Drug targets and mechanisms of resistance in the anaerobic protozoa

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated ruing laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.

Orally administered Giardia duodenalis extracts enhance an antigen-specific antibody response

We have identified novel adjuvant activity in specific cytosol fractions from trophozoites of Giardia isolate BRIS/95/HEPU/2041 (J. A. Upcroft, P. A. McDonnell, and P. Upcroft, Parasitol. Today, 14:281-284, 1998). Adjuvant activity was demonstrated in the systemic and mucosal compartments when Giardia extract was coadministered orally with antigen to mice. Enhanced antigen-specific serum antibody responses were demonstrated by enzyme-linked immunosorbent. assay to be comparable to those generated by the gold standard, mucosal adjuvant cholera toxin. A source of adjuvant activity was localized to the cytosolic component of the parasite. Fractionation of the cytosol produced fraction pools, some of which, when coadministered with antigen, stimulated an enhanced antigen-specific serum response. The toxic component of conventional mucosal adjuvants is associated with adjuvant activity; therefore, in a similar way, the toxin-like attributes of BRIS/95/HEPU/2041 may be responsible for its adjuvanticity. Complete characterization of the adjuvant is under way.

Towards a blood-stage vaccine for malaria: Are we following all the leads?

Although the malaria parasite was discovered more than 120 years ago, it is only during the past 20 years, following the cloning of malaria genes, that we have been able to think rationally about vaccine design and development. Effective vaccines for malaria could interrupt the life cycle of the parasite at different stages in the human host or in the mosquito. The purpose of this review is to outline the challenges we face in developing a vaccine that will limit growth of the parasite during the stage within red blood cells - the stage responsible for all the symptoms and pathology of malaria. More than 15 vaccine trials have either been completed or are in progress, and many more are planned. Success in current trials could lead to a vaccine capable of saving more than 2 million lives per year.

Apoptotic deletion of Th cells specific for the 19-kDa carboxyl-terminal fragment of merozoite surface protein 1 during malaria infection

Immunity induced by the 19-kDa fragment of merozoite surface protein 1 is dependent on CD4(+) Th cells. However, we found that adoptively transferred CFSE-labeled Th cells specific for an epitope on Plasmodium yoelii 19-kDa fragment of merozoite surface protein 1 (peptide (p)24), but not OVA-specific T cells, were deleted as a result of P. yoelii infection. As a result of infection, spleen cells recovered from infected p24-specific T cell-transfused mice demonstrated reduced response to specific Ag. A higher percentage of CFSE-labeled p24-specific T cells stained positive with annexin and anti-active caspase-3 in infected compared with uninfected mice, suggesting that apoptosis contributed to deletion of p24-specific T cells during infection. Apoptosis correlated with increased percentages of p24-specific T cells that stained positive for Fas from infected mice, suggesting that P. yoelii-induced apoptosis is, at least in part, mediated by Fas. However, bystander cells of other specificities also showed increased Fas expression during infection, suggesting that Fas expression alone is not sufficient for apoptosis. These data have implications for the development of immunity in the face of endemic parasite exposure.

The gene encoding the immunoregulatory signaling molecule CMRF-35A localized to human chromosome 17 in close proximity to other members of the CMRF-35 family

The immunoregulatory signaling (IRS) family includes several molecules, which play major roles in the regulation of the immune response. The CMRF-35A and CMRF-35H molecules are two new members of the IRS family of molecules, that are found on a wide variety of haemopoietic lineages. The extracellular functional interactions of these molecules is presently unknown, although CMRF-35H on initiate an inhibitory signal and is internalized when cross-linked. In this paper, we described the gene structure for the CMRF-35A gene and its localization to human chromosome 17. The gene consists of four exons spanning approximately 4.5 kb. Exon 1 encodes the 5' untranslated region and leader sequence, exon 2 encodes the immunoglobulin (Ig)-like domain, exon 3 encodes the membrane proximal region and exon 4 encodes the transmembrane region, the cytoplasmic tail and the 3' untranslated region. A region in the 5' flanking sequence of the CMRF-35A gene, that promoted expression of a reporter gene was identified. The genes for the CMRF-35A and CMRF-35H molecules are closely linked on chromosome 17. Similarity between the Ig-like exons and the preceding intron of the two genes suggests exon duplication was involved in their evolution. We also identified a further member of the CMRF-35 family, the CMRF-35J pseudogene. This gene appears to have arisen by gene duplication of the CMRF-35A gene. These three loci-the CMRF-35A, CMRF-35J and CMRF-35H genes-form a new complex of IRS genes on chromosome 17.

Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes

Flotillin-1 was recently shown to be enriched on detergent-resistant domains of the plasma membrane called lipid rafts. These rafts, enriched in sphingolipids and cholesterol, sequester certain proteins while excluding others. Lipid rafts have been implicated in numerous cellular processes including signal transduction, membrane trafficking and molecular sorting. In this study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes, These structures are enriched in flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1), The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes, Treatment with bafilomycin A1, a compound that inhibits the proton pump ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from endosomal organelles. A proteomic characterization of the lipid rafts of phagosomes indicates that actin, the alpha- and beta -subunits of heterotrimeric G proteins, as well as subunits of the proton pump V-ATPase are among the constituents of these domains. Remarkably, the intracellular parasite Leishmania donovani can actively inhibit the acquisition of flotillin-1-enriched lipid rafts by phagosomes and the maturation of these organelles. These results indicate that specialized functions required for phagolysosome biogenesis may occur at focal points on the phagosome membrane, and therefore represent a potential target of intracellular pathogens.

Time of treatment influences the appearance of drug-resistant parasites in Plasmodium falciparum infections

A deterministic mathematical model which predicts the probability of developing a new drug-resistant parasite population within the human host is reported, The model incorporates the host's specific antibody response to PfEMP1, and also investigates the influence of chemotherapy on the probability of developing a viable drug-resistant parasite population within the host. Results indicate that early, treatment, and a high antibody threshold coupled with a long lag time between antibody stimulation and activity, are risk factors which increase the likelihood of developing a viable drug-resistant parasite population. High parasite mutation rates and fast PfEMP1 var gene switching are also identified as risk factors. The model output allows the relative importance of the various risk factors as well as the relationships between them to be established, thereby increasing the understanding of the conditions which favour the development of a new drug-resistant parasite population.

Molecular clocks in reptiles: Life history influences rate of molecular evolution

Life history has been implicated as a determinant of variation in rate of molecular evolution amongst vertebrate species because of a negative correlation between bode size and substitution rate for many Molecular data sets. Both the generality and the cause of the negative bode size trend have been debated, and the validity of key studies has been questioned (particularly concerning the failure to account for phylogenetic bias). In this study, a comparative method has been used to test for an association between a range of life-history variables-such as body size age at maturity, and clutch size-and DNA substitution rate for three genes (NADH4, cytochrome b, and c-mos). A negative relationship between body size and rate of molecular evolution was found for phylogenetically independent pairs of reptile species spanning turtles. lizards. snakes, crocodile, and tuatara. Although this Study was limited by the number of comparisons for which both sequence and lite-history data were available, the results, suggest that a negative bode size trend in rate of molecular evloution may be a general feature of reptile molecular evolution. consistent with similar studies of mammals and birds. This observation has important implications for uncovering the mechanisms of molecular evolution and warns against assuming that related lineages will share the same substitution rate (a local molecular clock) in order to date evolutionary divergences from DNA sequences.

Cleavage of hemoglobin by hookworm cathespin D aspartic proteases and its potential contribution to host specificity

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.

Optimising cleaning behaviour: minimising the costs and maximising ectoparasite removal

Little is known of how client fish minimise the costs of cleaning behaviour while maximising ectoparasite removal by cleaner fish. Previous studies have found that abundance on fish and infestation behaviour of gnathiid isopods, the main parasite eaten by cleaner fish, varies diurnally. We examined whether reduced foraging is a cost of cleaning behaviour in clients and whether the behaviour of the client fish, the thick-lipped wrasse Hemigymnus melapterus, towards the cleaner fish Labroides dimidiatus varied diurnally to maximise ectoparasite removal, possibly in response to the diurnal changes in the abundance and infestation patterns of gnathiids. We found that during the midday and afternoon, client foraging rates were negatively related to the duration and frequency of inspections, suggesting that cleaning may, at some times of the day, be energetically costly to the client in terms of reduced foraging opportunities. Surprisingly, we found that the duration and frequency of inspections of clients by cleaners did not vary among diel time periods. A model of gnathiid dynamics on individual fish is proposed. It shows that the observed diurnal pattern in gnathiid abundance on fish can be generated with the constant duration and frequency of inspections that was observed in this study. Thus clients would not have more gnathiids removed by modifying their cleaning behaviour.

Testicular myxosporidiasis in anurans, with a description of Myxobolus fallax n. sp.

During studies of amphibian sperm cryopreservation, a new species of myxosporidean parasite (Myxozoa, Myxosporae) was observed in the testes of the Australian dwarf green tree frog Litoria fallax (Peters). Myxosporidiasis was found to have no affect on L. fallax body condition or sperm numbers. Myxobolus spores from L. fallax are morphologically distinct from Myxobolus hylae spores (infecting the sympatric Litoria aurea Lesson) and the three previously named (exotic to Australia) Myxobolus species found in anurans. Myxobolus fallax n. sp. is characterised by: pseudocyst white, spherical to ovoid, 141 x74 to 438 x337 mum in diameter (mature); plasmodium with spores loosely arranged within interior. Spores ovoid 13.4 +/- 0.5 (12.6-14.6) mum length, 9.5 +/- 0.4 (8.3-10.6) mum width, 6.8 +/- 0.4 (6.5-7.6) mum depth, 1.4 +/- 0.1 (1.3-1.6) length/width; polar capsules broadly pyriform and equal in size 4.2 +/- 0.3 (3.3-4.7) mum length, 2.4 +/- 0.2 (2.1-2.8) mum width; filament coils 7-8, wound tightly and perpendicular to the longitudinal axis of the capsule; polar filament 34 +/- 7.0 (18-50) mum length; intercapsular appendix and sutural ridge folds absent; and iodinophilous vacuole and mucous envelope lacking. In addition to this new species, data from archival samples of M. hylae are provided which show two morphologically distinct spore types. Both appeared rarely in the same pseudocysts and we cautiously retain the single species.

A molecular phylogeny of rainbow skinks (Scincidae : Carlia): taxonomic and biogeographic implications

The phylogenetic relationships amongst 29 species of Carlia and Lygisaurus were estimated using a 726-base-pair segment of the protein-coding mitochondrial ND4 gene. Results do not support the recent resurrection of the genus Lygisaurus. Although most Lygisaurus species formed a single clade, this clade is nested within Carlia and includes Carlia parrhasius. Due to this new molecular evidence, and the paucity of diagnostic morphological characters separating the genera, Lygisaurus de Vis 1884 is re-synonymised with Carlia Gray 1845. Our analysis is also inconsistent with a previous suggestion that Lygisaurus timlowi should be removed to Menetia, a genus that is distantly related relative to outgroups used here. Intraspecific variation in Carlia is, in several instances, greater than interspecific distance. The most strikingly divergent lineages are found within C. rubrigularis, which appears to be paraphyletic, with southern populations more closely related to C. rhomboidalis than to northern populations of C. rubrigularis. The two C. rubrigularis-C. rhomboidalis lineages form part of a major polytomy at an intermediate level of divergence. Lack of resolution at this level, however, does not appear to be due to saturation or loss of phylogenetic signal. Rather, the polytomy probably reflects a period of relatively rapid diversification that occurred sometime during the Miocene.

First record of the mite Hirstiella diolii Baker (Prostigmata : Pterygosomatidae) from Australia, with a review of mites found on Australian lizards

We report the First occurrence in Australia of an exotic mite parasite of lizards, Hirstiella diolii Baker (Prostigmata, Pterygosomatidae), and its association with iguanas at the Taronga Zoo in Sydney. We also report on the spread of the exotic snake mite Ophionyssus natricis (Gervais) to native populations of skinks, provide a key to the mites associated with Australian lizards and review how to distinguish chiggers from pterygosomatid mites.

The trematodes of groupers (Serranidae : Epinephelinae) knowledge, nature and evolution

Groupers (Epinephelinae) are prominent marine fishes distributed in the warmer waters of the world. Review of the literature suggests that trematodes are known from only 62 of the 159 species and only 9 of 15 genera; nearly 90% of host-parasite combinations have been reported only once or twice. All 20 families and all but 7 of 76 genera of trematodes found in epinephelines also occur in non-epihephelines. Only 12 genera of trematodes are reported from both the Atlantic-Eastern Pacific and the Indo-West Pacific. Few (perhaps no) species are credibly cosmopolitan but some have wide distributions across the Indo-West Pacific. The hierarchical 'relatedness' of epinephelines as suggested by how they share trematode taxa (families, genera, species) shows little congruence with what is known of their phylogeny. The major determinant of relatedness appears to be geographical proximity. Together these attributes suggest that host-parasite coevolution has contributed little to the evolution of trematode communities of epinephelines. Instead, they appear to have arisen through localized episodes of host-switching, presumably both into and out of the epinephelines. The Epinephelinae may well be typical of most groups of marine fishes both in the extent to which their trematode parasites are known and in that, apparently, co-evolution has contributed little to the evolution of their communities of trematodes.

The mitochondrial 12S gene is a suitable marker of populations of Sarcoptes scabiei from wombats, dogs and humans in Australia

We sequenced part of the mitochondrial 12S ribosomal RNA gene of 23 specimens of Sarcoptes scabiei from eight wombats, one dog and three humans. Twelve of the 326 nucleotide positions varied among these mites and there were nine haplotypes (sequences) that differed by 1-8 nucleotides. Phylogenetic analyses indicated that these mites were from two lineages: (1) mites from wombats from Victoria, Australia, and mites from the humans and dog from the Northern Territory, Australia (haplotypes 1-4, 9); and (2) mites from the humans and dog from the Northern Territory (haplotypes 5-8). Mites from the three different hosts (wombats, a dog and humans) had not diverged phylogenetically; rather, these mites had similar 12S sequences. Thus, we conclude that these mites from wombats, humans and a dog are closely related, and that they diverged from a common ancestor relatively recently. This conclusion is consistent with the argument that people and/or their dogs introduced to Australia the S. scabiei mites that infect wombats Australia. So, S. scabiei, which has been blamed for the extinction of populations of wombats in Australia, may be a parasitic mite that was introduced to Australia with people and/or their dogs. These data show that the mitochondrial 12S rRNA gene may be a suitable population marker of S. scabiei from wombats, dogs and humans in Australia.