Transmisión oral de Trypanosoma cruzi: una nueva situación epidemiológica de la enfermedad de Chagas en Colombia y otros países suramericanos / Oral transmission of Trypanosoma cruzi : a new epidemiological scenario for Chagas´ disease in Colombia and other South American countries

Durante la última década se han reportado numerosos casos de infección por Trypanosoma cruzi por vía oral, debidos a la contaminación de alimentos con heces de triatominos silvestres o con secreciones de reservorios en áreas donde los vectores domiciliados han sido controlados o no hay antecedentes de domiciliación. Con base en criterios epidemiológicos, clínicos y socioeconómicos, la Organización de las Naciones Unidas para la Agricultura y la Alimentación (FAO) y la Organización Mundial de la Salud (OMS) establecieron una clasificación de los parásitos transmitidos por contaminación de alimentos en diferentes regiones del mundo, en la cual T. cruzi ocupó el décimo lugar de importancia en un grupo de 24 parásitos. Los cambios ambientales, como la deforestación y el calentamiento global, han afectado los ecotopos y el comportamiento de los vectores y de los reservorios de T. cruzi , de manera que estos se han desplazado a nuevas zonas, generando una nueva forma de transmisión por contaminación de alimentos que requiere su evaluación en el país. La presente revisión aborda la transmisión oral de la enfermedad de Chagas con énfasis en los estudios orientados a identificar los factores de riesgo, las especies de triatominos involucrados, la fisiopatología de la infección oral y los genotipos del parásito que están implicados en esta forma de transmisión en Colombia y en otras regiones de América Latina, así como la necesidad de adoptar políticas para su control y vigilancia epidemiológica.

Many cases of infection caused by the oral transmission of Trypanosoma cruzi have been reported during the last decade. These have been due to the contamination of food by faeces from sylvatic triatomines or by leakage from reservoirs in areas where domiciliated vectors have been controlled or where there has been no prior background of domiciliation. The United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) have used epidemiological, clinical and socioeconomic criteria for ranking parasites transmitted by the contamination of food in different areas of the world; T. cruzi was placed tenth in importance amongst a group of 24 parasites in such ranking. Environmental changes such as deforestation and global warming have affected ecotopes and the behaviour of T. cruzi vectors and reservoirs so that these have become displaced to new areas, thereby leading to such new transmission scenario caused by the contamination of food, which requires evaluation in Colombia. The current review deals with the oral transmission of Chagas´ disease, emphasising studies aimed at identifying the pertinent risk factors, the triatomine species involved, the physiopathology of oral infection, the parasite´s genotypes implicated in this type of transmission in Colombia and other Latin American regions, as well as the need for ongoing epidemiological surveillance and control policies.

Immune responses to gp82 provide protection against mucosal Trypanosoma cruzi infection

The potential use of the Trypanosoma cruzi metacyclic trypomastigote (MT) stage-specific molecule glycoprotein-82 (gp82) as a vaccine target has not been fully explored. We show that the opsonization of T. cruzi MT with gp82-specific antibody prior to mucosal challenge significantly reduces parasite infectivity. In addition, we investigated the immune responses as well as the systemic and mucosal protective immunity induced by intranasal CpG-adjuvanted gp82 vaccination. Spleen cells from mice immunized with CpG-gp82 proliferated and secreted IFN-γ in a dose-dependent manner in response to in vitro stimulation with gp82 and parasite lysate. More importantly, these CpG-gp82-immunized mice were significantly protected from a biologically relevant oral parasite challenge.

Molecular mechanisms of Trypanosoma cruzi infection by oral route

Frequent reports on outbreaks of acute Chagas' disease by ingestion of food contaminated with parasites from triatomine insects illustrate the importance of this mode of transmission. Studies on oral Trypanosoma cruzi infection in mice have indicated that metacyclic trypomastigotes invade the gastric mucosal epithelium. A key molecule in this process is gp82, a stage-specific surface glycoprotein that binds to both gastric mucin and to target epithelial cells. By triggering Ca2+ signalling, gp82 promotes parasite internalisation. Gp82 is relatively resistant to peptic digestion at acidic pH, thus preserving the properties critical for oral infection. The infection process is also influenced by gp90, a metacyclic stage-specific molecule that negatively regulates the invasion process. T. cruzi strains expressing high gp90 levels invade cells poorly in vitro. However, their infectivity by oral route varies considerably due to varying susceptibilities of different gp90 isoforms to peptic digestion. Parasites expressing pepsin-susceptible gp90 become highly invasive against target cells upon contact with gastric juice. Such is the case of a T. cruzi isolate from an acute case of orally acquired Chagas' disease; the gp90 from this strain is extensively degraded upon short period of parasite permanence in the gastric milieu. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of Chagas' disease reported in outbreaks of oral infection.

Immunology and immunopathology of African trypanosomiasis

Major modifications of immune system have been observed in African trypanosomiasis. These immune reactions do not lead to protection and are also involved in immunopathology disorders. The major surface component (variable surface glycoprotein,VSG) is associated with escape to immune reactions, cytokine network dysfunctions and autoantibody production. Most of our knowledge result from experimental trypanosomiasis. Innate resistance elements have been characterised. In infected mice, VSG preferentially stimulates a Th 1-cell subset. A response of gd and CD8 T cells to trypanosome antigens was observed in trypanotolerant cattle. An increase in CD5 B cells, responsible for most serum IgM and production of autoantibodies has been noted in infected cattle. Macrophages play important roles in trypanosomiasis, in synergy with antibodies (phagocytosis) and by secreting various molecules (radicals, cytokines, prostaglandins,...). Trypanosomes are highly sensitive to TNF-alpha, reactive oxygen and nitrogen intermediates. TNF-alpha is also involved in cachexia. IFN-gamma acts as a parasite growth factor. These various elements contribute to immunosuppression. Trypanosomes have learnt to use immune mechanisms to its own profit. Recent data show the importance of alternative macrophage activation, including arginase induction. L-ornithine produced by host arginase is essential to parasite growth. All these data reflect the deep insight into the immune system realised by trypanosomes and might suggest interference therapeutic approaches.

Modificações importantes no sistema imune são observadas na tripanosomíase Africana. Essas reações imunológicas não protegem e estão envolvidas em distúrbios imunopatológicos. O principal componente de superfície (glicoproteína variante de superfície, VSG) está associado à evasão das respostas imune, às disfunções da rede de citocinas e à produção de autoanticorpos. Muitos de nossos conhecimentos resultam da tripanosomíase experimental. Componentes da imunidade inata estão sendo caracterizados. Em camundongos infectados, a VSG estimula preferencialmente células Th1. Uma resposta de gd e células T CD8 aos antígenos do tripanossoma foi observada em gado tripanotolerante. Um aumento em células B CD5, responsável por IgM sérica e produção de autoanticorpos, foi observado no gado infectado. Os macrófagos desempenham importantes funções na tripanosomíase, em sinergismo com anticorpos (fagocitose) e pela secreção de várias moléculas (radicais, citocinas, prostaglandinas). Tripanossomas são altamente sensíveis ao TNF-alfa, espécies reativas de oxigênio e nitrogênio. O TNF-alfa também está envolvido em caquexia. O IFN-gama atua como um fator de crescimento do parasita. Esses vários componentes contribuem para a imunossupressão. Os tripanossomas usam os mecanismos imunes para seu próprio benefício. Dados recentes mostram a importância da ativação alternativa de macrófagos, incluindo a indução pela arginase. A L-ornitina produzida pela arginase do hospedeiro é essencial para o crescimento do parasita. Todos esses dados mostram o envolvimento no sistema imune realizado pelos tripanossomas e sugerem a interferência de métodos terapêuticos.

Molecular basis of mammalian cell invasion by Trypanosoma cruzi

O estabelecimento da infecção por Trypanosoma cruzi, o agente da doença de Chagas, depende de uma série de eventos envolvendo interações de diversas moléculas do parasita com componentes do hospedeiro. Focalizamos aqui os mecanismos de invasão celular por tripomastigotas metacíclicos (TM) e por tripomastigotas de cultura de tecido (TCT). Durante a internalização de TM ou TCT, vias de transdução de sinal são ativadas tanto no parasita como na célula alvo, acarretando a mobilização de Ca2+. Para adesão, TM utiliza as glicoproteínas de superfície como a gp82 e gp35/50, que são moléculas indutoras de sinal de Ca2+. Em isolados de T. cruzi que entram na célula hospedeira de maneira dependente de gp82, a proteína tirosina quinase assim como a fosfolipase C do parasita são ativadas, e Ca2+ é liberado de reservatórios sensíveis a IP3, enquanto em isolados de T. cruzi que se ligam às células alvo através de gp35/50, a via de sinalização envolvendo adenilil ciclase parece ser estimulada, com liberação de Ca2+ de acidocalciossomos. Além disso, dependendo do isolado de T. cruzi, sinais inibitórios mediados por gp90 específica de TM podem ser desencadeados tanto na célula hospedeira como no parasita. O repertório de moléculas de TCT implicadas na invasão celular inclui glicoproteínas de superfície da família gp85, com membros contendo sitos de ligação à laminina e citoqueratina 18, enzimas como a cruzipaína, trans-sialidase, e uma oligopeptidase B que gera um agonista de Ca2+ a partir de uma molécula precursora. .

Vacinação genética na infecção experimental pelo Trypanosoma cruzi utilizando genes expressos em diferentes estágios do desenvolvimento do parasita / Genetic vaccination during experimental infection with Trypanosoma cruzi using genes expressed in different developmental stages of the parasite

A imunização de I camundongos, com plasmídios contendo genes de Trypanosoma cruzi induziu anticorpos específicos, células T CD4+ Thl, CD8+ Tcl e imunidade protetora contra infecção. Até o momento em que iniciamos este trabalho os plasmídios utilizados para vacinação com DNA continham genes codificando antígenos expressos por tripomastigotas, as formas não replicativas do parasita. No presente estudo exploramos a possibilidade de utilizar genes expressos, por amastigotas, as formas do parasita que se replicam dentro das células do hospedeiro, para vacinação experimental com DNA. Com este fim, selecionamos para nosso estudo um gene relacionado à proteína-2 de superficie de amastigotas (ASP-2), um antígeno reconhecido por anticorpos e células T de camundongos e indivíduos infectados utilizando oligonucleotídeos específicos para o gene asp-2, quatro grupos distintos de genes foram amplificados a partir do cDNA de formas amastigotas da cepa Y de T. cruzi. Ao nível de nucleotídeos, eles compartilharam 82,3 a 89,9 por cento de identidade com o gene asp-2 descrito previamente. Um gene denominado clone 9 apresentou o maior grau de identidade com o gene asp-2 e foi selecionado para estudos imunológicos. Anti-soro policlonal gerado contra a porção C-terminal da proteína recombinante expressa pelo gene clone 9 reagiu comi um, antígeno i de aproximadamente 83 kDa expresso nas formas amastigotas de T cruzi. A imunização de camundongos BALB/c com plasmídios de expressão em células eucarióticas contendo o gene clone 9 induziu anticorpos específicos e a secreção de IFN- dependente de células T CD4+. Quando desafiados com formas tripomastigotas, os camundongos imunizados com os plasmídios contendo o gene clone 9 exibiram parasitemias reduzidas e sobreviveram à infecção letal, Subseqüentemente, testamos a hipótese de que talvez pudéssemos incrementar a, imunidade protetora contra a infecção experimental pela indução simultânea de uma resposta imune contra antígenos expressos nas formas amastigotas e tripomastigotas do T cruzi, Com este intuito, camundongos altamente suscetíveis A/Sn foram imunizados com plasmídios contendo o gene clone 9 ou o gene da trans-sialidase...

Control of gene expression in Trypanosomatidae

The study of mechanisms which control gene expression in trypanosomatids has developed at an increasing rate since 1989 when the first successful DNA transfection experiments were reported. Using primarily Trypanosoma brucei as a model, several groups have begun to elucidate the basic control mechanisms and to define the cellular factors involved in mRNA transcription, processing and translation in these parasites. This review focuses on the most recent studies regarding a subset of genes that are expressed differentially during the life cycle of three groups of parasites. In addition to T. brucei, I will address studies on gene regulation in a few species of Leishmania and the results obtained by a much more limited group of laboratories studying gene expression in Trypanosoma cruzi. It is becoming evident that the regulatory strategies chosen by different species of trypanosomatids are not similar, and that for these very successful parasites it is probably advantageous to employ multiple mechanisms simultaneously. In addition, with the increasing numbers of parasite genes that have now been submitted to molecular dissection, it is also becoming evident that, among the various strategies for gene expression control, there is a predominance of regulatory pathways acting at the post-transcriptional level

Expressão da glicoproteína de superfície de 82kDa(GP82) de tripomastigotas metaciclícas de Trypanosoma cruzi em células de mamíferos e em forma epimastigotas do parasita / Metacyclic trypomastigotes of Trypanosama cruzi express a stage-specific surface glycoprotein of 82 kDa of the mammalian host cells by the parasite

Os tripomastigotas metacíclicos de Trypanosoma cruzi expressam uma glicoproteína de superficie de 82 kDa estágio-específica que tem sido implicada na invasao de células de mamíferos pelo parasita. A gp82 é uma molécula de adesao que induz aumento na concentraçao de Ca+2 , evento este fundamental no processo de invasao pelo T cruzi. A estrutura primária da gp82 foi recentemente deduzida a partir de um clone de cDNA (clone Jl8, GeneBank Ll4824) (Araya et al., 1994). Ela é constituída por um polipeptídeo de 516 aminoácidos que contém alguns motivos característicos da família das gp85/sialidases; incluindo-se os motivos Asp box, VTV e uma seqüência putativa para adiçao de âncora GPI. Contudo, a gp82 nao possue uma seqüência aminoterninal, comuns às proteínas de membrana. Apesar de vários aspectos da biosíntese de proteínas de superficie terem sido conservados na evoluçao, as exigências para translocaçao do peptídeo nascente através do retículo endoplasmático e a ligaçao através de âncora de glicosilfosfatidilinositol (GPI) pode nao ser os mesmos nos mamíferos e protozoários parasitas. Os objetivos do presente trabalho foram testar se os sinais presentes na gp82 funcionam nas células de mamíferos e estudar a expressao e processamento da gp82 em epimastigotas de T. cruzi, uma forma evolutiva que nao expressa esta proteína. Nós analisamos a expressao da gp82 em células Vero e Cos-7 transfectadas com o plasmídio pcDNA3 no qual introduziu-se a fase de leitura aberta completa do gene gp82. O cDNA da gp82 foi subclonado no plasmídio pcDNA3 (construçao pc-gp82) mantendo-se as seqüências potenciais para adiçao de âncora GPI e peptídeo sinal (seqüência NH2-terminal de clivagem). As células transfectadas expressaram 4 polipeptídeos de massa molecular inferior a 60 kDa sugerindo que o peptídeo sinal encontrado na gp82 nao foi reconhecido pelo sistema de processamento das células de mamíferos. Para testar esta hipótese, a seqüência codificadora do peptídeo sinal da hemaglutinina viral foi clonada em fase com o gene gp82 originando uma construçao denominada pc-gp82+PS. Nós comparamos a expressao da gp82 codificada pelas construçoes pc-gp82 e pc-gp82+PS através de "immunoblotting", imunofluorescência e inibiçao da N-glicosilaçao com tunicamicina. As células transfectadas com a construçao pc-gp82+PS expressaram uma proteína de 75 kDa que foi detectada pelo anticorpo monoclonal 3F6 (AcMo-3F6) específico para a gp82. Quando a transfecçao foi feita na presença ...(au)

Myristate exchange in glycolipid A and VSG of African trypanosomes

The variant surface glycoprotein (VSG) of T. brucei is anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor which is unique in that its fatty acids are exclusively myristate (a fourteen carbon saturated fatty acid). We showed that the myristate is added to the GPI precursor in a remodeling reaction involving deacylation and reacylation. We now demonstrate that trypanosomes have a second pathway of myristoylation for GPI anchors that we call "myristate exchange" which is distinct from the fatty acid remodeling pathway. We propose that this is an exchange of [3H]myristate into both sn-1 and sn-2 positions of glycolipid A, which already contains myristate, and have demonstrated this using inhibitors and a variety of other methods. We have partially characterized myristate exchange with respect to specificity and susceptibility to some inhibitors. The apparent Km for myristoyl CoA is 7 nM. This myristate-specific process may represent a proof-reading system to ensure that the fatty acids on VSG are exclusively myristate. Although myristate exchange was first discovered for glycolipid A, we now believe that VSG is the true substrate of this reaction. VSG is efficiently labeled by exchange in the presence of cycloheximide, which prevents anchoring of newly synthesized protein. Although its location is not yet know, we have evidence that exchange does not localize to either the endoplasmic reticulum or the plasma membrane. We will present data indicating that surface VSG may be internalized and undergo myristate exchange

The role of glycolipid C in the GPI biosynthetic pathway in Trypanosoma brucei bloodstream forms

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in Trypanosoma brucei bloodstream forms includes the formation of glycolipid C. This molecule is the inositol-acylated form of the GPI anchor precursor, glycolipid A. There is no evidence for the transfer of glycolipid C to protein in vivo and the role of glycolipid C is unclear. In this paper we show that glycolipid C is not synthesised in the presence of phenylmethylsulphonyl fluoride (PMSF) and that glycolipid C is not an obligatory intermediate on the pathway to the formation of glycolipid A. Using pulse-chase experiments we show that glycolipid A and glycolipid C are in a dynamic equilibrium and we suggest that only the forward reaction (glycolipid A conversion to glycolipid C) is inhibited by PMSF

Metabolism of GPIs in mammalian cells

In Trypanosoma brucei, glycosylphosphatidylinositol (GPI) anchors of proteins and free GPIs with identical structures have been characterized. This identity provides strong presumptive evidence that the free GPIs are in fact precursors of the GPI anchors on proteins. In mammalian tissues, however, rather consistent differences in the structures of free GPIs and GPI anchors are observed. The terminal GPIs produced by the mammalian biosynthetic pathway differ from GPI anchors in being almost exclusively fatty acid acylated on the inositol residue, having a greater number of phosphoethanolamine residues, and perhaps in containing a greater percentage of diacylglycerol components. While in principle these differences could be reconciled by remodeling reactions before or after attachment of GPI anchors, it is possible that some of the mammalian free GPIs play cellular roles other than as anchor precursors. We have approached this question by studying the lifetimes of the last three GPIs on the biosynthetic pathway, denoted H6, H7 and H8, in K562 cells and in K562 mutant designated class K that is devoid of GPI-anchored proteins. Pulse-chase metabolic labeling with [3H]-mannose indicated that H6 was a precursor of H7 and H8 and that the H8 lifetime was more than one hour in the parental cells and even longer in the mutant. Preliminary data indicated that the majority of each of the three GPIs was localized in the plasma membrane fraction rather than the endoplasmic reticulum. These observations argue that mammalian GPIs are not utilized exclusively as GPI anchor precursors

Topology of GPI biosynthesis in the endoplasmic reticulum

Glycosylphosphatidylinositol (GPI) anchors are constructed in the endoplasmic reticulum (ER) through the action of at least seven unique enzymes. Using cell-free systems, mainly derived from African trypanosomes, it has been experimentally possible to re-create many aspects of the GPI biosynthetic pathway in vitro and to obtain a series of glycosylated phosphatidylinositol structures that correspond to biosynthetic intermediates. This approach led to the identification of the biosynthetic donors of individual components of the GPI glycan, and the discovery of unusual fatty acid re-modelling reactions in the GPI pathway in trypanosomes. Despite this progress, questions remain concerning the enzymology of the pathway, particularly the topological distribution of the different assembly steps in the ER membrane. In the work described here we have attempted to define the transbilayer orientation of different GPI biosynthetic intermediates in the ER membrane bilayer. The experiments were performed with a microsomal fraction derived from bloodstream-form Trypanosoma brucei, and standard radiolabeling procedures. The orientation of GPIs was probed with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the jackbean lectin Concanavalin A. Contrary to expectations based on other ER glycosylation reactions, most notably the reactions involved in the dolichol pathway of N-glycosylation, our results suggest that non-inositol-acylated (PI-PLC-sensitive) GPIs are synthesized in the cytoplasmic leaflet of the ER membrane bilayer and that the final reaction product, a phosphoethanolamine-containing GPI, flips into the luminal leaflet for transfer to protein

GPI phospholipase C from Trypanosoma brucei causes a GPI-negative phenotype in Leishmania major: I. Implications for GPI-negative mammalian cells; II. Compartmentalization of two GPI biosynthetic pathways

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots, gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man (1Ó6)Man(1Ó4)GlcN(1Ó6)-myo-inositol-1-phospholipid, is cystolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma

The requirements for GPI-attachment are similar but not identical in mammalian cells and parasite protozoa

To test whether the requirements for GPI-attachment are the same in mammalian cells and parasitic protozoa, we expressed the GPI-linked variant surface glycoprotein (VSG) of Trypanosoma brucei (T. brucei) in COS cells. Although large amounts of VSG were produced, only a small fraction became GPI-linked. This impaired processing is not due to the VSG ectodomain since replacement of the VSG GPI-signal with that of decay accelerating factor (DAF) produced GPI-linked VSG. Further, whereas fusion of the DAF GPI-signal to the COOH-terminus of human growth hormone (hGH) produces GPI-linked hGH, an analogous fusion using the VSG GPI-signal does not, indicating that the VSG GPI-signal functions poorly in mammalian cells. By constructing chimeric VSG-DAF GPI-signals and fusing them to the COOH-terminus of hGH, we show that of the two critical elements that comprise the GPI-signal - the cleavage/attachment site and the hydrophobic domain - the former is responsible for the impaired activity of the VSG GPI-signal in COS cells. To confirm this, we show that the VSG GPI-signal can be converted to a viable signal for mammalian cells by altering the amino acid configuration at the cleavage/attachment site. We also show that when fused to hGH, the putative GPI-signal from the malaria circumsporozoite (CS) protein produces low levels of GPI-anchored hGH, suggesting that the CS protein is indeed GPI-linked, but that the CS protein-signal, like the VSG-signal, functions poorly in COS cells

Structure and function of GPI-anchored surface proteins of Trypanosoma brucei

While proteins modified at their COOH-terminal end by a glycosylphosphatidylinositol (GPI) membrane anchor have been found as minor components in many eukaryotic cells, they dominate surface constituents of several parasitic protozoa. In this article, GPI-anchored proteins of Trypanosoma brucei are discussed

The role of GPI-PLC in Trypanosoma brucei

The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG). However the evidence that it is involved in VSG metabolism in the living trypanosome is circunstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotomic lysis. Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease. To try to determine the role of the GPI-PLC, a homozygous mull mutant T. brucei has been constructed. The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes. The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype. The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be

GPI anchor-hydrolyzing phospholipases

Glycosilphosphatidylinositol(GPI) anchor-hydrolyzing phospholipases include C- and D- type phospholipases and have been described in a number of organisms including bacteria, protozoan parasites, plants, and mammals. Although these phospholipases efficiently cleave GPI structures in vitro, the physiological role of GPI hydrolysis by anchor-specific phospholipases is still unclear. In order to permit comparison of the known GPI anchor-hydrolyzing phospholipases, we studied the kinetic parameters of these enzymes and provide an overview of the currently available information

Further characterization of the acidic GPI-hydrolyzing phospholipase present in human sera

A phospholipase from human serum capable of hydrolyzing glycosylphosphatidylinositol membrane anchors was described and partially characterized by our group some years ago. This activity presented a pH optimum between 5.0 and 6.0 and was inhibited by EDTA, EGTA and 1,10-phenanthroline. Partial purification showed that the enzyme was a glycoprotein with an apparent molecular weight of 140 kDa as judged by gel filtration. Other investigators characterized at the same time a phospholipase D with similar properties but with a pH optimum near 7.5. We now confirm that the human serum enzyme is indeed a phospholipase D capable of hydrolyzing mfVSG and glycolipids A and C from T. brucei. Isoelectric focusing of whole sera suggests the presence of two isoforms, one with a pI of 4.7 which was the form previously purified by our group, and others with pI from 6.2 to 7.4