The occurrence in the body of Bemisia tabaci of a factor apparently related to the phenomenon of "periodic acquisition" of tomato yellow leaf curl virus.
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Insect Vectors↗
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The systemic infection of an aphid by a plant virus.
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Animals↗
Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties.
Defective interfering particles (DIPs) were generated upon continuous production of Autographa californica nuclear polyhedrosis virus (AcNPV) in bioreactors. This configuration mimicked the serial undiluted passaging of virus, which is known to result in plaque-morphology mutants. Restriction enzyme analysis of DIP-containing preparations of extracellular virus showed the presence of many DNA fragments in less than equimolar amounts. These fragments were colinear on the physical map of AcNPV and extended from map position 1.7 to 45. These DIPs thus lacked 43% of the genetic information of the standard virus, including the polyhedrin and DNA polymerase genes. The existence of DIPs was confirmed by electron microscopy, where virions were observed with reduced length. Among the less than equimolar fragments in DIP-containing preparations, fragments were observed linking sequences from map positions 1.7 and 45 via a TGTT linker of unknown origin. The DIPs could not be plaque-purified and needed standard (helper) virus to replicate; DIP-containing preparations interfered with standard virus replication in an interference assay, which explained the reduction in productivity of an AcNPV expression vector-insect cell system in continuous bioreactor operations. The origin of these DIPs and their possible generation mechanism are discussed.
Animals↗
Sequences homologous to the variant antigen mRNA spliced leader are located in tandem repeats and variable orphons in trypanosoma brucei.
We have examined the organization of genomic sequences homologous to the spliced leader of Trypanosoma brucei variant surface glycoprotein (VSG) mRNA, using a synthetic oligodeoxynucleotide probe. These sequences are highly reiterated in the trypanosome genome and most are located in 1.4 kb units arranged in a direct tandem repeat. However, some of the 1.4 kb sequences are dispersed from the cluster(s) of tandem repeats and are flanked by non-repeat DNA. The number and arrangement of these leader sequence orphons varies among different T. brucei stocks. Within the IsTat serodeme, the arrangement of three of four spliced leader orphons observed with Eco RV digestion was stable during a chronic infection and cyclic transmission through the insect vector. The fourth Eco RV orphon, however, undergoes rearrangement during antigenic variation and life-cycle differentiation.
Animals↗
Point mutations are associated with a gene duplication leading to the bloodstream reexpression of a trypanosome metacyclic VSG.
African trypanosomes evade the immune response of their hosts by sequentially expressing different variant surface glycoproteins (VSGs). We isolated a bloodstream trypanosome clone of Trypanosoma brucei rhodesiense that expresses a VSG normally present during the metacyclic stage of the parasite in the insect vector. Associated with the bloodstream reexpression of this metacyclic VSG is a gene conversion in which the duplicated, expressed gene of 1650 nt contains 11 scattered point mutations when compared with its donor gene. Analysis of an uncloned population of bloodstream trypanosomes revealed another VSG reexpressor of the same donor gene in which the coding region had undergone 24 point mutations. The mutations are unique to the duplicated gene and appear to be nontemplated. The generation of these mutations provides a way for the trypanosome to increase further its antigenic diversity.
Amino Acid Sequence↗
Monoclonal antibodies against the flagellar fraction of epimastigotes of Trypanosoma cruzi: complement-mediated lytic activity against trypomastigotes and passive immunoprotection in mice.
Trypanosoma cruzi, the causative agent of Chagas' disease, is widely spread in Central and South America. The present report describes three monoclonal antibodies (mAbs) directed against the flagellar fraction of epimastigotes (Ffe) of the parasite, Tulahuén strain. The three mAbs were of IgG1 isotype. Indirect immunofluorescence assays revealed that the three mAbs bind to epimastigotes, the FCH-F8-1 and -3 bind to blood trypomastigotes (BT) and FCH-F8-1 is the only one that binds to amastigotes. Three different proteins of the parasite were recognized by the mAbs in immunoprecipitation assays: an 85 kDa of BT with the FCH-F8-1 mAb, a 40 kDa of Ffe with the FCH-F8-2 mAb, and a 90 kDa of Ffe and of BT with the FCH-F8-3 mAb. Positive complement mediated lysis (CML) of BT and metacyclic forms, obtained from the insect vector feces, were shown by the FCH-F8-1, while FCH-F8-3 only showed CML against the metacyclic trypomastigotes. FCH-F8-2 did not mediate any CML activity. Passive transference of the mAbs to BALB/c mice conferred protection, in terms of survival, ranging from 50 (FCH-F8-2 and -3) to 80% (FCH-F8-1) against a challenge with 1 X 10(3) BT. These results suggest that FCH-F8-1 may be a useful tool to purify proteins in order to investigate their role in immunoprotection experiments.
Animals↗
Effect of temperature upon catalytic properties of alpha-hydroxyacid dehydrogenase from Trypanosoma cruzi.
A comparative study of the effect of temperature (10, 20, 30 and 37 degrees C) upon Km and V of alpha-hydroxyacid dehydrogenase (HADH), isozyme I and II, from Trypanosoma cruzi, a parasite whose life cycle comprises stages in an insect vector, and of another enzyme with analogous substrate specificity, the lactate dehydrogenase, isozyme X (LDH X) from mouse, a homeotherm, is presented. The Km for alpha-ketoisocaproate of HADH is markedly reduced as temperature decreases. This effect can compensate the reduction in thermal energy and produce stabilization of the reaction rate. This compensation does not occur with mouse LDH X. The activation energy for both HADH isozymes is about half the value determined for mouse LDH X. Results indicate that HADH from T. cruzi is able to adjust instantaneously to thermal changes of the environment, behaving as other enzymes of terrestrial poikilothermic animals.
Alcohol Oxidoreductases↗
The effect of swainsonine on the association of Trypanosoma cruzi with host cells.
The role of glycoprotein processing in Trypanosoma cruzi association with mammalian host cells (i.e., surface binding and internalization) was studied by using swainsonine - an inhibitor of glycoprotein processing. The presence of swainsonine in co-cultures of blood forms of T. cruzi with either mouse peritoneal macrophages or rat heart myoblasts markedly reduced parasite-host cell association as evidenced by significant decreases in both the percentage of cells associating with the organisms and the number of parasites per cell. This inhibition appeared to result from effects on both the parasite and the host cells since pretreatment of either one reduced its association with the untreated counterpart. The inhibitory effect of swainsonine on the host cells was demonstrable with trypomastigote forms derived from either infected mouse blood or the feces of infected insect vectors, and also with amastigotes. Conversely, treatment of the parasites with swainsonine inhibited their association with the host cells. Studies with blood trypomastigotes revealed that the swainsonine effect was reversible within 3 h. Two observations suggested that swainsonine had induced the parasite to produce defective oligosaccharides rich in mannose. First, swainsonine-treated organisms displayed a greater capacity to bind concanavalin than medium-treated organisms. Second, presence of alpha-methyl mannoside reduced such increased concanavalin A binding. Taken together, the present results suggest important roles for glycoprotein processing and oligosaccharide structures on the surface of both T. cruzi and its host cells in the initial stages of host cell infection.
Alkaloids↗
Characterization of the mechanism of protein glycosylation and the structure of glycoconjugates in tissue culture trypomastigotes and intracellular amastigotes of Trypanosoma cruzi.
Trypomastigote cells of Trypanosoma cruzi incubated with [U-14C]glucose accumulated dolichol-P-P-linked Man7GlcNAc2 and Man9GlcNAc2. Evidence is presented indicating that both oligosaccharides were transferred to asparagine residues in proteins. On the other hand, intracellular amastigotes behaved as epimastigotes, i.e., only Man9GlcNAc2 accumulated and was transferred to proteins under similar incubation conditions. Intracellular amastigotes differed, therefore, from amastigotes obtained from an axenic culture, which behaved as trypomastigotes. A similar processing of protein-linked Man9GlcNAc2 and Man8GlcNAc2 occurred in epimastigotes and trypomastigotes but the structure of the main Man7GlcNAc2 isomer produced by demannosylation of the above mentioned oligosaccharides differed from that of the Man7GlcNAc2 transferred in trypomastigotes and amastigotes from axenic cultures. The infective trypomastigote stage of the parasite showed, therefore, an alteration in the mechanism of protein N-glycosylation when compared to the other stages, namely epimastigote (insect vector stage) and amastigote (mammalian intracellular stage). Complex-type, asparagine-bound oligosaccharides were found to be synthesized in both epimastigotes and trypomastigotes but the amounts of those compounds were extremely low when compared to those of high mannose-type oligosaccharides.
Animals↗
Gene exchange in African trypanosomes: frequency and allelic segregation.
The existence of a system of genetic exchange in Trypanosoma brucei is now established, but the frequency with which mating occurs and the mechanisms by which genes are exchanged are still unknown. This paper presents the results of a study of one pair of trypanosome stocks, which show that mating is a non-obligatory but frequent event in a life-cycle stage within the insect vector. Analysis of ten progeny clones using a total of eleven markers (iso-enzymes and DNA probes detecting restriction fragment length polymorphisms) has indicated that segregation of alleles occurs at five of these loci. The segregation patterns of a polymorphic EcoRI site in the maxi-circle of the kinetoplast DNA (kDNA) show that the progeny inherit one or other of the parental kDNA types. These results demonstrate that segregation of alleles occurs and that new combinations of alleles at different loci are generated in the progeny clones. The implications of these findings for defining the mechanism of gene exchange are discussed in relation to a simple mendelian genetic system involving meiosis and syngamy.
Alleles↗
The major surface glycoprotein (GP63) is present in both life stages of Leishmania.
Leishmania exist as extracellular promastigotes which multiply in the gut of the sandfly insect vector and as intracellular amastigotes which divide in the phagolysosome of mononuclear phagocytic cells of the mammalian host. Promastigotes express a major surface glycoprotein of 63 kDa, referred to as GP63. The expression of GP63 in both Leishmania life stages was studied using rabbit antibodies against native GP63 as well as rabbit antibodies against recombinant GP63 that was synthesized in an Escherichia coli expression system. Immunofluorescence staining detected GP63 in intracellular amastigotes contained within a macrophage cell line and within freshly isolated lesion amastigotes. Western blot analysis using anti-recombinant GP63 antibodies also demonstrated that amastigotes synthesize GP63 which may undergo differential post-translational processing as compared to promastigote GP63.
Animals↗
Stage-specific differential polyadenylation of mini-exon derived RNA in African trypanosomes.
The differentiation of African trypanosomes through several distinct stages in their mammalian host and insect vector correlates with differential expression of some of the parasites' genes. In the search for genes from Trypanosoma brucei brucei involved in switching from the actively dividing long slender to the non-dividing short stumpy bloodstream forms, we have isolated cDNA clones which hybridise specifically to mRNA from short stumpy forms. All clones that were characterised contained similar sequences. Northern blot analysis showed that: (i) RNA transcripts which hybridise to these clones are barely detectable in the poly(A)+ fraction of RNA from long slender bloodstream forms and absent in procyclic culture forms, but are abundant in the poly(A)+ fraction of RNA from short stumpy forms; (ii) the RNA transcripts are abundant in the poly(A)- fraction of RNA from all life cycle stages of the trypanosomes, without significant differences and, (iii) three transcripts of 160, 280 and 400 nucleotides in size are detected in the poly(A)+ fraction of RNA, whereas only a single size-class of transcript of between 140 and 160 nucleotides is detectable in the poly(A)- fraction. Sequence analysis revealed that these clones correspond to mini-exon derived RNA containing a poly(A) tail at their 3' end. The polyadenylation of the transcript is a post-transcriptional event since sequences from genomic DNA could not be amplified in the polymerase chain reaction when mini-exon and oligo(dT) nucleotide sequences were used as primers. The differences in size of the transcripts detected can be accounted for by variations in the poly(A) tail length.
Animals↗
Combined occurrence of trypanosomal sialidase/trans-sialidase activities and leishmanial metalloproteinase gene homologues in Endotrypanum sp.
Endotrypanum (order Kinetoplastida: family Trypanosomatidae) is a parasite of forest dwelling tree sloths (Edentata: genera Choleopus and Bradypus). Unique among the haemoflagellates, this protozoan has an intraerythrocytic phase in the mammalian host. Nevertheless, many striking similarities exist between Endotrypanum and the human pathogen Leishmania that make it a useful model for epidemiological and evolutionary aspects of the biology of trypanosomatids. Importantly, Endotrypanum species share both the insect vector and host reservoir with certain species of Leishmania (subgenus Viannia). Because mixed infections with Endotrypanum and Leishmania are common in sloths and, therefore, likely to occur in the sandfly vector, there is a need for adequate biochemical markers to distinguish Endotrypanum from Leishmania infections. In this paper we show that Endotrypanum promastigotes possess sialidase and trans-sialidase activities, which are absent from Leishmania, and which are not closely related to the previously described trypanosomal sialidase/trans-sialidase enzyme. We also document the occurrence in Endotrypanum of homologues of the leishmanial surface metalloproteinase gp63 genes. The combined occurrence of sialidase/trans-sialidase activities and gp63 gene homologues in a unique organism has important ramifications for both field and laboratory studies on the biology of trypanosomatids, especially those related to host infection and evolution.
Animals↗
In situ detection of Pbs21 mRNA during sexual development of Plasmodium berghei.
The patterns of expression of ribosomal RNA and mRNA encoding the parasite surface antigen Pbs21 have been investigated during the sexual stages of development of the malaria parasite, Plasmodium berghei, using the technique of non-radioactive in situ RNA hybridisation. An RNA probe complementary to a region of the small subunit of P. berghei ribosomal RNA hybridised to parasites at all stages of development in a smear of blood taken from mice infected with P. berghei. Messenger RNA encoding Pbs21, in contrast, was detected only within parasites committed to sexual development within the vertebrate host and, furthermore, was shown to be expressed in a sex-specific manner, exclusively within female gametocytes. At later stages of sexual development, Pbs21 mRNA was detected at high levels in female gametes and ookinetes. We have previously shown that Pbs21 protein is first detectable only after the initiation of gametogenesis which occurs following transmission to the insect vector. These results suggest, therefore, that post-transcriptional mechanisms operate to regulate the translation of Pbs21 mRNA as it accumulates during female gametocytogenesis.
Animals↗
Intermediary metabolism of Leishmania.
In the course of their existence, parasites develop several metabolic pathways that differ significantly from those of their hosts. Despite the fairly close evolutionary kinship between Leishmania donovani and Trypanosoma brucei, the forms that live in the insect vectors have evolved different strategies for the disposition of available food resources. In this brief review, Joseph Blum will focus on the data available from studies on Leishmania spp and will largely ignore the information available from Trypanosoma spp.
Journal Article↗
Mechanisms of stage-regulated gene expression in kinetoplastida.
During their life cycle, trypanosomatid parasites of mammals encounter substantially different environments in their hosts and insect vectors, to which they must adapt by undergoing a series of differentiation processes. At the molecular level, these processes must be the direct result of an elaborate series of changes in stage-regulated expression of a wide range of gene products. How are these changes accomplished? In this review, Sheila Graham discusses some recent advances in understanding the mechanisms of gene expression in trypanosomatids, and examines some clues to some intriguingly complex means of regulating life cycle stage-specific gene expression.
Journal Article↗
Risk of transmission of HIV to home care and health care workers.
There are only three known routes of transmission of the human immunodeficiency virus (HIV): (1) exposure to blood via transfusion, sharing of contaminated needles by drug abusers, occupational needle stick or blood spill, unsterile needle injections; (2) sexual transmission; (3) perinatal exposure. There is no evidence for transmission by close interpersonal nonsexual contact or insect vectors. Health care workers are at risk for HIV transmission through accidental parenteral inoculation or extensive exposure to blood on nonintact skin or mucous membranes. Fortunately, population studies indicate that this risk is low and may be further reduced by adherence to simple infection control guidelines. The accumulated information on low rates of occupational transmission of HIV makes unwarranted the treatment of patients with acquired immunodeficiency syndrome (AIDS) or HIV infection as if they were highly contagious in the health care setting.
Acquired Immunodeficiency Syndrome↗
Aphid transmission and a polypeptide are specified by a defined region of the cauliflower mosaic virus genome.
Infection of young turnip leaves with an aphid-transmissible isolate, Cabb B-JI, of cauliflower mosaic virus (CaMV) causes synthesis of an Mr 18 000 polypeptide (p18) which co-purifies with virus inclusion bodies. This polypeptide is not detectable in leaves infected with either of two aphid non-transmissible isolates. Campbell and CM4-184. Construction in vitro, of hybrid genomes between Cabb B-JI and Campbell isolates demonstrates that aphid transmissibility and presence of p18 is dependent on the small genome fragment from the BstEII site to the XhoI site. A deletion made in this fragment within open reading frame (ORF) II causes loss of aphid transmissibility and also terminates production of p18. We conclude that aphid transmissibility and the presence of p18 are related to the expression of ORF II of the CaMV genome.
Animals↗