Trypanosoma brucei: A rapid “matching” method for estimating the host's parasitemia

A new technique for estimating the absolute level of parasitemias in trypanosome infections is described. At higher levels of infection this is achieved by matching microscopic fields of a wet blood film against charts and, where fewer organisms are present, by counting the number of trypanosomes in 5, 10, or 20 such microscope fields. Good estimates of the number of organisms per milliliter of blood can be made rapidly over the whole range of microscopically patent parasitemia, i.e., above antilog 5.4 (250,000) organisms/ml.     

Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity

The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.     

Early evolution within kinetoplastids (euglenozoa), and the late emergence of trypanosomatids

Many important relationships amongst kinetoplastids, including the position of trypanosomatids, remain uncertain, with limited taxon sampling of markers other than small subunit ribosomal RNA (SSUrRNA). We report gene sequences for cytosolic heat shock proteins 90 and/or 70 (HSP90, HSP70) from the potentially early-diverging kinetoplastids Ichthyobodo necator and Rhynchobodo sp., and from bodonid clades ‘2’ (Parabodonidae) and ‘3’ (Eubodonidae). Some of the new cytosolic HSP70 sequences represent a distinct paralog family (HSP70-B), which is related to yet another paralog known from trypanosomatids (HSP70-C). The (HSP70-B, HSP70-C) clade seemingly diverged before the separation between kinetoplastids and diplonemids. Protein phylogenies support the basal placement of Ichthyobodo within kinetoplastids. Unexpectedly, Rhynchobodo usually forms the next most basal group, separated from the clade ‘1’ bodonids with which it has been allied. Bootstrap support is often weak, but the possibility that Rhynchobodo represents a separate early-diverging lineage within core kinetoplastids deserves further testing. Trypanosomatids always fall remote from the root of kinetoplastids, forming a specific relationship with bodonid clades 2 (and 3), generally with strong bootstrap support. These protein trees with improved taxon sampling provide the best evidence to date for a ‘late’ emergence of trypanosomatids, contradicting recent SSUrRNA-based proposals for a relatively early divergence of this group.     

Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for “histone code” reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi.     

Increase in a heat-shock protein from blood cells in response of nestling house martins (Delichon urbica) to parasitism: an experimental approach

Heat-shock proteins (HSPs) are synthesized by animals and plants in response to various stressors. The level of the HSP60 stress protein was measured from the cell fraction of peripheral blood obtained from nestling house martins (Delichon urbica) to test whether ectoparasitism increased the concentration of stress protein. We assessed HSP from nestlings raised in nests previously treated with an insecticide or infested with 50 martin bugs (Oeciacus hirundinis). In addition, haematozoa infections were checked in blood smears. Nestlings from parasite-infested nests, or nestlings infected with trypanosomes, had increased levels of HSP in their blood cells. Nestling growth as determined from wing length was negatively related to HSP60 levels and within-brood variation in wing length increased with increasing levels of the stress protein independently of treatment and infection by trypanosomes. These results suggest HSPs may play a role in host-parasite interactions, and that they can be used reliably for measuring physiological responses to parasites. Received: 4 February 1998 / Accepted: 4 May 1998     

Antigens and Antigenic Variability of the African Trypanosomes*†

SYNOPSIS. The recent literature on the physical, chemical and biological characterization of antigens from the African trypanosomes is reviewed. The antigens are divided into three major groups: a) variant-specific antigens, b) common antigens, and c) host-like antigens. The variant-specific antigen(s) are relatively small molecular weight proteins located on the surface of the trypanosome, and are involved in protection and agglutination. It is suggested that there is more than a single variant-specific antigen on the cell surface. In contrast, the common antigens are internal or somatic antigens which are believed to have structural or enzymatic functions but are not involved in either protection or agglutination. In addition, evidence is also presented which suggests that there are host-like antigens within the surface coat and/or membranes of the trypanosomes. The importance of these three groups of antigens, and the mechanis(s) of antigenic variation are discussed in relationship to the immune response of the host to the African trypanosomes. Several possible approaches for future investigations are described.     

Fossil evidence of insect pathogens

The present report describes fossil evidence of insect pathogens, heretofore, almost non-existent, from six samples of amber ranging in age from 15 to 100 million years. They include a cytoplasmic polyhedrosis virus and trypanosomatid infection in an adult biting midge (Diptera: Ceratopogonidae), and a nuclear polyhedrosis virus in an adult sand fly (Diptera: Phlebotomidae), both from Early Cretaceous Burmese amber, several types of fungal thalli on the cuticle of an adult mosquito (Culicidae: Diptera), as well as a fungal growth on the prothorax of a fungus gnat (Mycetophilidae: Diptera) in Dominican amber and large tumors in the body cavity of a caterpillar (Lepidoptera) in Mexican amber. These discoveries suggest that insect polyhedrosis viruses were present 100 million years ago and present the possibility that vertebrate arboviruses (especially those in the family Reoviridae) could have evolved from cytoplasmic polyhedrosis viruses infecting biting insects. The flagellates in the Early Cretaceous biting midge represent the first fossil record of monogenetic trypanosomatid infections of arthropods.     

Development of a mathematical model for mechanical transmission of trypanosomes and other pathogens of cattle transmitted by tabanids

Mechanical transmission of pathogens by biting insects is a non-specific phenomenon in which pathogens are transmitted from the blood of an infected host to another host during interrupted feeding of the insects. A large range of pathogens can be mechanically transmitted, e.g. hemoparasites, bacteria and viruses. Some pathogens are almost exclusively mechanically transmitted, while others are also cyclically transmitted. For agents transmitted both cyclically and mechanically (mixed transmission), such as certain African pathogenic trypanosomes, the relative impact of mechanical versus cyclical transmission is essentially unknown. We have developed a mathematical model of pathogen transmission by a defined insect population to evaluate the importance of mechanical transmission. Based on a series of experiments aimed at demonstrating mechanical transmission of African trypanosomes by tabanids, the main parameters of the model were either quantified (host parasitaemia, mean individual insect burden, initial prevalence of infection) or estimated (unknown parameters). This model allows us to simulate the evolution of pathogen prevalence under various predictive circumstances, including control measures and could be used to assess the risk of mechanical transmission under field conditions. If adjustments of parameters are provided, this model could be generalized to other pathogenic agents present in the blood of their hosts (Bovine Leukemia virus, Anaplasma, etc.) or other biting insects such as biting muscids (stomoxyines) and hippoboscids.     

Vinyl sulfone-based inhibitors of trypanosomal cysteine protease rhodesain with improved antitrypanosomal activities

The number of reported cases of Human African Trypanosmiasis (HAT), caused by kinetoplastid protozoan parasite Trypanosoma brucei, is declining in sub-Saharan Africa. Historically, such declines are generally followed by periods of higher incidence, and one of the lingering public health challenges of HAT is that its drug development pipeline is historically sparse. As a continuation of our work on new antitrypanosomal agents, we found that partially saturated quinoline-based vinyl sulfone compounds selectively inhibit the growth of T. brucei but displayed relatively weak inhibitory activity towards T. brucei’s cysteine protease rhodesain. While two nitroaromatic analogues of the quinoline-based vinyl sulfone compounds displayed potent inhibition of T. brucei and rhodesain. The quinoline derivatives and the nitroaromatic-based compounds discovered in this work can serve as leads for ADME-based optimization and pre-clinical investigations.