Noel B. Murphy

Prohibitin and RACK homologues are up-regulated in trypanosomes induced to undergo apoptosis and in naturally occurring terminally differentiated forms.

Two genes have been identified as up-regulated late during ConA-induced apoptosis in procyclic form Trypanosoma brucei rhodesiense. The first represents a homologue of prohibitin, a proto-oncogene originally described in mammals and subsequently in yeast, which is involved in cell-cycle control and senescence. The Trypanosoma prohibitin homologue appears to contain within it a putative death domain. The second gene, homologous to a family of regulatory proteins which are receptors for activated protein kinase C (RACKs), is also shown to be up-regulated in terminally differentiated bloodstream form trypanosomes. These are the first endogenous genes to be identified as up-regulated in programmed cell death (PCD) in unicellular organisms.

Three genes and two isozymes: Gene conversion and the compartmentalization and expression of the phosphoglycerate kinases of Trypanosoma (Nannomonas) congolense

The glycosome, a microbody organelle found only in kinetoplastid protozoa, compartmentalizes the first six enzymes of glycolysis. In order to better understand the regulation and targeting of glycolytic enzymes in trypanosomes, we have cloned and analyzed the three genes of the phosphoglycerate kinase (PGK) complex of Trypanosoma (Nannomonas) congolense. The organization of the genes within the complex is similar to that of Trypanosoma brucei brucei. The nucleotide and amino-acid sequences, including those of the novel high-molecular-weight 56PGK, show substantial cross-species similarity. However, the two downstream genes, c1PGK and c2PGK, encode identical isozymes in T. congolense, while they encode distinct glycosomal and cytoplasmic isozymes in T. brucei. Western analysis also indicated that there are only two isozymes in T. congolense and that these are constitutively expressed. Differential digitonin solubilization of the trypanosomes indicated that 56PGK is primarily localized to the glycosome, as expected, and that c1/c2PGK is cytoplasmic. Northern analysis demonstrates that while 56PGK is constitutively expressed, c1PGK and c2PGK mRNAs are differentially expressed in the T. congolense developmental stages. This work demonstrates that T. congolense has only one PGK isozyme, 56PGK, that is predominantly localized in glycosomes.

Serum xanthine oxidase: origin, regulation, and contribution to control of trypanosome parasitemia.

African trypanosomiasis is caused by Salivarian trypanosomes, tsetse fly-transmitted protozoa that inhabit the blood plasma, lymph and interstitial fluids, and, in the case of Trypanosoma brucei species, also the cerebrospinal fluid of mammal hosts. Trypanosomiasis in people and domestic animals manifests as recurring waves of parasites in the blood and is typically fatal. In contrast, trypanosomiasis in Cape buffaloes, which are naturally selected to resist the disease, is characterized by the development of only one or a few waves of parasitemia, after which the infection becomes cryptic, being maintained by the presence of 1-20 mammal-infective organisms/ml of blood. The control of the acute phase of parasitemia in Cape buffaloes correlates with a decline in blood catalase activity and the generation of trypanocidal H(2)O(2) in serum during the catabolism of endogenous purine by xanthine oxidase. Here we review features of this response, and of trypanosome metabolism, that facilitate H(2)O(2)-mediated killing of the parasites with minimal damage to the host. We also discuss the origin and regulation of serum xanthine oxidase and catalase, and show how recovery of serum catalase in infected Cape buffaloes precludes a role for H(2)O(2) in the long-term, stable suppression of trypanosome parasitemia.

Recombinant Tumor Necrosis Factor Alpha Does Not Inhibit the Growth of African Trypanosomes in Axenic Cultures

ABSTRACT Mice whose tumor necrosis factor alpha (TNF-α) genes were disrupted developed higher levels of parasitemia than wild-type mice following infection with Trypanosoma congolense IL1180 or T. brucei brucei GUTat3.1, confirming the results of earlier studies. To determine whether TNF-α directly affects the growth of these and other bloodstream forms of African trypanosomes, we studied the effects of recombinant mouse, human, and bovine TNF-α on the growth of two isolates of T. congolense, IL1180 and IL3338, and two isolates of T. brucei brucei, GUTat3.1 and ILTat1.1, under axenic culture conditions. The preparations of recombinant TNF-α used were biologically active as determined by their capacity to kill L929 cells. Of five recombinant TNF-α lots tested, one lot of mouse TNF-α inhibited the growth of both isolates of T. brucei brucei and one lot of bovine TNF-α inhibited the growth of T. brucei brucei ILTat1.1 but only at very high concentrations and without causing detectable killing of the parasites. The other lots of mouse recombinant TNF-α, as well as human TNF-α, did not affect the growth of any of the test trypanosomes even at maximal concentrations that could be attained in the culture systems (3,000 to 15,000 U of TNF-α/ml of medium). These results suggest that exogenously added recombinant TNF-α generally does not inhibit the growth of African trypanosomes under the culture conditions we used. The impact of TNF-α on trypanosome parasitemia may be indirect, at least with respect to the four strains of trypanosomes reported here.

Programmed cell death in procyclic Trypanosoma brucei rhodesiense is associated with differential expression of mRNAs.

Procyclic Trypanosoma brucei rhodesiense have a cell death mechanism which can be activated by an external signal, the lectin ConA, in vitro. ConA has been shown to cause profound changes in cellular morphology and induce fragmentation of nuclear DNA in T.b. rhodesiense which are characteristic of apoptosis, a form of programmed cell death (PCD) in other eukaryotic cells. RNA analysis of trypanosomes induced to undergo PCD revealed that RNA remains intact up to 48 h into the process, a time when nuclear DNA fragmentation has already started. Using the randomly amplified differentially expressed sequences polymerase chain reaction method, ConA-induced cell death in T.b. rhodesiense is shown to be associated with differential expression of mRNAs, including up regulation of mRNAs late in the death process. The results demonstrate that trypanosomes actively participate in their own destruction through a PCD process and confirm that cell death in trypanosomes is associated with de novo gene expression.

Innate and acquired control of trypanosome parasitaemia in Cape buffalo

The review discusses the roles of serum xanthine oxidase, serum catalase and trypanosome-specific immune responses in the regulation of the level of trypanosome parasitaemic waves in Cape buffalo.

INNATE AND ACQUIRED RESISTANCE TO AFRICAN TRYPANOSOMIASIS

The review discusses the current field status of human and bovine trypanosomiases, and focuses on the molecular basis of innate and acquired control of African trypanosomes in people, cattle, and Cape buffalo.

Towards a Trypanosomiasis Vaccine

This essay examines efforts at vaccine development for trypanosomiasis. We contrast antibodies with other trypanocidal materials of host origin, concluding that the goal of the trypanosome vaccine hunter should be to identify conserved trypanosome antigens that elicit a trypanocidal or trypanostatic antibody response that can be boosted by natural infection. In the search for conserved and surface accessible trypanosome antigens, on-going research indicates that tomato lectin-purified trypanosome components, which include growth factor receptors, elicit host protective antibody responses.

Rapid Identification of Differentially Expressed Genes in Trypanosomes

Members of the genus Trypanosoma, within the order Kinetoplastida, are unicellular parasitic organisms which are a major threat to human health and livestock productivity in large areas of the developing world. Despite their relatively small genome size, these primitive organisms undergo complex life cycles, are capable of controlling their rates of proliferation at different stages, can modulate the immune system of their mammalian hosts, and, more recently, have been shown to undergo programmed cell death (see Ameisen et al. 1995; Vickerman 1985; Zilberstein and Shapira 1994). Many of the molecular features and processes of these organisms provide paradigms for eukaryotic biology (see Adler and Hajduk 1994; Bonen 1993; Gonners-Ampt and Borst 1995; Pays et al. 1994; Simpson and Maslov 1994; Tschudi and Ullu 1994). To gain a better understanding of the molecular mechanisms involved in the control of differentiation of these intriguing and deadly organisms we have developed a differential display PCR method, dubbed randomly amplified differentially expressed sequences (RADES), for the rapid identification of differentially expressed trypanosome or leishmania genes (Murphy and Pelle 1994).