Evolutionarily conserved proteins as prominent immunogens during Leishmania infections

Many Leishmania antigens have been identified as members of conserved protein families, such as the acidic ribosomal proteins, the histones and the heat-shock proteins; despite this, they elicit specific immune responses. Furthermore, homologues of many of these antigens are immune targets in other infectious diseases and systemic autoimmune diseases. Here, Jose Mar a Requena, Carlos Alonso and Manuel Soto review this class of widely distributed antigens, which they call 'panantigens'. They also propose a model to explain the prominent immunogenicity of these antigens during Leishmania infection, on the basis of the fact that many panantigens are constituents of multicomponent complexes in the cell. The elucidation of the pathways by which Leishmania antigens are processed and presented to effector cells from the host immune system will shed light on the immunopathology of leishmaniasis and help in the development of protective immunotherapies.     

Molecular and functional analyses of a novel class I secretory nuclease from the human pathogen, Leishmania donovani

The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc(s), a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc(s) chimeric construct was used in conjunction with an anti-LdNuc(s) peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single- and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen.     

The active site of the DNA repair endonuclease XPF-ERCC1 forms a highly conserved nuclease motif

XPF-ERCC1 is a structure-specific endonuclease involved in nucleotide excision repair, interstrand crosslink repair and homologous recombination. So far, it has not been shown experimentally which subunit of the heterodimer harbors the nuclease activity and which amino acids contribute to catalysis. We used an affinity cleavage assay and located the active site to amino acids 670-740 of XPF. Point mutations generated in this region were analyzed for their role in nuclease activity, metal coordination and DNA binding. Several acidic and basic residues turned out to be required for nuclease activity, but not DNA binding. The separation of substrate binding and catalysis by XPF-ERCC1 will be invaluable in studying the role of this protein in various DNA repair processes. Alignment of the active site region of XPF with proteins belonging to the Mus81 family and a putative archaeal RNA helicase family reveals that seven of the residues of XPF involved in nuclease activity are absolutely conserved in the three protein families, indicating that they share a common nuclease motif.     

Aspartic proteases of Plasmodium vivax are highly conserved in wild isolates

The plasmepsins are the aspartic proteases of malaria parasites. Treatment of aspartic protease inhibitor inhibits hemoglobin hydrolysis and blocks the parasite development in vitro suggesting that these proteases might be exploited their potentials as antimalarial drug targets. In this study, we determined the genetic variations of the aspartic proteases of Plasmodium vivax (PvPMs) of wild isolates. Two plasmepsins (PvPM4 and PvPM5) were cloned and sequenced from 20 P. vivax Korean isolates and two imported isolates. The sequences of the enzymes were highly conserved except a small number of amino acid substitutions did not modify key residues for the function or the structure of the enzymes. The high sequence conservations between the plasmepsins from the isolates support the notion that the enzymes could be reliable targets for new antimalarial chemotherapeutics.     

Viscerotropic growth pattern of Leishmania tropica in BALB/c mice is suggestive of a murine model for human viscerotropic leishmaniasis

Leishmania (L.) tropica is a causative agent of cutaneous leishmaniasis, and occasionally of visceral or viscerotropic leishmaniasis in humans. Murine models of Leishmania infection have been proven to be useful for elucidation of mechanisms for pathogenesis and immunity in leishmaniasis. The aim of this study was to establish a murine model for human viscerotropic leishmaniasis, and the growth pattern of L. tropica was studied in different tissues of BALB/c mice in order to find out whether the parasite visceralizes in this murine model. L. major was used as a control as this species is known to cause a progressive infection in BALB/c mice. L. tropica or L. major was injected into the footpad of mice, and thickness of footpad, parasite loads in different tissues, and the weight of the spleen and lymph node were determined at different intervals. Results showed that L. tropica visceralizes to the spleen and grows there while its growth is controlled in footpad tissues. Dissemination of L. tropica to visceral organs in BALB/c mice was similar to the growth patterns of this parasite in human viscerotropic leishmaniasis. The BALB/c model of L. tropica infection may be considered as a good experimental model for human diseases.     

Kinetoplast DNA minicircles of Leishmania donovani express a protein product

We describe an unprecedented finding of an open reading frame present in the variable region in one of the minicircle sequence classes of a human pathogenic strain of Leishmania donovani (MHOM/IN/90/RMRI 68) which is transcribed and translated. The encoded protein showed homologies to known transport proteins.     

Functional significance of a highly conserved glutamate residue of the human noradrenaline transporter

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of hE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in Vmax values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.     

Experimental transmission of Leishmania infantum by two major vectors: a comparison between a viscerotropic and a dermotropic strain

We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Both L. infantum strains studied, dermotropic CUK3 and viscerotropic IMT373, developed well in Phlebotomus perniciosus and Lutzomyia longipalpis. They produced heavy late-stage infection and colonized the stomodeal valve, which is a prerequisite for successful transmission. Infected sand fly females, and especially those that transmit parasites, feed significantly longer on the host (1.5-1.8 times) than non-transmitting females. Quantitative PCR revealed that P. perniciosus harboured more CUK3 strain parasites, while in L. longipalpis the intensity of infection was higher for the IMT373 strain. However, in both sand fly species the parasite load transmitted was higher for the strain with dermal tropism (CUK3). All but one sand fly female infected by the IMT373 strain transmitted less than 600 promastigotes; in contrast, 29% of L. longipalpis and 14% of P. perniciosus infected with the CUK3 strain transmitted more than 1000 parasites. The parasite number transmitted by individual sand flies ranged from 4 up to 4.19×10(4) promastigotes; thus, the maximal natural dose found was still about 250 times lower than the experimental challenge dose used in previous studies. This finding emphasizes the importance of determining the natural infective dose for the development of an accurate experimental model useful for the evaluation of new drugs and vaccines.     

Diverse cellular localizations of secretory phospholipase A2 enzymes in several human tissues

The secretory phospholipase A2 (sPLA2) family in mammals contains more than 10 enzymes. In this study, we examined by immunohistochemistry the localization of six sPLA2s (IIA, IID, IIE, IIF, V and X) in human heart, kidney, liver and stomach. In normal hearts, sPLA2-IIA was detected in coronary vascular smooth muscle cells (VSMC) and sPLA2-V in cardiomyocytes beneath the endocardium. In infarcted hearts, expression of these two enzymes was markedly increased in damaged cardiomyocytes, and expression of sPLA2-IID and-IIE, which was undetectable in normal hearts, was elevated in damaged cardiomyocytes and VSMC, respectively. In infarcted kidneys, sPLA2-IIA and-V were markedly induced in the uriniferous tubular epithelium. In livers affected by viral hepatitis, sPLA2-IIA and-V were expressed in hepatocytes with fatty degeneration. In the gastric glands exhibiting intestinal metaplasia, sPLA2-IIA was localized in the glandular base, sPLA2-IID and-V in the glandular body epithelium, sPLA2-IIE and-IIF in goblet cells in the foveolar epithelium, and sPLA2-X in both glandular body epithelial cells and foveolar epithelial goblet cells. In the gastric submucosal tissues, sPLA2-IIA and-IIE were located in VSMC and sPLA2-V was in the interstitial fibroblasts. In addition, sPLA2-IIA,-IIE,-IIF and-X were highly expressed in gastric signet ring cell carcinoma. Thus, individual sPLA2s exhibit unique cellular localizations in each tissue, suggesting their distinct roles in pathophysiology.     

Exons encoding the highly conserved part of human glutaminyl-tRNA synthetase

Summary Aminoacyl-tRNA synthetases are important components of the genetic apparatus. In spite of common catalytic properties, synthetases with different amino acid specificities are widely diverse in their primary structures, subunit sizes, and subunit composition. However, synthetases with given amino acid specificities are well conserved throughout evolution. We have been studying the human glutaminyl-tRNA synthetase possessing a sequence of about 400 amino acid residues (the core region) that is very similar to sequences in the corresponding enzymes from bacteria and yeast. The conserved sequence appears to be essential for the basic function of the enzyme, the charging of tRNA with glutamine. As a first step to a better understanding of the evolution of this enzyme, we determined the coding region for the conserved part of the human glutaminyl-tRNA synthetase. The coding region is composed of eight exons. It appears that individual exons encode defined secondary structural elements as parts of functionally important domains of the enzyme. Evolution of the gene by assembly of individual exons seems to be a viable hypothesis; alternative pathways are discussed.Offprint requests to: R. Knippers     

Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon

The nucleotide sequences of the rRNA genes and the 5 flanking region were determined for R. salmoninarum ATCC 33209^T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5 leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S–23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S–5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries.     

Molecular cloning of human uracil-DNA glycosylase, a highly conserved DNA repair enzyme.

Uracil-DNA glycosylase is the DNA repair enzyme responsible for the removal of uracil from DNA, and it is present in all organisms investigated. Here we report on the cloning and sequencing of a cDNA encoding the human uracil-DNA glycosylase. The sequences of uracil-DNA glycosylases from yeast, Escherichia coli, herpes simplex virus type 1 and 2, and homologous genes from varicella-zoster and Epstein-Barr viruses are known. It is shown in this report that the predicted amino acid sequence of the human uracil-DNA glycosylase shows a striking similarity to the other uracil-DNA glycosylases, ranging from 40.3 to 55.7% identical residues. The proteins of human and bacterial origin were unexpectedly found to be most closely related, 73.3% similarity when conservative amino acid substitutions were included. The similarity between the different uracil-DNA glycosylase genes is confined to several discrete boxes. These findings strongly indicate that uracil-DNA glycosylases from phylogenetically distant species are highly conserved.     

mRNA decay during herpesvirus infections: interaction between a putative viral nuclease and a cellular translation factor

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5' end of mRNAs prior to the 3' end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using the Saccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathione S-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.     

Developmentally regulated expression of a cell surface class I nuclease in Leishmania mexicana

Leishmania mexicana, like other trypanosomatid parasites, is a purine auxotroph and must obtain these essential nutrients from its sandfly and mammalian hosts. A single copy gene encoding its unique externally oriented, surface membrane, purine salvage enzyme 3'-nucleotidase/nuclease, was isolated. Structural features of the deduced protein included: an endoplasmic reticulum-directed signal peptide, several conserved class I catalytic and metal co-factor (Zn(2+)) binding domains, transmembrane anchor sequence and a C-terminal cytoplasmic tail. 3'-Nucleotidase/nuclease gene (mRNA) and protein (enzyme activity) expression were examined in three different L. mexicana developmental forms: procyclic promastigotes, metacyclic promastigotes and amastigotes. Results of both approaches demonstrated that the 3'-nucleotidase/nuclease was a stage-specific enzyme, being expressed by promastigote forms (stages restricted to the insect vector), but not by amastigotes (which produce disease in mammalian hosts). Starvation of these parasites for purines resulted in the significant up-regulation of both 3'-nucleotidase/nuclease mRNA and enzyme activity in promastigotes, but not in amastigotes. These results underscore the critical role that the 3'-nucleotidase/nuclease must play in purine salvage during the rapid multiplicative expansion of the parasite population within its insect vector. To our knowledge, the L. mexicana 3'-nucleotidase/nuclease is the first example of a nutrient-induced and developmentally regulated enzyme in any parasitic protozoan.     

Identification, characterization, and expression of a unique secretory lipase from the human pathogen Leishmania donovani

Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a ~33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.     

Partial characterization of a highly conserved aspartyl kinase (AK) in normal human liver

Subcellular fractions from human liver were assayed for aspartyl kinase (AK) activity measured by standard spectrophotometric methods. Along the purification procedure three different fractions displayed the expected enzyme activity. Interestingly, two of these fractions were specifically recognized by antibodies raised against E. coli aspartate kinases, suggesting a high degree of evolutionary conservation for these ubiquitous enzymes for prokaryotes. Since their known function in bacteria is not strictly required in eukaryotes, these observation imply that the presence and activity of aspartyl kinase(s) in mammals might represent putative regulatory roles for these enzymes in eukaryotic cell metabolism.     

The preference of the mitochondrial endonuclease for a conserved sequence block in mitochondrial DNA is highly conserved during mammalian evolution.

Endonuclease activity identified in crude preparations of rat and human heart mitochondria has each been partially purified and characterized. Both the rat and human activities purify as a single enzyme that closely resembles the endonuclease of bovine-heart mitochondria (Cummings, O.W. et. al. (1987) J. Biol. Chem. 262:2005-2015). All three enzymes, for example elute similarly during gel filtration and DNA-cellulose chromatography, and exhibit similar enzymatic properties. Although the nucleotide sequences of the mtDNAs indicate that there has occurred an unusual degree of divergence in the displacement-loop region during mammalian evolution, the nucleotide specificities of the mt endonucleases appear highly conserved and show a striking preference for an evolutionarily-conserved sequence tract that is located upstream from the heavy (H)-strand origin of DNA replication (OriH).     

The temporal distribution of gene duplication events in a set of highly conserved human gene families

Using a data set of protein translations associated with map positions in the human genome, we identified 1520 mapped highly conserved gene families. By comparing sharing of families between genomic windows, we identified 92 potentially duplicated blocks in the human genome containing 422 duplicated members of these families. Using branching order in the phylogenetic trees, we timed gene duplication events in these families relative to the primate-rodent divergence, the amniote-amphibian divergence, and the deuterostome-protostome divergence. The results showed similar patterns of gene duplication times within duplicated blocks and outside duplicated blocks. Both within and outside duplicated blocks, numerous duplications were timed prior to the deuterostome-protostome divergence, whereas others occurred after the amniote-amphibian divergence. Thus, neither gene duplication in general nor duplication of genomic blocks could be attributed entirely to polyploidization early in vertebrate history. The strongest signal in the data was a tendency for intrachromosomal duplications to be more recent than interchromosomal duplications, consistent with a model whereby tandem duplication-whether of single genes or of genomic blocks-may be followed by eventual separation of duplicates due to chromosomal rearrangements. The rate of separation of tandemly duplicated gene pairs onto separated chromosomes in the human lineage was estimated at 1.7 x 10(-9) per gene-pair per year.     

Gamma-tubulin is a highly conserved component of the centrosome.

We have cloned and characterized gamma-tubulin genes from both X. laevis and S. pombe, and partial genes from maize, diatom, and a budding yeast. The proteins encoded by these genes are very similar to each other and to the original Aspergillus protein, indicating that gamma-tubulins are an ubiquitous and highly conserved subfamily of the tubulin family. A null mutation of the S. pombe gene is lethal. gamma-tubulin is a minor protein, present at less than 1% the level of alpha- and beta-tubulin, and is limited to the centrosome. In particular, gamma-tubulin is associated with the pericentriolar material, the microtubule-nucleating material of the centrosome. gamma-Tubulin remains associated with the centrosome when microtubules are depolymerized, suggesting that it is an integral component that might play a role in microtubule organization.