resulting in a tight clustering of both regulatory and coding sequences, which is unprecedented for genome organisation of PLASMODIUM: In total, six putative genes were identified, three of which are transcribed in gametocytes, the precursor cells of gametes. At least in the case of two multiple exon genes, alternative splicing and alternative transcription initiation sites contribute to a flexible use of the dense information content of this locus. The data of the small sample presented here indicate the value of a comparative approach for Plasmodium to elucidate structure, organisation and gene content of complex genomic loci and emphasise the need to integrate biological data of all Plasmodium species into the P.falciparum genome database and associated projects such as PlasmodB to further improve their annotation.

Assay of gamma-glutamylcysteine synthetase activity in Plasmodium berghei by liquid chromatography with electrochemical detection.

This work describes a high-performance liquid chromatography (HPLC) method to determine gamma-glutamylcysteine (gamma-GC), the intermediate product of glutathione biosynthesis. Separation relies on isocratic reversed-phase chromatography using a Symmetry C18 HPLC column, particle size 5 microm, 4.6 x 250 mm i.d. The mobile phase is methanol-dibasic sodium phosphate (pH 6.6; 2.8 mM) (10:90, v/v) at the flow-rate of 0.5 ml/min and detection is operated electrochemically (+200 and +550 mV) with a pre-column derivatisation reaction using ortho-phthalaldehyde (OPA) as reagent. Under these conditions the calibration range of gamma-GC was 0.3-10 microg/ml; the limit of quantification was 0.3 microg/ml; accuracy, expressed as %Bias, was <10 and precision (%CV) was <6. The proposed HPLC assay was used to quantitate the gamma-glutamylcysteine produced by the gamma-glutamylcysteine synthetase of the rodent malaria parasite Plasmodium berghei in an in vitro enzymatic assay.

Targeted terminal deletions as a tool for functional genomics studies in Plasmodium.

We describe a transfection system that induces terminal deletions at specific chromosome ends in malaria parasites using a linear construct containing telomeric repeats at one end and plasmodial sequences able to drive homologous recombination at the other. A site-specific deletion was generated at one extremity of chromosome 5 of Plasmodium berghei, which was stably maintained in the parasite population selected after transfection. The telomeric repeat array introduced with the construct reached the average length observed in natural telomeres of Plasmodium, indicating that in vivo telomere addition occurred at the newly formed extremity. The expression of a mutant dhfr/ts gene conferring pyrimethamine resistance, used as a selectable marker, was not affected by the proximity to the telomeric sequences, either in the presence or absence of drug pressure. In addition, no transcriptional silencing was observed on insertion of the mutant dhfr/ts gene either in subtelomeric or internal positions that are transcriptionally silent in blood-stage parasites. This suggests that the activity of its promoter is not affected by the chromatin organization of the chromosomal context.

The putative gene for the first enzyme of glutathione biosynthesis in Plasmodium berghei and Plasmodium falciparum.

The putative gene for gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, has been characterized both in Plasmodium berghei and Plasmodium falciparum. Protein sequence comparison between these two species reveals large conserved regions sharing more than 80% similarity, separated by less conserved portions. When the comparison is extended to known gamma-glutamylcysteine synthetases from other eukaryotes, a number of high similarity blocks are observed which may help in identifying sequence essential for protein function.

Genome plasticity and sexual differentiation in Plasmodium.

Spontaneous subtelomeric deletions of Plasmodium chromosomes have been observed both in natural infections and in laboratory maintained parasites. In the latter case, functions dispensable for asexual parasite multiplication and encoded at the extremities of the chromosomes are easily lost. In particular, spontaneous subtelomeric deletions have been characterised which affect gametocytogenesis both in Plasmodium berghei maintained in laboratory animals and in Plasmodium falciparum propagated in in vitro cultures. In order to identify these genetic determinants, and, potentially, other genes located subtelomerically, we designed a transfection system able to induce and select for controlled, site-specific subtelomeric deletions.

Developmental regulation of a Plasmodium gene involves the generation of stage-specific 5' untranslated sequences.

The B7 gene of Plasmodium berghei, highly conserved within the genus Plasmodium, encodes a nuclear protein most likely involved in chromatin assembly. In this study we describe the transcription pattern of B7 during asexual multiplication and sexual differentiation of the parasites in the blood of the vertebrate host. Two alternative transcripts have been identified: one, 1.4 kb in length is specific for asexual blood stages; the other, 1.8 kb in length is specific for sexually differentiated cells (gametocytes). The processed mRNAs are identical in their coding region and differ only in their 5' untranslated regions (5' UTRs). We show here that the differences in 5' UTRs are the result of two mechanisms: (1) the use of alternative transcription initiation sites mapped at least 1.4 kb apart, which imply the existence of separate, stage-specific promoters; (2) the splicing of a 765 bp gametocyte-specific intron at the 5' UTR of the 1.8 kb transcript.

A chromatin-associated protein is encoded in a genomic region highly conserved in the Plasmodium genus.

A single copy gene, pbB7, encoding a putative 26 kDa acidic protein has been isolated from Plasmodium berghei and appears to be part of a genomic region well conserved within the Plasmodium genus. The deduced amino acid sequence exhibits significant blocks of similarity with nucleosome assembly proteins from yeast and man. The nuclear localization of the natural protein and its close association with chromatin during the entire erythrocytic cycle of the parasite have been demonstrated using specific monoclonal antibodies against the pbB7 product expressed in Escherichia coli. These results suggest an involvement of this nuclear factor in the dynamics of chromatin packaging.

Isolation of a distally located gene possibly correlated with gametocyte production ability.

Previous studies were focussed on the attempt to correlate observable variations in the size of Plasmodium berghei chromosomes with the loss of ability to produce viable gametocytes. A temporal coincidence between the appearance of a subtelomeric deletion on P. berghei chromosome 5 and the loss of the ability to produce viable gametocytes was observed in a clone (HPE) directly derived from the high gametocyte-producer clone 8417 during mechanical passages. Interestingly enough, three P. berghei sexual-specific genes have already been mapped on internal fragments of this chromosome. A novel gene, clone 150, isolated from a genomic library of clone 8417 using a probe enriched for sexual-specific transcripts, maps on chromosome 5 within 100kb from the telomere. Subtelomeric deletions of chromosome 5 affecting two non-producer clones involve part of the transcribed region of this gene.

Isolation of a distally located gene possibly correlated with gametocyte production ability

Previous studies were focussed on the attempt to correlate observable variations in the size of Plasmodium berghei chromosomes with the loss of ability to produce viable gametocytes. A temporal coincidence between the appearance of a subtelomeric deletion on P. berghei chromosome 5 and the loss of the ability to produce viable gametocytes was observed in a clone (HPE) directly derived from the high gametocyte-producer clone 8417 during mechanical passages. Interestingly enough, three P. berghei sexual-specific genes have already been mapped on internal fragments of this chromosome. A novel gene, clone 150, isolated from a genomic library of clone 8417 using a probe enriched for sexual-specific transcripts, maps on chromosome 5 within 100kb from the telomere. Subtelomeric deletions of chromosome 5 affecting two non-producer clones involve part of the transcribed region of this gene.

Bent helical structure in Trypanosoma lewisi minicircles.

By gel retardation assay and computational analysis we demonstrated a bent region in Trypanosoma lewisi, localized in two different classes of minicircles. We showed that in each minicircle this bent region is unique, adjacent to one of two highly conserved regions and characterized by adenine stretches. The same properties are conserved in the majority of minicircles from Trypanosomes tested so far. Therefore, we suggest that the genetic information could be located in a definite structure of minicircle DNA molecules rather than in the nucleotide sequence.

Two identical symmetrical regions in the minicircle structure of Trypanosoma lewisi kinetoplast DNA.

Two minicircles of Trypanosoma lewisi kinetoplast DNA, linearized by different restriction enzymes, have been cloned in M13 derivative vectors. Their nucleotide sequences share a DNA fragment which accounts for 10% of the minicircle total length. This highly conserved region (constant region) is present twice within the same minicircle in a direct orientation and in an almost symmetrical location. Comparison of the T. lewisi constant region with those of T. brucei and T. equiperdum shows identical short nucleotide blocks which are shared by all the minicircles so far analyzed.

Isolation of a single messenger RNA and of the corresponding gene in Plasmodium berghei.

A genomic library of Plasmodium berghei DNA was constructed using lambda 47.I as a vector. It represents 90% of Plasmodium genome. Genes expressed during the intraerythrocytic stage of P. berghei were isolated among the recombinant clones of the library using labelled cDNA complementary to the polyA + Plasmodium mRNA extracted during this stage. The purified coding strand of an expressed clone was utilized to catch the corresponding mRNA(s). The hybridized mRNA fraction was eluted and in vitro translated. Translation products were analyzed by gel electrophoresis; the gel fluorography revealed a single protein band of 32.500 daltons of molecular weight, corresponding to a 900bp coding region in the examined clone.

The glycoproteins of the complex surface coat of Trypanosoma lewisi.

Blood stream forms of Trypanosoma lewisi from rats previously infected were labelled in vitro by galactose-oxidase oxidation followed by NaB3H4 reduction and subjected to 7.5% SDS-polyacrylamide gel analysis, by this procedure 8 bands were detected on SDS polyacrylamide gel electrophoresis. We conclude that surface composition of Trypanosoma lewisi resulted more complex than that of Trypanosoma so far studied, suggesting a possible relation with induced immunity and reduced ability of the parasite to survive in its host.

Mosquito infectivity is directly related to the proportion of repetitive DNA in Plasmodium berghei.

A strain of Plasmodium berghei (NK 65) was followed during syringe transmission in mice for over 120 passages after the last complete cycle, while the following parameters were monitored: (a) capacity to infect mosquitoes, inducing oocyst formation; (b) presence in the peripheral blood of morphologically identifiable gametocytes; (c) presence of a repetitive component in the DNA extracted from intraerythrocytic population. The suggestion of a possible role of this component in gametogenesis came from an earlier work (Dore, E., Birago, C., Frontali, C. and Battaglia, P.A. (1980) Mol. Biochem. Parasitol. 1, 199-208). Present results confirm the correlation between proportion of repetitive DNA and infectivity towards mosquitoes with a correlation coefficient r = 0.92-0.07+0.04. A parallel decrease of the two quantities is observed in the course of syringe transmission. A limited number of cloned lines, derived from strain NK 65 at different times during syringe transmission, shared the infectivity properties of the parent strain at the moment of cloning, thus confirming that in the infective stage single asexual parasites from the schizogonic cycle are able to originate the whole cycle. The above arguments and results suggest that differentiation into active gametocytes involves amplification of a portion of the genome.