The extended tubulin superfamily.

Although most eukaryotic cells can express multiple isotypes of alphabeta-tubulin, the significance of this diversity has not always been apparent. Recent data indicate that particular alphabeta-tubulin isotypes, both genome encoded and those derived by post-translational modification, can directly influence microtubule structure and function--thus validating ideas originally proposed in the multitubulin hypothesis over 25 years ago. It has also become increasingly evident over the past year that some (but intriguingly not all) eukaryotes encode several other tubulin proteins, and to date five further members of the tubulin superfamily, gamma, delta, epsilon, zeta and eta, have been identified. Although the role of gamma-tubulin in the nucleation of microtubule assembly is now well established, far less is known about the functions of delta-, epsilon-, zeta- and eta-tubulin. Recent work has expanded our knowledge of the functions and localisation of these newer members of the tubulin superfamily, and the emerging data suggesting a restricted evolutionary distribution of these 'new' tubulin proteins, conforms to established knowledge of microtubule cell biology. On the basis of current evidence, we predict that delta-, epsilon-, zeta- and eta-tubulin all have functions associated with the centriole or basal body of eukaryotic cells and organisms.

Phenotypic changes associated with deletion and overexpression of a stage-regulated gene family in Leishmania.

The LmcDNA16 locus of Leishmania major contains three highly related genes HASPA1, HASPA2 and HASPB, encoding hydrophilic, acylated surface proteins and a tandem pair of unrelated sequences, SHERP1 and SHERP2, coding for a small, hydrophilic protein that localizes to the endoplasmic reticulum and outer mitochondrial membrane. Differential regulation of these genes results in expression of a subset of the HASP proteins and SHERP only in infective stage parasites. To assess the contribution of these molecules to parasite virulence, the diploid LmcDNA16 gene locus has been removed by targeted gene deletion. Homozygous null mutants have precise deletions of both alleles and exhibit no HASP or SHERP expression. They are at least as virulent as wild-type parasites in macrophage invasion and intracellular survival assays, both in vitro and in vivo. Conversely, null mutants engineered to overexpress the entire LmcDNA16 gene locus are unable to survive within the intramacrophage environment despite their differentiation into infective metacyclic parasites. Both null and overexpressing null parasites show increased sensitivity to complement-mediated lysis, suggesting perturbation of their surface architecture. Avirulence in overexpressing parasites correlates with selective depletion of a specific lipid species, decreased expression of the major surface glycoprotein GP63, but no significant downregulation of the glycoconjugate lipophosphoglycan.

Identification and characterisation of a RAD51 gene from Leishmania major.

The RAD51 gene is a homologue of Escherichia coli recA which plays a central role in homologous recombination and DNA repair. This paper describes the identification of the RAD51 gene from the trypanosomatid parasite Leishmania major. The LmRAD51 gene codes for a 377 amino acid polypeptide with a predicted molecular mass of 41259 Da that is highly homologous to the Rad51 family of proteins. Recombinant L. major Rad51 protein (LmRad51) was over-expressed in a bacterial expression system, purified to homogeneity and shown to bind DNA and exhibit DNA-stimulated ATPase activity, consistent with previously reported biochemical characteristics of Rad51 protein. Although LmRad51 expression is below the level of detection in exponentially growing cultures of Leishmania, high levels of LmRad51 mRNA and protein expression can be detected following exposure to the DNA-damaging agent phleomycin. LmRAD51 is one of the first examples of a DNA damage-inducible gene to be characterised in Leishmania, and will be invaluable in studying the contribution of homologous recombination to Leishmania virulence.

Characterization of a differentially expressed protein that shows an unusual localization to intracellular membranes in Leishmania major.

The SHERP genes are found as a tandem pair within the differentially regulated LmcDNA16 locus of Leishmania major. The SHERP gene product (small hydrophilic endoplasmic reticulum-associated protein) is unusual in its small size (6.2 kDa), its acidic pI (4.6) and its exclusive, high-level expression ( approximately 100000 copies per cell) in infective non-replicative parasite stages. No homologues have been found to date. Secondary-structure predictions suggest that SHERP contains an amphiphilic alpha-helix that is presumably involved in protein-protein interactions. SHERP has been localized to the endoplasmic reticulum as well as to the outer mitochondrial membrane in both wild-type and over-expressing parasites. Given the absence of an N-terminal signal sequence, transmembrane-spanning domains or detectable post-translational modifications, it is likely that this hydrophilic molecule is a peripheral membrane protein on the cytosolic face of intracellular membranes. This weak membrane association has been confirmed in cell-fractionation assays, in which SHERP redistributes from the cytoplasmic to the membrane fraction after in vivo cross-linking. SHERP does not appear to be involved in rearrangements of the cytoskeleton or conservation of organelle morphology during parasite differentiation. The role of this novel protein, presumed to be part of a protein complex, in infective parasites that are nutrient-deficient and pre-adapted for intracellular survival in the mammalian host is under investigation.

Diversity in repeat-containing surface proteins of Leishmania major.

The gene B protein (GBP) is one of the products of the LmcDNA16 gene family, a cluster of related but non-identical genes that are differentially-expressed during the Leishmania life cycle. This protein, which is found on the surface of infective stage parasites, contains an extensive region of proline-rich amino acid repeats, constituting 45% of the total protein. The structure and stability of these repeats have been investigated in a number of L. major strains by polymerase chain reaction (PCR) amplification and Southern blotting. Data reported in this paper demonstrate variability between strains with respect to the number of repeats encoded by GBP, although those strains isolated within adjacent geographical regions have conserved repeat structures. The data also reveal that some parasite lines have additional repeat sequences within a second, related gene in the LmcDNA16 array. Western blotting experiments have established that these sequences are expressed in vivo, indicating that L. major strains are heterogeneous in their surface complement of gene B repeat-containing proteins.

Characterisation of a second protein encoded by the differentially regulated LmcDNA16 gene family of Leishmania major.

The LmcDNA16 gene family of Leishmania major contains five genes: three highly related sequences, genes A, B and C, and a tandem pair of unrelated sequences, genes D1 and D2. Previous studies have demonstrated that gene B codes for a novel, hydrophilic protein that is present on the surface of infective parasite stages at approximately 10(5) copies per call. This paper describes the identification and characterisation of a second protein encoded by this gene array: the 7.6 kDa A/C protein. This molecule shares considerable amino acid identity with the gene B protein (GBP) but lacks the characteristic proline rich amino acid repeat region. Like GBP, the A/C protein is expressed on the surface of infective metacyclic parasites, despite the lack of conventional signal and anchor sequences. It has previously been suggested that the GBP repetitive sequence plays a role in mediating protein attachment to the parasite surface. It now appears more likely that the conserved amino- and/or carboxyl-terminal domains of the A/C and B proteins are involved in this process.

A single gene copy merozoite surface antigen and immune evasion?

During the course of chronic malaria infection antigenic variants of a parasite antigen are expressed and exposed on the surface of infected erythrocyte membranes. There also exists a number of apparently invariant single gene copy blood-stage antigens, exposed or non-exposed, which have been shown to afford immunity under experimental conditions. To determine why the host, presented with invariant 'protective' antigens, is unable to control infections effectively, immunity to a representative single gene copy antigen, the merozoite surface protein 1 (MSP1) was investigated in Plasmodium chabaudi chabaudi AS, a murine model of chronic malaria. Immunization with monoclonal antibody affinity purified native MSP1 resulted in enhanced control of parasitaemia on challenge, irrespective of the parasite inoculum size; challenge with a single parasite, however, suggested that expansion of resistant parasite subpopulations was not occurring. Challenge of mice immunized with recombinant fusion proteins encoding N- or C-terminal regions of the P.c. chabaudi AS MSP1 produced inconsistent effects, often parasitaemias were indistinguishable from controls despite significant anti-MSP1 antibody responses. The not unlikely contamination of MSP1 native preparations with erythrocyte (E) components was considered. Immunization with a mixture of the MSP1 C-terminus recombinant polypeptide and a Triton X-100 solubilized lysate of normal E resulted in enhanced control of parasitaemia, however, no effect was seen after administration of either component on its own. Co-immunization of E with the N-terminus polypeptide reversed the inhibition seen, on this occasion with this construct alone.

Processing of the Plasmodium chabaudi chabaudi AS merozoite surface protein 1 in vivo and in vitro.

Processing of the Plasmodium merozoite surface protein 1 (MSP-1) has been described for parasites maintained under in vitro conditions. We have now demonstrated, using CBA/Ca mice infected with Plasmodium chabaudi chabaudi AS, that MSP-1 processing also occurs in vivo. The major proteolytic cleavage sites and a processing scheme were deduced from N-terminal amino-acid sequences of the MSP-1 breakdown products. Comparison of MSP-1 processing in P. falciparum and P.c. chabaudi indicates a degree of conservation and in two cases the position of protease cleavage appears identical. Significant amounts of MSP-1 polypeptides are found in plasma during schizogony. Various aspects of MSP-1 processing including immunological and physiological reactions in the host during the critical period of schizogony can now be examined in vivo.

Nucleotide sequence analysis and epitope mapping of the merozoite surface protein 1 from Plasmodium chabaudi chabaudi AS.

The complete nucleotide sequence of the gene encoding the merozoite surface protein 1 (MSP-1) from the rodent malaria parasite Plasmodium chabaudi chabaudi AS has been determined by direct sequencing of overlapping PCR derived fragments. Comparison of the P. c. chabaudi AS nucleotide sequence with the previously published P. c. chabaudi IP-PC1 sequence indicates that although the MSP-1 gene of these two P. c. chabaudi strains is highly conserved, with sequence identity often approaching 100%, interspersed throughout the molecule are 5 regions of divergence. This is at variance with published data which suggested that the P. c. chabaudi AS and P. c. chabaudi IP-PC1 MSP-1 sequences are largely identical. Epitope mapping studies with a panel of anti-P. c. chabaudi AS MSP-1 monoclonal antibodies demonstrate that whilst most of these mAbs recognise epitopes at the N-terminus of the MSP-1 molecule, two mAbs, including one capable of inhibiting challenge infections in mice in an in vivo passive transfer assay, recognise epitopes which map to the C-terminus.

A single amino acid determines the specificity of a monoclonal antibody which inhibits Plasmodium chabaudi AS in vivo.

The in vivo inhibitory action of NIMP23, a monoclonal antibody raised against the rodent parasite Plasmodium chabaudi chabaudi AS, has previously been shown to be strain-specific, capable of delaying significantly the onset of P. c. chabaudi AS but not a P. c. chabaudi CB challenge parasitaemia. The epitope to which this mAb binds has been mapped to the second of two epidermal growth factor-like domains located at the C-terminus of the merozoite surface protein 1 (MSP-1) of P. c. chabaudi AS. The C-terminus region of the MSP-1 of P. c. chabaudi is a region of heterogeneity with AS and CB strain parasites showing only 78% identity at the amino acid level. The critical amino acid substitution which accounts for the strain specificity of the NIMP23 monoclonal antibody has now been identified. Polymerase chain reaction directed mutagenesis experiments demonstrate that a single proline to asparagine substitution at position 1722 in the primary amino acid sequence is sufficient to convert NIMP23-negative P. c. chabaudi CB expression constructs into NIMP23-positive clones whilst the converse substitution of an asparagine for a proline residue converts P. c. chabaudi AS expression constructs into NIMP23-negative clones.

Antigenic cross-reactivity between Necator americanus and Ascaris lumbricoides in a community in Papua New Guinea infected predominantly with hookworm.

Sero-epidemiological data are presented in which antigenic cross-reactivity between Necator americanus and Ascaris lumbricoides has been investigated in a community in Papua New Guinea infected predominantly with N. americanus. It is our contention that the antigenic cross-reactivity which undoubtedly exists between these species accounted for (i) a peak in antibody levels against N. americanus in 10-13 years old children (driven by infection with A. lumbricoides), and (ii) the maintenance of apparent antibody levels against A. lumbricoides in older age groups (driven by infection with N. americanus in the absence of overt infection with A. lumbricoides). Cross-reactivity was analysed further, and apparently N. americanus-specific epitopes identified, by immunoblotting. These observations could have considerable bearing on the interpretation of data from sero-epidemiological studies which failed to take account of concurrent infection with these parasites.

Necator americanus secretory acetylcholinesterase and its purification from excretory-secretory products by affinity chromatography.

Acetylcholinesterase (AChE) secretion by adult N. americanus was enhanced in vitro by incorporating insoluble collagen rafts into culture dishes. Enzyme produced in this way had preferential substrate specificity for acetylthiocholine iodide (ATC), and its activity was inhibited by eserine (1.1 x 10(-8) M). Ancylostoma ceylanicum, another hookworm species, failed to produce comparable amounts of AChE in culture. AChE was efficiently purified from culture medium by affinity chromatography on edrophonium sepharose; 81% of the AChE activity was retained by the affinity matrix, although this fraction contained only 4.3% of the protein loaded. Antisera raised against purified AChE in rabbits immunohistochemically stained the oesophageal glands of the parasite, and reacted with molecules of 32, 60, 80, 140 and 220 kDa in reduced adult ES products on Western blotting, although differential activity was observed against worm homogenates and earlier developmental stages. On IEF, purified AChE resolved predominantly with a pl of 3.55; proteins with a similar pl were recognized by rabbit anti-AChE. IgG preparations of this antiserum inhibited AChE activity in ES products, and inhibited AChE secretion by adult worms in culture. The availability of this immunological probe will allow definitive experiments to be conducted on the role of this enigmatic enzyme in the host-parasite relationship.

Hookworm (Necator americanus) infection and storage iron depletion.

The relationship between iron status and the intensity of infection with hookworm was investigated in a rural population on Karkar Island, Mandang Province, Papua New Guinea. There was a significant negative correlation between plasma ferritin level and hookworm burden, which was strongest in males. In contrast, there was no correlation between plasma ferritin and hookworm egg count, and no consistent correlation between haemoglobin level or haematocrit and either measure of hookworm intensity. The results suggest that the role of hookworm in the aetiology of anaemia may be difficult to assess without the accurate measurement of hookworm burden.

An immunological and biochemical comparison of hookworm species.

The successful development of diagnostic tests capable of distinguishing human hookworm species will ultimately depend on an increase in our knowledge of their comparative biochemistry. Here, David Pritchard, Paul McKean and Gerry Schad describe some aspects of the comparative biochemistry of Necator americanus and Ancylostoma duodenale, and emphasize differences between the species that could form the basis of discriminatory diagnostic tests.

The identification of a species-specific antigen from Necator americanus.

A 17 kD protein of Necator americanus was isolated by SDS-PAGE and used to raise monospecific antisera in rabbits. ELISA and Western blotting against a range of parasite extracts demonstrated the species specificity of this protein. It is expressed at all stages of the life-cycle, appears to be accumulated through the larval stages to adulthood, and can be localized in the oesophageal glands and cuticle of the adult parasite. The possible nature and diagnostic potential of this protein is discussed.

Epidemiology and immunology of Necator americanus infection in a community in Papua New Guinea: humoral responses to excretory-secretory and cuticular collagen antigens.

Baseline data from an immuno-epidemiological study of hookworm infection in a rural village in Madang Province, Papua New Guinea are reported. Necator americanus was found to be the commonest helminth infection, with a prevalence of near 100% and intensity of 40 worms per host in adults. Enterobius vermicularis, Ascaris lumbricoides and Trichuris trichiura were also present, at prevalences of 53, 10 and 3% respectively; Ancylostoma duodenale was absent. The frequency distribution of N. americanus was highly over-dispersed, and was well described by a negative binomial distribution with aggregation parameter, k, of 0.370. Intensity of infection was significantly related to host age, but did not differ between the sexes. Haemoglobin levels and haematocrit values were indicative of anaemia in the community, but were unrelated to hookworm infection. Levels of antibodies (IgG, IgA and IgM combined) against adult Necator cuticular collagen and excretory-secretory (ES) products were determined. Serum concentrations of the two types of antibody were significantly correlated with each other. Significant positive correlations were found between anti-ES antibody levels and hookworm egg production, and between anti-collagen antibody levels and host age. It is suggested that the level of anti-collagen antibodies may reflect cumulative exposure to infection, whereas levels of anti-ES antibodies may be more dependent on current worm burden. No evidence was found to suggest that either antibody response is important in regulating parasite population growth. Similarly, the presence of a positive correlation between eosinophil concentration and infection intensity in adults indicates that eosinophilia reflects, rather than determines, the host's worm burden.

The action of a mast cell protease on the cuticular collagens of Necator americanus.

Collagen-like proteins, thought to be responsible for maintaining the structural integrity of the nematode cuticle, were isolated from adult Necator americanus and shown to be susceptible to digestion by purified mast cell proteases. Although these collagens would appear normally to be masked by superficially expressed (surface) antigens, it is suggested that a sufficiently avid and specific immune response could remove this potentially protective coat, rendering the structurally important underlying layers open to immune attack.