Functional genetic identification of PRP1, an ABC transporter superfamily member conferring pentamidine resistance in Leishmania major

Pentamidine (PEN) is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance is not well understood. Here, we used a genetic strategy to search for loci able to mediate PEN resistance (PENr) when overexpressed in Leishmania major. A shuttle cosmid library containing genomic DNA inserts was transfected into wild-type promastigotes and screened for PEN-resistant transfectants. Two different cosmids identifying the same locus were found, which differed from other known Leishmania drug resistance genes. The PENr gene was mapped by deletion and transposon mutagenesis to an open reading frame (ORF) belonging to the P-glycoprotein (PGP)/MRP ATP-binding cassette (ABC) transporter superfamily that we named pentamidine resistance protein 1 (PRP1). The predicted PRP1 protein encodes 1,807 amino acids with the typical dimeric structure involving 10 transmembrane domains and two nucleotide-binding domains (NBDs). PRP1-mediated PENr could be reversed by verapamil and PRP1 overexpressors showed cross-resistance to trivalent antimony but not to pentavalent antimony (glucantime). Although the degree of PENr was modest (1.7- to 3.7-fold), this may be significant in clinical drug resistance given the marginal efficacy of PEN against Leishmania.     

Leishmania lipophosphoglycan (LPG) activates NK cells through toll-like receptor-2

Toll-like receptors (TLRs) mediate the cellular response to conserved molecular patterns shared by microorganisms. We report that TLR-2 on human NK cells is upregulated and stimulated by Leishmania major lipophosphoglycan (LPG), a phosphoglycan belonging to a family of unique Leishmania glycoconjugates. We found that purified L. major LPG upregulates both mRNA and the membrane expression of TLR-2 in NK cells. Additionally, IFN-gamma and TNF-alpha production and nuclear translocation of NF-kappaB was enhanced. The activation effect was more intense with LPG purified from infectious metacyclic parasites than from noninfectious procyclic Leishmania. Since the difference between the molecules derived from these two stages of the parasite growth cycle lies exclusively in the number of phosphosaccharide repeat domains and in the composition of glycan side chains that branch off these domains, we propose that TLR-2 possibly distinguishes between phosphorylated glycan repeats on LPG molecules. The effect of LPG on cytokine production and on membrane expression of TLR-2 could be blocked with F(ab')2 fragments of the mAb against LPG (WIC 79.3). Confocal microscopy demonstrated the co-localization of LPG and TLR-2 on the NK cell membrane. Binding of LPG to TLR-2 in NK cells was demonstrated by immunoprecipitations done with anti-TLR-2 and anti-LPG mAb followed by immunoblotting with anti-LPG and anti-TLR-2, respectively. Both antibodies recognized the immune complexes. These results suggest that NK cells are capable of recognition of, and activation by, Leishmania LPG through TLR-2, enabling them to participate autonomously in the innate immune system and thereby increasing the effective destruction of the parasite.     

Characterization of plasma membrane bound inorganic pyrophosphatase from Leishmania donovani promastigotes and amastigotes

Background

Currently, a major problem in the management of visceral leishmaniasis or kala-azar, especially in the Indian subcontinent, is the growing unresponsiveness to conventional antimonial therapy. Membrane bound pyrophophatase (PPases) do not exist in plasma membrane from mammals. Thus, H⁺-PPases from Leishmania plasma membrane might be potential target in rational chemotherapy of the disease caused by Leishmania parasites.

Objective

To characterize the activities of inorganic pyrophophatase (PPase) in the plasma membrane of Leishmania donovani promastigote and amastigote.

Methods

Culture method of promastigote and amastigote were developed. We assayed PPase activity in isolated plasma membrane of L. donovani.

Results

We characterized K⁺-PPase present in the plasma membrane of Leishmania donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K⁺ ions and sodium orthovanadate, inhibited by pyrophosphate analogs imidodiphosphate and alendronate, KF, DCCD, thiol reagent parachloromercuribenzenesulfonate (PCMBS), N-ethylmaliemide (NEM), phenylarsineoxide, ABC superfamily transport modulator verapamil and was also by F₁F_o-ATPase inhibitor quercetin.

Conclusion

We conclude that there are significant differences within promastigote, amastigote and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as putative targets for rational drug design.     

Complement C3 is required for the progression of cutaneous lesions and neutrophil attraction in<Emphasis Type="Italic"> Leishmania major</Emphasis> infection

To elucidate the role of complement-mediated uptake in Leishmania major infection in vivo, transgenic BALB/c mice that express the cobra venom factor (CVF) under control of the 1-antitrypsin promoter were infected. CVF expression in these mice leads to a continuous activation and subsequent consumption of complement C3 in the serum. In contrast to susceptible non-transgenic BALB/c mice, CVF-transgenic mice are highly resistant to L. major infection and show a significantly reduced parasite dissemination. Transient depletion of C3 in wild-type BALB/c mice delays progression of lesions for some days. Both CVF-transgenic and non-transgenic mice exhibit similar T cell responses upon infection. However, in CVF-transgenic mice, no infiltration of neutrophils, which were the prominent infiltrating cells at the site of infection in normal susceptible mice, could be detected. We conclude that C3 cleavage is required for the attraction of neutrophils that participate in parasite dissemination.     

Structure and expression of two genes encoding secreted acid phosphatases under phosphate-deficient conditions in Pholiota nameko strain N2

Twenty-three polypeptides secreted in response to a deficiency of inorganic phosphate (Pi) were previously found by two-dimensional polyacrylamide gel electrophoresis analysis in mycelia of Pholiota nameko strain N2. In this study, N-terminal sequencing revealed three of them to be identical to known acid phosphatases of P. nameko strain N114. Two cDNAs and the corresponding genomic DNAs of genes PNAP1 and PNAP2 which encode two of the three acid phosphatases were cloned. The deduced amino acid sequences of PNAP1 and PNAP2 showed high similarity to other fungal acid phosphatases and contained a putative catalytic active site of acid phosphatase. PNAP1 and PNAP2 are comprised of five and seven exons interrupted by four and six introns, respectively. Their promoter regions include two cis-acting elements found in Pi deficiency-inducible genes of Saccharomyces cerevisiae, together with several known functional elements such as a TATA box. Northern blot analysis showed that PNAP1 and PNAP2 are expressed in response to a deficiency of Pi.     

Pentose phosphate metabolism in Leishmania mexicana

The metabolism of pentose phosphates was studied in Leishmania mexicana promastigotes. Each of the enzymes of the classical pentose phosphate pathway (PPP) has been identified and specific activities measured. Functioning of the PPP was demonstrated in non-growing cells by measuring the evolution of 14CO2 from [1-14C]D-glucose and [6-14C]D-glucose under normal conditions and also under selective stimulation of the PPP by exposure to methylene blue. The proportion of glucose which passes through the PPP increases in the latter condition, thus suggesting a protective role against oxidant stress. The incorporation into nucleic acids of ribose 5-phosphate provided via either glucose or free ribose was also determined. Results indicate that the PPP enables glucose to serve as a source of ribose 5-phosphate in nucleotide biosynthesis. Moreover, free ribose is incorporated efficiently, implying the presence of a ribose uptake system and also of ribokinase. Ribose was shown to be accumulated by a carrier mediated process in L. mexicana promastigotes and ribokinase activity was also measured in these cells.     

Expression of infection-related genes in parasites and host during murine experimental infection with Leishmania (Leishmania) amazonensis

Leishmania parasites are able to interfere with host immune responses on many levels, as T cell responses balance, as observed in the murine model of infection. In the present study, we analyzed genes expression in both host and parasite during the progression of infection. Host genes associated to T-lymphocytes responses, MHC classes I and II, as well as parasite enzymes genes, cysteine-proteinases(CP) B and C, were examined in mice along evolution of infection by Leishmania (Leishmania) amazonensis. Murine strains with distinct levels of susceptibility to infection presented different patterns of MHC genes expression: MHC class I genes tend to have higher expression levels in CBA mice, whereas MHC class II genes expression predominates in BALB/c mice. CPB genes expression in the parasites was shown to predominate over CPC in both mice strains tested. Understanding genes expression patterns during infection may lead to new and more efficient treatments for leishmaniasis.     

Antibodies against Leishmania cross-react with Crithidia luciliae: indirect immunofluorescence and Dot-ELISA study in dogs

Leismaniasis is zoonotic parasitic disease distributed in many areas of Mediterranean basin. Because of its pathogenicity and difficulty to grow in vitro, we used Leishmania infantum as primary and Crithidia luciliae as secondary source of antigen. We compared 100 canine sera by indirect immunofluorescence and Dot-ELISA. Both atigens showed same results.     

Histopathologic and electron microscopic characterization of cutaneous leishmaniasis in <Emphasis Type="Italic">Tatera indica</Emphasis> and <Emphasis Type="Italic">Gerbillus</Emphasis> spp. infected with <Emphasis Type="Italic">Leishmania major</Emphasis>

Cutaneous leishmaniasis (CL) is a zoonotic disease transmitted between rodents and canines, mainly by Phlebotomus sand flies and man. In southern Iran, the incidence of this protozoan disease has doubled over the last decade. The present study deals with histopathological features of CL in Tatera indica and Gerbillus spp. that participate in the epidemiology of CL in southern Iran. Thirty-two trapped rodents were evaluated for any Leishmania infection using enzyme electrophoresis and polymerase chain reaction and were concomitantly studied for any histopathological changes. Histopathological studies showed that bone marrow was the tissue of choice for light and electron microscopic study of Leishmania, demonstrating the macrophages infected with the amastigote form of the parasite. This is the first report of the histopathological detection of L. major in naturally infected T. indica and Gerbillus spp in the Larestan region.     

Isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae by functional complementation in yeast

Summary This paper describes the isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae. Two pools of plasmids consisting of BamHI and Sa1GI yeast DNA inserts into the bifunctional yeast — Escherichia coli vector pLC544 (Kingsman et al. 1979) were constructed in E. coli and used for the isolation of the two genes by selection for functional complementation of trp2 and trp3 mutations, respectively, in yeast.The TRP2 gene was isolated on a 6.2 kb BamHl and a 5.8 kb Sa1GI yeast DNA fragment which shared an identical 4.5 kb BamHI-SaIGI fragment. The TRP3 gene was located on a 5.2 kb BamHl fragment.By physical, genetic and physiological experiments it could be shown that the cloned yeast DNA fragments contained the whole structural sequences as well as the regulatory regions of the TRP2 and the TRP3 genes.     

Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags

We report on the development of a new PCR technique for the isolation of genomic fragments that flank known DNA sequences. This technique, single oligonucleotide nested (SON)-PCR, relies on only two amplification reactions with two or three nested sequence-specific primers. It allows the isolation of DNA regions located on either side of a known DNA sequence, with high specificity. DNA products of 2 kb in size can be generated that all contain one copy of the same primer at both ends. Sequence analysis of these products indicates that the binding of the primers to non-specific DNA sites mainly depends on their overall complementarity to the target sequence. Moreover, analysis shows that short extensions of the primers can occur during the first amplification reaction and that a 2-bp overlap between subsequent primers can target their annealing to their predecessors sequence. Ninety percent of the DNA products larger than 0.5 kb correspond to fragments of interest and we obtained successful results with various templates and primer sets. SON-PCR therefore seems a very efficient and widely applicable method for the rapid identification of large unknown DNA regions. Based on available expressed sequence tags, this technique was applied to isolate the palH and pacC genes of the phytopathogenic fungus Botrytis cinerea, with their 5 or 3 flanking regions.     

Oligopeptidase B-2 from <Emphasis Type="Italic">Leishmania amazonensis</Emphasis> with an unusual C-terminal extension

The oligopeptidase B serine protease is an important virulence factor and therapeutic target in Trypanosoma infections. Recently, the Leishmania major Genome Project identified a new oligopeptidase B that was denominated oligopeptidase B-like, herein named oligopeptidase B-2. In this study, a complete open reading frame of oligopeptidase B-2 from Leishmania amazonensis (PH8 strain) was amplified by PCR using primers designed for the oligopeptidase B-2 gene of L. major. The 2,715 bp fragment coded for a protein of 905 amino acids with a predicted molecular mass of 103,918.9 Da and theoretical pI of 5.82. The encoded protein displayed ∼96% identity with L. major and ∼75% identity with Trypanosoma cruzi and T. brucei oligopeptidases B-2, and ∼21% identity with Escherichia coli and L. amazonensis classical oligopeptidase B. An unusual C-terminal extension was found in relation to the classical trypanosomatid oligopeptidase B. By sequence alignment, we determined a catalytic triad (Ser 629, Asp 717 and His 758), S1 subsite (Glu 674 and Glu 676) and suggest a difference in the S2 subsite of L. amazonensis oligopeptidase B-2. We also found that the oligopeptidase B-2 gene is expressed in all cycle stages of L. amazonensis. A phylogenetic analysis indicated that oligopeptidase B-2 is a new member of oligopeptidase B.     

用高通量实时荧光PCR技术研究低浓度MNNG诱发的细胞基因应答反应

目的：研究低浓度 N-甲基-N'-硝基-N-甲基亚硝基胍对人羊膜FL细胞部分基因表达的影响，以助于阐明MNNG引起细胞应答反应的基因及其调控机制。方法：用ABI公司的高通量实时荧光定量PCR方法，检测FL细胞在0.2 μmol/L MNNG处理后基因表达发生的改变。数据用ABI公司的SDS 2.1软件分析。结果：MNNG处理后，在检测的95个基因中，29个基因表达发生改变，其中14个基因下调2倍以上，15个基因下调在1.5-2倍之间；其中有4个基因与细胞周期相关，6个基因与信号转导相关，6个基因与转录调节相关。结论：在低浓度 MNNG攻击后，FL细胞的基因表达发生了显著的变化。     

Either one of the two yeast EF-1α genes is required for cell viability

Summary Two genes,TEF1 andTEF2, encode the protein elongation factor EF-1 in the yeastSaccharomyces cerevisiae. We have generated yeast haploid strains containing eitherTEF1 orTEF2 interrupted by insertion of a large piece of foreign DNA. Cells which contain either one functional copy of the EF-1 genes are viable. In contrast, attempts to isolate a yeast haploid strain with bothTEF1 andTEF2 inactivated have failed suggesting that the double gene disruption is a lethal event.     

Genome-wide analysis of differentially expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source

Penicillium chrysogenum is an economically important ascomycete used as industrial producer of penicillin. However, with the exception of penicillin biosynthesis genes, little attention has been paid to the genetics of other aspects of the metabolism of this fungus. In this article we describe the first attempt of systematic analysis of expressed genes in P. chrysogenum, using a suppression subtractive hybridization approach to clone and identify sequences of genes differentially expressed in media with glucose or lactose as carbon source (penicillin-repressing or non-repressing conditions). A total of 167 clones were analysed, 95 from the glucose condition and 72 from the lactose condition. Genes differentially expressed in the glucose condition encode mainly proteins involved in the mitochondrial electron transport chain and primary metabolism. Genes expressed differentially in lactose-containing medium include genes for secondary metabolism (pcbC, isopenicillin N synthase), different hydrolases and a gene encoding a putative hexose transporter or sensor. The results provided information on how the metabolism of this fungus adapts to different carbon sources. The expression patterns of some of the genes support the hypothesis that glucose induces higher rates of respiration in P. chrysogenum while repressing secondary metabolism.