Complete sequences were obtained for the coding portions of the mitochondrial (mt) genomes of Schistosoma mansoni (NMRI strain, Puerto Rico; 14 415 bp), S. japonicum (Anhui strain, China; 14 085 bp) and S. mekongi (Khong Island, Laos; 14 072 bp). Each comprises 36 genes: 12 protein-encoding genes (cox1-3, nad1-6, nad4L, atp6 and cob); two ribosomal RNAs, rrnL (large subunit rRNA or 16S) and rrnS (small subunit rRNA or 12S); as well as 22 transfer RNA (tRNA) genes. The atp8 gene is absent. A large segment (9.6 kb) of the coding region (comprising 14 tRNAs, eight complete and two incomplete protein-encoding genes) for S. malayensis (Baling, Malaysian Peninsula) was also obtained. Each genome also possesses a long non-coding region that is divided into two parts (a small and a large non-coding region, the latter not fully sequenced in any species) by one or more tRNAs. The protein-encoding genes are similar in size, composition and codon usage in all species except for cox1 in S. mansoni (609 aa) and cox2 in S. mekongi (219 aa), both of which are longer than homologues in other species. An unexpected finding in all the Schistosoma species was the presence of a leucine zipper motif in the nad4L gene. The gene order in S. mansoni is strikingly different from that seen in the S. japonicum group and other flatworms. There is a high level of identity (87-94% at both the nucleotide and amino acid levels) for all protein-encoding genes of S. mekongi and S. malayensis. The identity between genes of these two species and those of S. japonicum is less (56-83% for amino acids and 73-79% for nucleotides). The identity between the genes of S. mansoni and the Asian schistosomes is far less (33-66% for amino acids and 54-68% for nucleotides), an observation consistent with the known phylogenetic distance between S. mansoni and the other species.
Eight strains of a lizard Leishmania species, L. tarentolae, were compared with four other saurian species [L. hoogstrali, L. adleri, L. agamae and Leishmania sp. LizS], with L. major from man and with Trypanosoma platydactyli, a putative lizard trypanosome, in terms of kinetoplast DNA minicircle and maxicircle sequences and in terms of nuclear chromosome patterns on orthogonal gel electrophoresis. The L. tarentolae strains fell into two major groups, one (group A) consisting of the L. tarentolae strains, UC, Krassner and Trager, derived from an Algerian gecko isolate and the other (group B) consisting of five L. tarentolae LEM strains isolated from geckos in southern France. T. platydactyli TPCL2, which was postulated by Wallbanks et al. to represent the lizard form of a French L. tarentolae strain, was closely related to the UC strain and not to the LEM strains, in all respects analyzed. Leishmania sp. LizS from a Mongolian gecko and L. hoogstrali from a Sudanese gecko showed some sequence similarities to the L. tarentolae strains, but the leishmanias said to be L. adleri from a Kenyan lacertid and L. agamae from an Israeli agamid showed no minicircle sequence similarities with lizard Leishmania and in fact were probably the same species. The maxicircle divergent region was larger in the group B strains than in the group A strains, but there were sequences in common with both groups, and not with L. hoogstrali and L. major. Four strains of L. tarentolae, the four other supposed saurian Leishmania species, three mammalian leishmanias, T. platydactyli and four other trypanosomes, T. cyclops (Malaysian macaque), T. conorrhini (Hawaiian reduviid bug), T. cruzi (man) and T. lewisi (feral rat) were analyzed for their contents of sterols and phosphoglyceride fatty acyl groups. T. platydactyli TPCL2 contained a sterol (5-dehydroepisterol), a phosphatidylcholine fatty acyl group (alpha-linolenic acid) and a phosphatidylethanolamine fatty acyl group (dihydrosterculic acid) characteristic of members of the genus Leishmania and not the genus Trypanosoma. The proportions of those lipids in the free sterol and phosphoglyceride fractions of T. platydactyli TPCL2 most closely resembled those seen in the Leishmania strains from Algerian, French, Mongolian and Sudanese geckos.
Random amplification of polymorphic DNA (RAPD) was used to analyse genomic DNA from virgin females and males of Brugia malayi, with a view to identifying sex-specific differences predicted by an XX/XY system of chromosomal sex determination. A product of 2338 bp, amplified with the arbitrary primer 5' GTTGCGATCC 3', was obtained exclusively from males. Primers based on the sequence of this product amplified a DNA fragment of the expected size from each of two independent isolates of B. malayi (from Malaysia and Indonesia) by PCR. No reaction product was obtained from the closely related species Brugia pahangi. In a genetic cross between B. malayi males and B. pahangi females, F1 hybrid microfilariae were PCR-positive, indicating that the locus is paternally-inherited. Southern blotting demonstrated that the target sequence resides in the high molecular weight fraction of genomic DNA, confirming that it is of chromosomal, rather than mitochondrial, origin. Sequencing of the locus revealed significant similarity with members of a family of reverse transcriptase-like genes in Caenorhabditis elegans. In-frame stops indicate that the gene is non-functional, but multiple bands of hybridisation in Southern blots suggest that the RT sequence may be the relic of a transposable element. Multiple repeats of the dinucleotide AT occurred in another region of the sequence. These varied in number between the two isolates of B. malayi in the manner of a microsatellite, surprisingly the first to be described from the B. malayi genome. Because of its association with the Y chromosome, we have given the locus the acronym TOY (Tag On Y). Identification of this chromosome-specific marker confirms the XX/XY heterogametic karyotype in B. malayi and opens the way to elucidation of the role of Y in sex determination.
Recognition sites for nine different restriction endonucleases were mapped on rDNA genes of fasciolid species. Southern blots of digested DNA from individual worms were probed sequentially with three different probes derived from rDNA of Schistosoma mansoni and known to span between them the entire rDNA repeat unit in that species. Eighteen recognition sites were mapped for Fasciola hepatica, and seventeen for Fasciola gigantica and Fascioloides magna. Each fasciolid species had no more than two unique recognition sites, the remainder being common to one or both of the other two species. No intraspecific variation in restriction sites was noted in F. hepatica (individuals from 11 samples studied; hosts were sheep, cattle and laboratory animals; geographical origins. Australia, New Zealand, Mexico, U.K., Hungary and Spain), or in F. gigantica (two samples; Indonesia and Malaysia). Only one sample of F. magna was available. One specimen of Fasciola sp. from Japan (specific identity regarded in the literature as uncertain) yielded a restriction map identical to that of F. gigantica. Almost all recognition sites occurred in or near the putative rRNA coding regions. The non-transcribed spacer region had few or no cut sites despite the fact that this region is up to about one half of the entire repeat unit in length. Length heterogeneity was noted in the non-transcribed spacer, even within individual worms.
The intestinal tract of schistosomes opens at the mouth and leads into the foregut or oesophageal region that is lined with syncytium continuous with the apical cytoplasm of the tegument. The oesophagus is surrounded by a specialised gland, the oesophageal gland. This gland releases materials into the lumen of the oesophagus and the region is thought to initiate the lysis of erythrocytes and neutralisation of immune effectors of the host. The oesophageal region is present in the early invasive schistosomulum, a stage potentially targetable by anti-schistosome vaccines. We used a 44k oligonucleotide microarray to identify highly up-regulated genes in microdissected frozen sections of the oesophageal gland of male worms of S. mansoni. We show that 122 genes were up-regulated 2-fold or higher in the oesophageal gland compared with a whole male worm tissue control. The enriched genes included several associated with lipid metabolism and transmembrane transport as well as some micro-exon genes. Since the oesophageal gland is important in the initiation of digestion and the fact that it develops early after invasion of the mammalian host, further study of selected highly up-regulated functionally important genes in this tissue may reveal new anti-schistosome intervention targets for schistosomiasis control.
Comparing patterns of genetic variation at multiple loci in the genome of a species can potentially identify loci which are under selection. The large number of polymorphic microsatellites in the malaria parasite Plasmodium falciparum are available markers to screen for selectively important loci. The Pfs48/45 gene on Chromosome 13 encodes an antigenic protein located on the surface of parasite gametes, which is a candidate for a transmission blocking vaccine. Here, genotypic data from 255 P. falciparum isolates are presented, which show that alleles and haplotypes of five single nucleotide polymorphisms (SNPs) in the Pfs48/45 gene are exceptionally skewed in frequency among different P. falciparum populations, compared with alleles at 11 microsatellite loci sampled widely from the parasite genome. Fixation indices measuring inter-population variance in allele frequencies (F(ST)) were in the order of four to seven times higher for Pfs48/45 than for the microsatellites, whether considered (i) among populations within Africa, or (ii) among different continents. Differing mutational processes at microsatellite and SNP loci could generally affect the population structure at these different types of loci, to an unknown extent which deserves further investigation. The highly contrasting population structure may also suggest divergent selection on the amino acid sequence of Pfs48/45 in different populations, which plausibly indicates a role for the protein in determining gamete recognition and compatibility.
Schistosomes are parasitic blood flukes that infect approximately 250 million people worldwide. The disease known as schistosomiasis, is the second most significant tropical parasitic disease after malaria. Praziquantel is the only effective drug currently licensed for schistosomiasis and there are concerns about resistance to the drug. There has been much effort to develop vaccines against schistosomiasis to produce long-term protection in endemic regions. Surface-associated proteins, and in particular, those expressed in the body wall, or tegument, have been proposed as potential vaccine targets. Of these, annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the structural and immunobiochemical characterization of four homologous annexins namely annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni. Bioinformatics analysis showed that there was no signal peptide predicted for any annexin in this study. Further analysis showed that each of all four annexin protein possesses a primary structure consisting of a short but variable N-terminal region and a long C-terminal core containing four homologous annexin repeats (I-IV), which contain five alpha-helices. The life cycle expression profile of each annexin was assessed using quantitative PCR. The results showed that the overall transcript levels of the each of four homologous annexins were relatively low in the egg stage, but increased gradually after the transition of cercariae (the invasive schistosome larvae) to schistosomula (the post-invasive larvae). Circular dichroism (CD) demonstrated that rAnnexin B30, rAnnexin B5a and rAnnexin 7a were folded, showing a secondary structure content rich in alpha-helices. The membrane binding affinity was enhanced when rAnnexin B30, rAnnexin B5a and rAnnexin 7a was incubated in the presence of Ca2+. All annexin members evaluated in this study were immunolocalized to the tegument, with immunoreactivity also occurring in cells and in muscle of adult parasites. All four recombinant annexins were immunoreactive and they were recognized by the sera of mice infected with S. mansoni. In conclusion, the overall results present the molecular characterization of annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni in host-parasite interactions and strongly suggest that the molecules could be useful candidates for vaccine or diagnostic development.
The present study aimed to examine the genetic diversity of human malaria parasites (i.e., P. falciparum, P. vivax and P. knowlesi) in Malaysia and southern Thailand targeting the 19-kDa C-terminal region of Merozoite Surface Protein-1 (MSP-119). This region is essential for the recognition and invasion of erythrocytes and it is considered one of the leading candidates for asexual blood stage vaccines. However, the genetic data of MSP-119 among human malaria parasites in Malaysia is limited and there is also a need to update the current sequence diversity of this gene region among the Thailand isolates. In this study, genomic DNA was extracted from 384 microscopy-positive blood samples collected from patients who attended the hospitals or clinics in Malaysia and malaria clinics in Thailand from the year 2008 to 2016. The MSP-119 was amplified using PCR followed by bidirectional sequencing. DNA sequences identified in the present study were subjected to Median-joining network analysis with sequences of MSP-119 obtained from GenBank. DNA sequence analysis revealed that PfMSP-119 of Malaysian and Thailand isolates was not genetically conserved as high number of haplotypes were detected and positive selection was prevalent in PfMSP-119, hence questioning its suitability to be used as a vaccine candidate. A novel haplotype (Q/TNG/L) was also detected in Thailand P. falciparum isolate. In contrast, PvMSP-119 was highly conserved, however for the first time, a non-synonymous substitution (A1657S) was reported among Malaysian isolates. As for PkMSP-119, the presence of purifying selection and low nucleotide diversity indicated that it might be a potential vaccine target for P. knowlesi.
Protistan parasitic infections contribute significantly to morbidity and mortality, causing more than 2 billion human infections annually. However, current treatments are often limited; due to ineffective drugs and drug resistance, thus better options are urgently required. In the present context, theranostics agents are those that offer simultaneous detection, diagnosis and even treatment of protistan parasitic diseases. "Nanotheranostics" is the term used to describe such agents, that are around 100 nm or less in size. Anti-parasitic activity of nanoparticles (NPs) has been reported, and many have useful intrinsic imaging properties, but it is perhaps their multifunctional nature that offers the greatest potential. NPs may be used as adapters onto which various subunits with different functions may be attached. These subunits may facilitate targeting parasites, coupled with toxins to eradicate parasites, and probe subunits for detection of particles and/or parasites. The modular nature of nano-platforms promises a "mix and match" approach for the construction of tailored agents by using combinations of these subunits against different protistan parasites. Even though many of the subunits have shown promise alone, these have not yet been put together convincingly enough to form working theranostics against protistan parasites. Although the clinical application of nanotheranostics to protistan parasitic infections in humans requires more research, we conclude that they offer not just a realisation of Paul Ehrlich's long imagined "magic bullet" concept, but potentially are magic bullets combined with tracer bullets.
The transmission-blocking vaccine candidate Pfs48/45 from the human malaria parasite Plasmodium falciparum is known to be difficult to express in heterologous systems, either as full-length protein or as correctly folded protein fragments that retain conformational epitopes. In this study we express full-length Pfs48/45 in the rodent parasite P. berghei. Pfs48/45 is expressed as a transgene under control of the strong P. berghei schizont-specific msp1 gene promoter (Pfs48/45@PbMSP1). Pfs48/45@PbMSP1 schizont-infected red blood cells produced full-length Pfs48/45 and the structural integrity of Pfs48/45 was confirmed using a panel of conformation-specific monoclonal antibodies that bind to different Pfs48/45 epitopes. Sera from mice immunized with transgenic Pfs48/45@PbMSP1 schizonts showed strong transmission-reducing activity in mosquitoes infected with P. falciparum using standard membrane feeding. These results demonstrate that transgenic rodent malaria parasites expressing human malaria antigens may be used as means to evaluate immunogenicity and functionality of difficult to express malaria vaccine candidate antigens.