Darren R. Brooks

Roles of cysteine proteinases of trypanosomes and Leishmania in host-parasite interactions

Trypanosomes and Leishmania contain an abundance of stage-regulated cysteine proteinases encoded by several gene families. Analysis of parasites rendered defective in cysteine proteinase function, either through genetic manipulation or through the use of specific inhibitors, has revealed roles for the enzymes in parasite virulence, in modulation of the hosts immune response and in parasite differentiation.

The multiple cpb cysteine proteinase genes of Leishmania mexicana encode isoenzymes that differ in their stage regulation and substrate preferences.

The cpb genes ofLeishmania mexicana encode stage-regulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3–cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation in the COOH-terminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection ofcpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into thecpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the isoenzymes implicates five residues located in the mature domain (Asn18, Asp60, Asn61, Ser64, and Tyr84) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct roles in the parasite’s interaction with its host. This complexity reflects the adaptation of cathepsin L-like cysteine proteinases to diverse functions in parasitic protozoa.

Expression of Multiple CPB Genes Encoding Cysteine Proteases Is Required for Leishmania mexicana Virulence In Vivo

ABSTRACT Leishmania mexicana mutants deficient in the multicopy CPB gene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specific CPB2.8 or metacyclic stage-specific CPB2 gene into a CPB-deficient mutant L. mexicana failed to restore either a Th2 response or sustained virulence. However, reexpression of multiple CPB genes from a cosmid significantly restored virulence. This was characterized by increased lesion and parasite growth and the acquisition of a Th2 response, as determined by measuring interleukin-4 production and immunoglobulin G1 (IgG1) and IgE levels. These studies confirm that L. mexicana cysteine proteases are important virulence factors and provide an explanation for the presence in L. mexicana of a multicopy tandem array of CPB genes.

An Essential Role in Molting and Morphogenesis of Caenorhabditis elegans for ACN-1, a Novel Member of the Angiotensin-converting Enzyme Family That Lacks a Metallopeptidase Active Site

Genome sequence analyses predict many proteins that are structurally related to proteases but lack catalytic residues, thus making functional assignment difficult. We show that one of these proteins (ACN-1), a unique multi-domain angiotensin-converting enzyme (ACE)-like protein from Caenorhabditis elegans, is essential for larval development and adult morphogenesis. Green fluorescent protein-tagged ACN-1 is expressed in hypodermal cells, the developing vulva, and the ray papillae of the male tail. The hypodermal expression of acn-1 appears to be controlled by nhr-23 and nhr-25, two nuclear hormone receptors known to regulate molting in C. elegans. acn-1(RNAi) causes arrest of larval development because of a molting defect, a protruding vulva in adult hermaphrodites, severely disrupted alae, and an incomplete seam syncytium. Adult males also have multiple tail defects. The failure of the larval seam cells to undergo normal cell fusion is the likely reason for the severe disruption of the adult alae. We propose that alteration of the ancestral ACE during evolution, by loss of the metallopeptidase active site and the addition of new protein modules, has provided opportunities for novel molecular interactions important for post-embryonic development in nematodes.

The Caenorhabditis elegans orthologue of mammalian puromycin-sensitive aminopeptidase has roles in embryogenesis and reproduction

Mammals possess membrane-associated and cytosolic forms of the puromycin-sensitive aminopeptidase (PSA; EC 3.4.11.14). Increasing evidence suggests the membrane PSA is involved in neuromodulation within the central nervous system and in reproductive biology. The functional roles of the cytosolic PSA are less clear. The genome of the nematode Caenorhabditis elegans encodes an aminopeptidase, F49E8.3 (PAM-1), that is orthologous to PSA, and sequence analysis predicts it to be cytosolic. We have determined the spatio/temporal gene expression pattern of pam-1 by using the promoter region of F49E8.3 to control expression in the nematode of a second exon translational fusion of the aminopeptidase to green fluorescent protein. Cytosolic fluorescence was observed throughout development in the intestine and nerve cells of the head. Neuronal expression was also observed in the tail of adult males. Recombinant PAM-1, expressed and purified from Escherichia coli, hydrolyzed the N-terminal amino acid from peptide substrates. Favored substrates had positively charged or small neutral amino acids in the N-terminal position. Peptide hydrolysis was inhibited by the metal-chelating agent 1,10-phenanthroline and by the aminopeptidase inhibitors actinonin, amastatin, and leuhistin. However, the enzyme was ∼100-fold less sensitive toward puromycin (IC50, 135 μm) than other PSA homologues. Following inactivation of the enzyme, aminopeptidase activity was recovered with Zn2+, Co2+, and Ni2+. Silencing expression of pam-1 by RNA interference resulted in 30% embryonic lethality. Surviving adult hermaphrodites deposited large numbers of oocytes throughout the self-fertile period. The overall brood size was, however, unaffected. We conclude that pam-1 encodes an aminopeptidase that clusters phylogenetically with the PSAs, despite attenuated sensitivity toward puromycin, and that it functions in embryo development and reproduction of the nematode.

Functional genomics of parasitic worms: The dawn of a new era

The study of gene function in parasitic worms is technically demanding due to difficulties associated with life-cycle propagation and, hence, molecular genetics. Exploitation of the free-living nematode, Caenorhabditis elegans, coupled with recent major advances in molecular studies of parasitic nematodes, have opened up new avenues for understanding the biology of these parasites and present opportunities for novel strategies of therapeutic intervention and control.

The PAM-1 aminopeptidase regulates centrosome positioning to ensure anterior–posterior axis specification in one-cell C. elegans embryos

In the one-cell Caenorhabditis elegans embryo, the anterior-posterior (A-P) axis is established when the sperm donated centrosome contacts the posterior cortex. While this contact appears to be essential for axis polarization, little is known about the mechanisms governing centrosome positioning during this process. pam-1 encodes a puromycin sensitive aminopeptidase that regulates centrosome positioning in the early embryo. Previously we showed that pam-1 mutants fail to polarize the A-P axis. Here we show that PAM-1 can be found in mature sperm and in cytoplasm throughout early embryogenesis where it concentrates around mitotic centrosomes and chromosomes. We provide further evidence that PAM-1 acts early in the polarization process by showing that PAR-1 and PAR-6 do not localize appropriately in pam-1 mutants. Additionally, we tested the hypothesis that PAM-1s role in polarity establishment is to ensure centrosome contact with the posterior cortex. We inactivated the microtubule motor dynein, DHC-1, in pam-1 mutants, in an attempt to prevent centrosome movement from the cortex and restore anterior-posterior polarity. When this was done, the aberrant centrosome movements of pam-1 mutants were not observed and anterior-posterior polarity was properly established, with proper localization of cortical and cytoplasmic determinants. We conclude that PAM-1s role in axis polarization is to prevent premature movement of the centrosome from the posterior cortex, ensuring proper axis establishment in the embryo.

Gastrointestinal helminths of pipistrelle bats (Pipistrellus pipistrellus/Pipistrellus pygmaeus) (Chiroptera: Vespertilionidae) of England.

Although bats are one of the most successful and diverse of mammalian orders, studies that focus upon bat endoparasites are limited. To further knowledge of bat parasitology, pipistrelle bats (Pipistrellus pipistrellus and P. pygmaeus) were acquired from across the Greater Manchester and Lancashire region of England and examined for gastrointestinal helminths using morphological and molecular analyses. Sixty-eight of 90 adult/juvenile bats (76% prevalence) were infected with at least 1 species of helminth and mean helminth abundance was 48·2 (+/-7·0). All helminths were digenean trematodes and the following species were identified in 51 P. pipistrellus specimens (prevalence in parentheses): Lecithodendrium linstowi (80·4%), L. spathulatum (19·6%), Prosthodendrium sp. (35·3%), Plagiorchis koreanus (29·4%) and Pycnoporus heteroporus (9·8%). Statistical analyses, incorporating multifactorial models, showed that male bats exhibited a significantly more aggregated helminth distribution and lower abundance than females. Positive associations were observed between L. linstowi and L. spathulatum, Prosthodendrium sp. and P. heteroporus and between L. spathulatum and P. koreanus. A revised phylogeny of bat-associated Lecithodendriidae, incorporating novel L. spathulatum and Prosthodendrium sp. 28S rRNA sequences, separated the controversial clade formed by L. linstowi and P. hurkovaae. Further studies are likely to assist the understanding of bat-parasite/pathogen relationships, helminth infracommunity structures and phylogenetics.

Toxoplasma gondii: Prevalence in species and genotypes of British bats (Pipistrellus pipistrellus and P. pygmaeus)

Few studies have investigated Toxoplasma gondii infections in bat populations and none have reported its presence in protected British bat species. Using a collection of dead/euthanased bats collected from Lancashire, UK, two species of bats (Pipistrellus pipistrellus and Pipistrellus pygmaeus) were tested using a highly sensitive SAG1-PCR method specific for detection of T. gondii DNA (n=77; 71 P. pipistrellus and 6 P. pygmaeus). Whilst some potential bias may exist in the sampling strategy, an overall prevalence of 10.39% (±6.06%; 95%CI) was detected. All P. pipistrellus, were also genotyped using eleven polymorphic microsatellite loci to determine their local population structure. The programme STRUCTURE revealed that the majority of individuals (83%) were derived from one interbreeding population, and the remaining individuals (17%) had mixed genetic origins. There was no significant difference in the frequency of T. gondii infection or geographical distribution between subclusters. As all British bats are insectivorous, the routes of infection with T. gondii remain elusive. However, the locally large and panmictic gene pool suggests that intraspecies transmission could be applicable.

Bat Endoparasites: A UK Perspective

Studies have shown that bats are infected with a rich community of endoparasites. However, detailed investigations are lacking; not least because of the challenges of working with hosts that are protected by legislation. Below, we review the status of bat endoparasite studies in the UK, giving due consideration to a significant body of classical parasitological investigations on haematozoa (trypanosomes, the piroplasm Babesia vesperuginis and the haemosporidian Polychromophilus murinus) carried out in the mid-1980s on almost 500 hosts and encompassing 12 of the 17 bat species known to breed in the UK. Of these parasites, only B. vesperuginis-infected bats showed any adverse health impacts, including elevated reticulocyte and white blood cell counts, reduced haemoglobin levels, haemoglobinuria and splenomegaly. More recently, molecular-based analyses of UK bat haematozoa have contributed not only to enriching survey data but also importantly to a wider understanding of evolutionary relationships amongst parasites, which in turn has provided insight into historic movements of the hosts. We also discuss gastrointestinal parasite infections and highlight the lack of published studies on UK bat coccidians and helminths. As such, morphological and molecular analyses carried out in our laboratory, on a population of pipistrelle bats in South Lancashire and Greater Manchester, are providing baseline data on these infections in UK bats. With regard to helminths, we find that pipistrelle bats are commonly infected with digenean trematodes (prevalence = 76 %; mean abundance = 48.2 ± 7), particularly lecithodendriids (e.g. Lecithodendrium linstowi). Moreover, helminth infections were significantly more aggregated and also less abundant in male bats compared to females, an interesting and perhaps surprising parasite response to the host sex hormones. DNA sequencing of the 28S rRNA gene of representative lecithodendriid specimens has offered new insight into evolutionary relationships amongst the Lecithodendriidae, specifically, separating a controversial clade between L. linstowi and Prosthodendrium hurkovaae. Finally, we highlight recent work that utilises PCR-based detection to implicate bats as potentially important reservoir hosts of the apicomplexan Toxoplasma gondii (prevalence = 10 %).