Toxoplasma gondii dense granule protein 3 (GRA3) is a type I transmembrane protein that possesses a cytoplasmic dilysine (KKXX) endoplasmic reticulum (ER) retrieval motif.

Studies using antibodies to immunolocalize the Toxoplasma gondii dense granule protein GRA3, have shown that this protein associates strongly with the parasitophorous vacuole membrane (PVM). However, as there was no predicted membrane-spanning domain this highlighted an unanswered paradox. We demonstrate that the previously published sequence for GRA3 is actually an artificial chimera of 2 proteins. One protein, of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank. The second, with a predicted molecular weight of 24 kDa shares the N-terminal region, is recognized by the monoclonal antibody 2H11 known to react with the dense granules of T. gondii and is therefore the authentic GRA3. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM. Antibodies to recombinant GRA3 recognize a protein of 24 kDa in T. gondii excretory-secretory antigen preparations. The signal peptide is necessary and sufficient to target GFP to the dense granules and parasitophorous vacuole. A homologue was identified in Neospora caninum. Finally, GRA3 possesses a dilysine 'KKXX' endoplasmic reticulum (ER) retrieval motif that rationalizes its association with PVM and possibly the host cell ER.

A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation.

The shikimate pathway is essential for survival of the apicomplexan parasites Plasmodium falciparum, Toxoplasma gondii and Cryptosporidium parvum. As it is absent in mammals it is a promising therapeutic target. Herein, we describe the genes encoding the shikimate pathway enzymes in T. gondii. The molecular arrangement and phylogeny of the proteins suggests homology with the eukaryotic fungal enzymes, including a pentafunctional AROM. Current rooting of the eukaryotic evolutionary tree infers that the fungi and apicomplexan lineages diverged deeply, suggesting that the arom is an ancient supergene present in early eukaryotes and subsequently lost or replaced in a number of lineages.

Delivery of antimicrobials into parasites.

To eliminate apicomplexan parasites, inhibitory compounds must cross host cell, parasitophorous vacuole, and parasite membranes and cyst walls, making delivery challenging. Here, we show that short oligomers of arginine enter Toxoplasma gondii tachyzoites and encysted bradyzoites. Triclosan, which inhibits enoyl-ACP reductase (ENR), conjugated to arginine oligomers enters extracellular tachyzoites, host cells, tachyzoites inside parasitophorous vacuoles within host cells, extracellular bradyzoites, and bradyzoites within cysts. We identify, clone, and sequence T. gondii enr and produce and characterize enzymatically active, recombinant ENR. This enzyme has the requisite amino acids to bind triclosan. Triclosan released after conjugation to octaarginine via a readily hydrolyzable ester linkage inhibits ENR activity, tachyzoites in vitro, and tachyzoites in mice. Delivery of an inhibitor to a microorganism via conjugation to octaarginine provides an approach to transporting antimicrobials and other small molecules to sequestered parasites, a model system to characterize transport across multiple membrane barriers and structures, a widely applicable paradigm for treatment of active and encysted apicomplexan and other infections, and a generic proof of principle for a mechanism of medicine delivery.

Transport mechanisms in Plasmodium-infected erythrocytes: lipid rafts and a tubovesicular network.

The mature human erythrocyte is a simple cell that is devoid of intracellular organelles and does not show endocytic or phagocytic activity at the plasma membrane. However, following infection by Plasmodium, the erythrocyte undergoes several morphological and functional changes. Parasite-derived proteins are exported into the erythrocyte cytoplasm and to the membrane, while several proteins are localised to the parasitophorous vacuolar membrane and to the tubovesicular membranous network structures surrounding the parasite. Recent evidence indicates that multiple host proteins, independent of the type of their membrane anchor, that exist in detergent-resistant membrane (DRM) rafts or microdomains enter this apicomplexan vacuole. The internalised host components along with the parasite-encoded transmembrane protein PfEXP1 can be detected as DRM rafts in the vacuole. It appears that in Plasmodium-infected erythrocytes lipid rafts may play a role in endovacuolation and macromolecular transport.

The role of cholesterol and glycosylphosphatidylinositol-anchored proteins of erythrocyte rafts in regulating raft protein content and malarial infection.

Human erythrocytes are terminally differentiated, nonendocytic cells that lack all intracellular organelles. Here we show that their plasma membranes contain detergent-resistant membrane rafts that constitute a small fraction (4%) of the total membrane protein, with a complex mixture of proteins that differentially associate with rafts. Depletion of raft-cholesterol abrogates association of all proteins with no significant effect on cholesterol:protein ratios in the rest of the membrane, lipid asymmetry, deformability, or transport properties of the bilayer, indicating that cholesterol is critical for protein assembly into rafts and suggesting that rafts have little influence on several erythrocyte functions. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, which lack glycosylphosphatidylinositol-anchored proteins, show significant elevation in raft-cholesterol but no increase in raft protein association, suggesting that raft assembly does not require glycosylphosphatidylinositol-anchored proteins, raft proteins do not bind directly to cholesterol, and only threshold levels of raft-cholesterol are critical for protein recruitment. Loss of glycosylphosphatidylinositol-anchored proteins had no effect on erythrocytic infection by malarial parasite or movement of raft markers into the parasite's vacuole. However, infection is blocked following raft-cholesterol disruption, suggesting that erythrocyte rafts can be functionally exploited and providing the first evidence for the involvement of host rafts in an apicomplexan infection.

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I.

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

Erythrocytic vacuolar rafts induced by malaria parasites.

Studies in the past year displaced long-standing dogmas and provided many new molecular insights into how proteins and solutes move between the erythrocyte plasma membrane and the malarial vacuole. Highlights include a demonstration that (1) detergent-resistant membrane (DRM) rafts exist in the red cell membrane and their resident proteins are detected as rafts in the plasmodial vacuole, (2) a voltage-gated channel in the infected red cell membrane mediates uptake of extracellular nutrient solutes, and (3) intraerythrocytic membranes transport a parasite-encoded adherence antigen to the red cell surface.

Vacuolar uptake of host components, and a role for cholesterol and sphingomyelin in malarial infection.

Erythrocytes, which are incapable of endocytosis or phagocytosis, can be infected by the malaria parasite Plasmodium falciparum. We find that a transmembrane protein (Duffy), glycosylphosphatidylinositol (GPI)-anchored and cytoplasmic proteins, associated with detergent-resistant membranes (DRMs) that are characteristic of microdomains in host cell membranes, are internalized by vacuolar parasites, while the major integral membrane and cytoskeletal proteins are not. The internalized host proteins and a plasmodial transmembrane resident parasitophorous vacuolar membrane (PVM) protein are detected in DRMs associated with vacuolar parasites. This is the first report of a host transmembrane protein being recruited into an apicomplexan vacuole and of the presence of vacuolar DRMs; it establishes that integral association does not preclude protein internalization into the P.FALCIPARUM: vacuole. Rather, as shown for Duffy, intracellular accumulation occurs at the same rate as that seen for a DRM-associated GPI-anchored protein. Furthermore, novel mechanisms regulated by the DRM lipids, sphingomyelin and cholesterol, mediate (i) the uptake of host DRM proteins and (ii) maintenance of the intracellular vacuole in the non-endocytic red cell, which may have implications for intracellular parasitism and pathogenesis.

Targeted reduction of nucleoside triphosphate hydrolase by antisense RNA inhibits Toxoplasma gondii proliferation.

Nucleoside triphosphate hydrolase (NTPase) is a very abundant protein secreted by the obligate intracellular parasite Toxoplasma gondii shortly after invasion of the host cell. When activated by dithiols, NTPase is one of the most potent apyrases known to date, but its physiological function remains unknown. The genes encoding NTPase have been cloned (Bermudes, D., Peck, K. R., Afifi-Afifi, M., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260). We have recently shown that the enzyme is tightly controlled within the vacuolar space and may influence parasite exit from the host cell (Silverman, J. A., Qi, H., Riehl, A., Beckers, C., Nakaar, V., and Joiner, K. A (1998) J. Biol. Chem. 273, 12352-12359). In the present study, we have generated an antisense NTP RNA construct in which the 3'-untranslated region is replaced by a hammerhead ribozyme. The constitutive synthesis of the chimeric antisense RNA-ribozyme construct in parasites that were stably transfected with this construct resulted in a dramatic reduction in the steady-state levels of NTPase. This inhibition was accompanied by a decrease in the capacity of the parasites to replicate. The reduction in parasite proliferation was due to a specific effect of antisense NTP RNA, since a drastic inhibition of hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) expression by a chimeric antisense HXGPRT RNA-ribozyme construct did not alter NTPase expression nor compromise parasite replication. These data implicate NTPase in an essential parasite function and suggest that NTPase may have more than one function in vivo. These results also establish that it is possible to study gene function in apicomplexan parasites using antisense RNA coupled to ribozymes.

Immune response of infants to fractional doses of intradermally administered inactivated poliovirus vaccine.

Seventy eight infants aged 6-8 weeks received either two doses of 0.1 ml of inactivated poliovirus vaccine (IPV) intradermally 8 weeks apart (group A) or three doses 4 weeks apart (group B). Pre- and 4 weeks post-immunization serum samples were tested for the presence and titer of neutralizing antibody to poliovirus types 1, 2 and 3. The seroconversion rates to poliovirus types 1, 2 and 3 were 90, 70 and 97%, respectively, among infants in group A and 90, 80 and 98%, respectively, in group B; in children without pre-existing maternal antibody, seroconversion rates were 100% to all three poliovirus serotypes in both groups. These rates were comparable to those in children receiving five doses of OPV or two doses of intramuscular IPV. Intradermal administration of fractional doses of IPV may be a less expensive alternative for use in developing countries.

The pregnant traveler.

The care of the pregnant traveler is both challenging and rewarding. It requires clinical information and skills that are derived from many disciplines. This article reviews preparatory guidelines for safe travel by the pregnant mother and her most important travel companion, the developing fetus. Issues considered are pretravel risk assessment, immunizations, and prevention of travelers' diarrhea and hepatitis. The safety and efficacy of malaria chemoprophylaxis in the present context of widespread multidrug-resistant malaria is discussed, and guidelines are offered for both prevention and treatment. A safety profile of commonly used travel medications, antibiotics, and antiparasitic drugs is reviewed.

Prevalence and presentation of hepatitis C related chronic liver disease in southern India.

To determine the importance of hepatitis C virus (HCV) infection in the aetiology of chronic liver disease in southern India, the prevalence of HCV antibodies and HBV markers was estimated in 100 patients with chronic liver disease and in 56 patients with a variety of other gastrointestinal and liver diseases who served as controls. HCV antibody was measured by a second-generation ELISA. HBsAg, anti-HBc, anti-HBs and anti-D were also estimated. HCV antibodies were detected in 26/100 patients with chronic liver disease compared to 0/56 controls. HBV markers were present in 72 of 100 patients with chronic liver disease compared to 21/56 (37.5%) controls. Anti-D was noted in 4/100 patients with chronic liver disease and in none of the controls. Many patients had serological evidence of both B and C infection; 73% of those with anti-HCV also tested positive for HBV markers. HCV related disease presented at a median age of 60 years compared to HBV related disease which presented at a median age of 40. There was no significant difference between HCV and HBV positive patients in symptomatology, but encephalopathy was uncommon and cirrhosis the usual finding at histology in HCV positive individuals, while chronic active hepatitis was found in 30% of biopsied HBV related disease. HCV is a significant cause of chronic liver disease in this geographic region, although HBV infection continues to account for the largest proportion of cases.

A comparison of low vs standard dose intramuscularly administered hepatitis B vaccine.

In a randomised, controlled, single-blind trial we compared different doses of a yeast-derived recombinant hepatitis B vaccine: a low dose intramuscular (i.m.) regimen (using 2 micrograms at first followed by 1 microgram each at 1, 2 and 6 months) with the standard dose (10 micrograms each at 0, 1 and 6 months) for post vaccination anti-HBs seropositivity. At the end of 7 months, only 78% of volunteers on the low dose (n = 77) tested positive for anti-HBs whereas 100% of volunteers on the standard dose (n = 43) were seropositive. Therefore, low dose regimen is not satisfactory for primary HBV vaccination.

Experimental poliomyelitis in bonnet monkey. Clinical features, virology and pathology.

We have investigated the distribution of virus and the pathology in the spinal cords of bonnet monkeys (Macaca radiata) with experimental paralytic poliomyelitis induced by inoculating poliovirus type 1 (Mahoney) into the right ulnar nerve. Viraemia and alimentary tract infection due to the inoculated virus were found in all monkeys. Between two and seven days after the onset of limb paralysis, the animals were killed and the spinal cord examined. Virus was isolated from the cervical enlargement, thoracic region and lumbar enlargement in titres ranging from 10(2.5) to 10(6.4) TCID50/gram of tissue. Cellular infiltrate, perivascular cuffing, early and late neuronal damage such as chromatolysis, nuclear pyknosis, retraction of the cytoplasm and neuronophagia were seen distributed throughout the spinal cord. This experimental animal model resembles human paralytic poliomyelitis clinically and pathologically.

Ultrastructural specialization of intestinal epithelium over Peyer's patches in the bonnet monkey, Macaca radiata.

The follicle associated epithelium (FAE) which separates the lymphoid follicle of Peyer's patch from the gut lumen is known to have specialized cells called M cells or "microfold" cells in man and certain animals. These cells are considered to be involved in antigen uptake and transport. Our light microscopic study of the small intestine of bonnet monkeys suggested the presence of such specialised cells in FAE. We have confirmed the presence of M cells in bonnet monkey FAE having ultrastructural features very similar to those of human M cells.

Microscopic anatomy of small intestine of Macaca radiata with special reference to gut associated lymphoid tissue.

The hitherto unreported microscopic anatomy of the small intestine of the bonnet monkey (M. radiata) was studied. There was a striking similarity in the general structure and cellular composition of the small intestine of the animal and that of humans. Of special interest was our observation of the occurrence of cells interspersed in the follicle associated epithelium which were morphologically similar to human M cells. These cells were pale, devoid of a brush border and frequently had lymphocytes apparently enclosed within them.

Ulnar nerve inoculation of poliovirus in bonnet monkey: a new primate model to investigate neurovirulence.

A new monkey model of poliovirus neurovirulence has been developed avoiding the currently used intraspinal injection route which traumatizes the spinal cord. Poliovirus type 1 (0.1 ml) was inoculated into the ulnar nerve of bonnet monkeys (Macaca radiata) at the right elbow. Five monkeys were inoculated with 10(7) TCID of LSc/2ab (Sabin vaccine strain); none developed any illness. Limb paralysis, clinically resembling spinal poliomyelitis in children, developed in all four monkeys given greater than or equal to 10(5) TCID50 of Mahoney strain, and in three of four monkeys given 10(4) or 10(3) TCID50. Higher functions and cranial nerves were not affected. Paralysis occurred more frequently in the lower limbs (11 limbs in seven monkeys) than in upper limbs (six limbs in seven monkeys). The incubation period, from inoculation to onset of paralysis, ranged from 5 to 12 days. Further progression of paralysis to other limbs occurred within 2 to 6 days. No illness developed in two monkeys given 10(2) TCID50 of Mahoney virus. All monkeys given LSc/2ab and those given greater than 10(2) TCID50 Mahoney virus developed humoral antibody response; however, infection of the gastrointestinal tract was detected by virus isolation from throat swabs and stools only in monkeys given Mahoney virus, but not in those given LSc/2ab. Thus, intraneural spread of Mahoney virus to the spinal cord, neurovirulence of Mahoney but not of LSc/2ab and retrograde gastrointestinal infection with Mahoney but not with LSc/2ab are the features of this experimental model.

Low prevalence of hepatitis D (delta) virus infection in a nephrology unit in south India.

Fifty-five consecutive patients with end-stage renal disease entering haemodialysis programmes over a two-month period and 48 consecutive recipients of renal allografts during a period of 6 months were investigated for hepatitis B virus (HBV) and hepatitis D virus (delta) infection. HBV markers were present in 25 of the former and 40 of the latter. Of the 65 patients with HBV infection, 12 were not available for delta antibody screening. HBV infection was present for a mean of 2.5 months and 45.3% of those infected had clinical hepatitis; none had fulminant hepatitis. All the patients tested were negative for antidelta antibody. An additional patient on dialysis with delta superinfection and hepatic encephalopathy is also reported.