The Leishmania major RNA polymerase II largest subunit lacks a carboxy-terminus heptad repeat structure and its encoding gene is linked with the calreticulin gene

The gene encoding the RNA polymerase II largest subunit (RPOIILS) has been isolated and sequenced from the kinetoplastid protozoan, Leishmania (Leishmania) major. The RPOIILS gene was shown to be present as a single copy and is composed of an uninterrupted open reading frame of 4.99 kb, specifying a protein 1663 aa in length with a predicted molecular mass of approximately 185 kDa. The carboxy terminus domain (CTD) of the RPOIILS from L. (L.) major, typical of the more evolutionary primitive protozoa, lacked a heptad repeat structure which is present in higher eukaryotes and some other protozoan phyla. Comparison of the predicted aa composition of the CTD from a diverse range of eukaryotic species revealed the abundance of Ser and Pro residues as the only discernible evolutionary conservative feature. A putative ATG start codon for an additional expressed sequence was located 1.1 kb downstream of the L. (L.) major RPOIILS gene stop codon. Nucleic acid database searches revealed the identity of this gene as that encoding the calcium binding protein calreticulin (CLT). The close proximity of the RPOIILS and CLT genes in L. (L.) major raises the possibility that these genes are transcribed as part of the same polycistronic unit.     

Isolation and detection of Leishmania species among naturally infected Rhombomis opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran

In Iran, three species of Leishmania have been incriminated as the causative agents of human leishmaniasis, Leishmania (L.) major, Leishmania tropica, and Leishmania infantum.Rhombomis opimus have been incriminated as a principal reservoirs of the parasitic protozoan Leishmania major, the causative agent of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran. Rodents captured and examined to find Leishmania species using conventional methods including direct impression smear and microscopic observation inoculation samples to Balb/c and culture in NNN medium. Also molecular method was employed to detect Leishmania in rodents by amplifying a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA–ITS2) using Nested PCR. Leshmania species were specified by DNA sequences. 36 (38.3%) of R. opimus were Leishmania positive using at least one conventional methods. Many more ITS-rDNA fragments were amplified from R. opimus but only 65 out of 74 PCR products contained enough DNA for direct sequencing or readable sequences. The PCR assays detected in Iranian R. opimus not only Leishmania major in 59 (79.7%) rodents but also Leishmania turanica in 6 (8.1%) rodents, another parasite of the great gerbil. These parasites were found in Turkemen Sahara, North East of Iran, in a focus of rural (ZCL). L. major and L. turanica in R. opimus firmly identified from Turkemen Sahara. Nine rodents with Leishmania infections unidentified which some were unreadable sequences, these could be mixed infections of L. major, L. turanica, Leishmania gerbillisensu lato and Leishmania close to L. gerbilli or a related species reported in sandflies previously from this location. The haplotypes of L. major and L. turanica were found to be identical to that of isolates of L. major and L. turanica from Iran and in GenBank elsewhere. R. opimus is probably the key reservoir in this ZCL focus because of its abundance and its infection rates with both L. major and L. turanica.     

Screening Leishmania donovani‐specific genes required for visceral infection

Comparison of the Leishmania infantum genome with Leishmania braziliensis and Leishmania major genomes has identified 25 L. infantum species‐specific genes that are absent or pseudogenes in L. major and L. braziliensis. To determine whether these L. infantum species‐specific genes are involved in visceral Leishmania infection, we cloned the orthologues of 14 L. infantum species‐specific genes from the genetically closely related Leishmania donovani and introduced them into L. major. Two of these L. donovani species‐specific genes were found to significantly increase L. major survival in visceral organs in BALB/c mice. One (orthologue of LinJ28_V3.0340; Ld2834) of these two genes was further investigated. The L. donovani Ld2834 null mutants displayed dramatically reduced virulence in BALB/c mice and were unable to survive in axenic amastigote culture conditions arguing that Ld2834 plays a crucial role in enabling L. donovani survive at the increased temperature typically associated with visceral organs. Ld2834 encodes a 50 kDa protein that is localized in the cytoplasma and has no significant sequence similarity with other known genes. This study validates the importance of comparative genomics for understanding Leishmania species pathology and argues that Leishmania species‐specific genes play important roles in tissue tropism and virulence.     

Overexpression of a single Leishmania major gene enhances parasite infectivity in vivo and in vitro

We identified a Leishmania major‐specific gene that can partly compensate for the loss of virulence observed for L. major HSP100 null mutants. The gene, encoding a 46 kD protein of unknown function and lineage, also enhances the virulence of wild type L. major upon overexpression. Surprisingly, the approximately sixfold overexpression of this protein also extends the host range of L. major to normally resistant C57BL/6 mice, causing persisting lesions in this strain, even while eliciting a strong cellular immune response. This enhanced virulence in vivo is mirrored in vitro by increased parasite burden inside bone marrow‐derived macrophages. The localization of the protein in the macrophage cytoplasm suggests that it may modulate the macrophage effector mechanisms. In summary, our data show that even minor changes of gene expression in L. major may alter the outcome of an infection, regardless of the host's genetic predisposition.     

Stage-Specific Differential Gene Expression of Glutathione Peroxidase in Leishmania Major and Leishmania Tropica

Background:Leishmania (L) major and L. tropica are the etiological agents of cutaneous leishmaniosis. Leishmania species cause a board spectrum of phenotypes. A small number of genes are differentially expressed between them that have likely an important role in the disease phenotype. Procyclic and metacyclic are two morphological promastigote forms of Leishmania that express different genes. The glutathione peroxidase is an important antioxidant enzyme that essential in parasite protection against oxidative stress and parasite survival. This study aimed to compare glutathione peroxidase (TDPX) gene expression in procyclic and metacyclic and also interspecies in Iranian isolates of L. major and L. tropica. Methods:The samples were cultured in Novy-Nicolle-Mc Neal medium to obtain the promastigotes and identified using PCR-RFLP technique. They were then grown in RPMI1640 media for mass cultivation. The expression level of TDPX gene was compared by Real-time PCR.Results:By comparison of expression level, up-regulation of TDPX gene was observed (5.37 and 2.29 folds) in L. major and L. tropica metacyclic compared to their procyclic, respectively. Moreover, there was no significant difference between procyclic forms of isolates, while 3.05 folds up-regulation in metacyclic was detected in L. major compared L. tropica.Conclusion:Our data provide a foundation for identifying infectivity and high survival related factors in the Leishmania spp. In addition, the results improve our understanding of the molecular basis of metacyclogenesis and development of new potential targets to control or treatment and also, to the identification of species-specific factors contributing to virulence and pathogenicity in the host cells.Key Words: Glutathione peroxidase, Leishmania, L. major, L. tropica, Quantitative Real-time PCR     

Systems analysis of metabolism in the pathogenic trypanosomatid Leishmania major

Systems analyses have facilitated the characterization of metabolic networks of several organisms. We have reconstructed the metabolic network of Leishmania major, a poorly characterized organism that causes cutaneous leishmaniasis in mammalian hosts. This network reconstruction accounts for 560 genes, 1112 reactions, 1101 metabolites and 8 unique subcellular localizations. Using a systems‐based approach, we hypothesized a comprehensive set of lethal single and double gene deletions, some of which were validated using published data with approximately 70% accuracy. Additionally, we generated hypothetical annotations to dozens of previously uncharacterized genes in the L. major genome and proposed a minimal medium for growth. We further demonstrated the utility of a network reconstruction with two proof‐of‐concept examples that yielded insight into robustness of the network in the presence of enzymatic inhibitors and delineation of promastigote/amastigote stage‐specific metabolism. This reconstruction and the associated network analyses of L. major is the first of its kind for a protozoan. It can serve as a tool for clarifying discrepancies between data sources, generating hypotheses that can be experimentally validated and identifying ideal therapeutic targets.     

两种梧桐叶绿体基因组密码子使用偏性分析

为了分析美丽梧桐、云南梧桐叶绿体基因组密码子的使用偏性,该研究通过筛选美丽梧桐、云南梧桐叶绿体基因组中各52条蛋白编码序列,并利用CodonW、CUSP和SPSS软件对其密码子使用模式及偏性进行了分析。结果表明:(1)美丽梧桐、云南梧桐的GC含量分别为38.12%、38.05%,表明叶绿体基因组内富含A/T碱基。(2)有效密码子数(ENC)范围为36.91～56.46、36.55～58.04,表明多数密码子偏性较弱。(3)相对同义密码子(RSCU)分析显示,RSCU>1的密码子各有29个,其中28个以A、U结尾。(4)中性绘图显示,GC₃与GC₁₂的相关性不显著,回归曲线斜率分别为0.195和0.304,说明密码子偏好性主要受到自然选择的影响。(5) ENC-plot分析中大部分基因分布于曲线的周围和下方,ENC比值多分布于-0.04～0.10之间,表明突变会影响密码子偏性的形成。此外,17、18个密码子分别被鉴定为美丽梧桐、云南梧桐的最优密码子。以上结果说明美丽梧桐、云南梧桐叶绿体基因组的密码子使用偏性可能受选择和突变共同作用,且使用...     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.     

Synonymous Codon Usage, Accuracy of Translation, and Gene Length in Caenorhabditis elegans

In many unicellular organisms, invertebrates, and plants, synonymous codon usage biases result from a coadaptation between codon usage and tRNAs abundance to optimize the efficiency of protein synthesis. However, it remains unclear whether natural selection acts at the level of the speed or the accuracy of mRNAs translation. Here we show that codon usage can improve the fidelity of protein synthesis in multicellular species. As predicted by the model of selection for translational accuracy, we find that the frequency of codons optimal for translation is significantly higher at codons encoding for conserved amino acids than at codons encoding for nonconserved amino acids in 548 genes compared between Caenorhabditis elegans and Homo sapiens. Although this model predicts that codon bias correlates positively with gene length, a negative correlation between codon bias and gene length has been observed in eukaryotes. This suggests that selection for fidelity of protein synthesis is not the main factor responsible for codon biases. The relationship between codon bias and gene length remains unexplained. Exploring the differences in gene expression process in eukaryotes and prokaryotes should provide new insights to understand this key question of codon usage. Received: 18 June 2000 / Accepted: 10 November 2000     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

Conservation of the mammalian RNA polymerase II largest-subunit C-terminal domain

Summary We have isolated and sequenced a portion of the gene encoding the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II from three mammals. These mammalian sequences include one rodent and two primate CTDs. Comparisons of the new sequences to mouse and Chinese hamster show a high degree of conservation among the mammalian CTDs. Due to synonymous codon usage, the nucleotide differences between hamster, rat, ape, and human result in no amino acid changes. The amino acid sequence for the mouse CTD appears to have one different amino acid when compared to the other four sequences. Therefore, except for the one variation in mouse, all of the known mammalian CTDs have identical amino acid sequences. This is in marked contrast to the situation among more divergent species. The present study suggests that there is a strong evolutionary pressure to maintain the primary structure of the mammalian CTD. Offprint requests to: J.L. Corden     

Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite Leishmania major

Leishmania major possesses, apparently uniquely, four families of ATG8-like genes, designated ATG8, ATG8A, ATG8B and ATG8C, and 25 genes in total.  L. major ATG8 and examples from the ATG8A, ATG8B and ATG8C families are able to complement a Saccharomyces cerevisiae ATG8-deficient strain, indicating functional conservation. Whereas ATG8 has been shown to form putative autophagosomes during differentiation and starvation of L. major, ATG8A primarily form puncta in response to starvation - suggesting a role for ATG8A in starvation-induced autophagy. Recombinant ATG8A was processed at the scissile glycine by recombinant ATG4.2 but not ATG4.1 cysteine peptidases of L. major and, consistent with this, ATG4.2-deficient L. major mutants were unable to process ATG8A and were less able to withstand starvation than wild type cells. GFP-ATG8-containing puncta were less abundant in ATG4.2 over-expression lines, in which unlipidated ATG8 predominated, which is consistent with ATG4.2 being an ATG8-deconjugating enzyme as well as an ATG8A-processing enzyme. In contrast, recombinant ATG8, ATG8B and ATG8C were all processed by ATG4.1, but not by ATG4.2. ATG8B and ATG8C both have a distinct subcellular location close to the flagellar pocket, but the occurrence of the GFP-labelled puncta suggest that they do not have a role in autophagy. L. major genes encoding possible ATG5, ATG10 and ATG12 homologues were found to complement their respective S. cerevisiae mutants, and ATG12 localised in part to ATG8-containing puncta, suggestive of a functional ATG5-ATG12 conjugation pathway in the parasite. L. major ATG12 is unusual as it requires C-terminal processing by an as yet unidentified peptidase.     

Golgi-Located NTPDase1 of Leishmania major Is Required for Lipophosphoglycan Elongation and Normal Lesion Development whereas Secreted NTPDase2 Is Dispensable for Virulence

Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.     

Members of a unique histidine acid phosphatase family are conserved amongst a group of primitive eukaryotic human pathogens

Recently, we identified and characterized the genes encoding several distinct members of the histidine-acid phosphatase enzyme family from Leishmania donovani, a primitive protozoan pathogen of humans. These included genes encoding the heavily phosphorylated/glycosylated, tartrate-sensitive, secretory acid phosphatases (Ld SAcP-1 and Ld SAcP-2) and the unique, tartrate-resistant, externally-oriented, surface membrane-bound acid phosphatase (Ld MAcP) of this parasite. It had been previously suggested that these enzymes may play essential roles in the growth, development and survival of this organism. In this report, to further examine this hypothesis, we assessed whether members of the L. donovani histidine-acid phosphatase enzyme family were conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Such phylogenetic conservation would clearly indicate an evolutionary selection for this family of enzymes and strongly suggest and support an important functional role for acid phosphatases to the survival of these parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that homologs of both the Ld SAcPs and Ld MAcP were present in each of the visceral and cutaneous Leishmania species examined (i.e. isolates of L. donovani, L. infantum,L. tropica, L. major and L. mexicana, respectively). Further, results of enzyme assays showed that all of these organisms expressed both tartrate-sensitive and tartrate-resistant acid phosphatase activities. In addition, homologs of both the Ld SAcPs and Ld MAcP genes and their corresponding enzyme activities were also identified in two Crithidia species (C. fasciculata and C. luciliae) and in Leptomonas seymouri. In contrast, Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens had only very low levels of such enzyme activities. Cumulatively, results of this study showed that homologs of the Ld SAcPs and Ld MAcP are conserved amongst all pathogenic Leishmania sps. suggesting that they may play significant functional roles in the growth, development and survival of all members of this important group of human pathogens.     

The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.