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1.  Changes in the transcriptome of the malaria parasite Plasmodium falciparum during the initial phase of transmission from the human to the mosquito 
BMC Genomics  2013;14:256.
The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector.
To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence.
For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using quantitative confocal microscopy.
The obtained transcriptome data demonstrate the regulations of gene expression immediately following malaria parasite transmission to the mosquito. Our findings support the identification of proteins important for sexual reproduction and further development of the mosquito midgut stages and provide insights into the genetic basis of the rapid adaption of Plasmodium to the insect vector.
PMCID: PMC3640944  PMID: 23586929
Malaria; Plasmodium falciparum; Gametocyte; Gametogenesis; Transmission; Mosquito; Transcriptome
2.  Methods to identify enzymes that degrade the main extracellular polysaccharide component of Staphylococcus epidermidis biofilms 
Virulence  2013;4(3):260-270.
The production of extracellular poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by Staphylococcus epidermidis is the principal determinant of biofilm formation on indwelling medical devices. Enzymes that degrade PNAG therefore provide an attractive strategy for biofilm removal and for the manufacture of biofilm-resistant coatings. Here we present methods that allow the identification of PNAG-degrading enzymes with the ability to detach biofilms. Our protocol includes the preparation of soluble PNAG from S. epidermidis cultures, the incubation of soluble PNAG with candidate enzymes and assays that detect the release of N-acetyl-d-glucosamine using high-pH anion-exchange chromatography (HPAEC) followed in parallel by pulsed amperometric detection (PAD) and online electrospray ionization mass spectrometry (ESI-MS). We validated our procedures using dispersin B, which is currently the only known PNAG-degrading enzyme.
PMCID: PMC3711985  PMID: 23357872
Staphylococcus epidermidis; biofilm; exopolysaccharide; high-pH anion-exchange chromatography; poly-β-1,6-N-acetyl-d-glucosamine
3.  Harmonine, a defence compound from the harlequin ladybird, inhibits mycobacterial growth and demonstrates multi-stage antimalarial activity 
Biology Letters  2011;8(2):308-311.
The harlequin ladybird beetle Harmonia axyridis has been introduced in many countries as a biological control agent, but has become an invasive species threatening the biodiversity of native ladybirds. Its invasive success has been attributed to its vigorous resistance against diverse pathogens. This study demonstrates that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), which is present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited. Harmonine displays gametocytocidal activity, and inhibits the exflagellation of microgametocytes and zygote formation. In an Anopheles stephensi mosquito feeding model, harmonine displays transmission-blocking activity.
PMCID: PMC3297383  PMID: 21937493
Harmonia axyridis; insect immunity; harmonine; antimicrobial activity
4.  Fosmidomycin Uptake into Plasmodium and Babesia-Infected Erythrocytes Is Facilitated by Parasite-Induced New Permeability Pathways 
PLoS ONE  2011;6(5):e19334.
Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii.
Methodology/Principal Findings
Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite.
Our findings provide an explanation for the observed differences in activity of fosmidomycin and FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.
PMCID: PMC3087763  PMID: 21573242
5.  RlmN and Cfr are Radical SAM Enzymes Involved in Methylation of Ribosomal RNA 
Posttranscriptional modifications of ribosomal RNA (rRNA) nucleotides are a common mechanism of modulating the ribosome’s function and conferring bacterial resistance to ribosome-targeting antibiotics. One such modification is methylation of an adenosine nucleotide within the peptidyl transferase center of the ribosome mediated by the indigenous methyltransferase RlmN and its evolutionary-related resistance enzyme Cfr. These methyltransferases catalyze methyl transfer to aromatic carbon atoms of the adenosine within a complex 23S rRNA substrate to form the 2,8-dimethylated product. RlmN and Cfr are members of the Radical SAM superfamily, and contain the characteristic cysteine rich CX3CX2C motif. We demonstrate that both enzymes are capable of accommodating the requisite [4Fe-4S] cluster. S-adenosylmethionine (SAM) is both the methyl donor and the source of a 5′-deoxyadenosyl radical, which activates the substrate for methylation. Detailed analyses of the rRNA requirements show that the enzymes can utilize protein-free 23S rRNA as a substrate, but not the fully-assembled large ribosomal subunit, suggesting that the methylations take place during the assembly of the ribosome. The key recognition elements in the 23S rRNA are helices 90–92 and the adjacent single stranded RNA that encompasses A2503. To our knowledge, this study represents the first in vitro description of a methyl transfer catalyzed by a member of Radical SAM superfamily, and it expands the catalytic repertoire of this diverse enzyme class. Furthermore, by providing information on both the timing of methylation and its substrate requirements, our findings have important implications for the functional consequences of Cfr-mediated modification of rRNA in acquisition of antibiotic resistance.
PMCID: PMC2859901  PMID: 20184321
7.  Analysis of the Isoprenoid Biosynthesis Pathways in Listeria monocytogenes Reveals a Role for the Alternative 2-C-Methyl-d-Erythritol 4-Phosphate Pathway in Murine Infection▿  
Infection and Immunity  2008;76(11):5392-5401.
Most bacteria synthesize isoprenoids through one of two essential pathways which provide the basic building block, isopentyl diphosphate (IPP): either the classical mevalonate pathway or the alternative non-mevalonate 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. However, postgenomic analyses of the Listeria monocytogenes genome revealed that this pathogen possesses the genetic capacity to produce the complete set of enzymes involved in both pathways. The nonpathogenic species Listeria innocua naturally lacks the last two genes (gcpE and lytB) of the MEP pathway, and bioinformatic analyses strongly suggest that the genes have been lost through evolution. In the present study we show that heterologous expression of gcpE and lytB in L. innocua can functionally restore the MEP pathway in this organism and confer on it the ability to induce Vγ9Vδ2 T cells. We have previously confirmed that both pathways are functional in L. monocytogenes and can provide sufficient IPP for normal growth in laboratory media (M. Begley, C. G. Gahan, A. K. Kollas, M. Hintz, C. Hill, H. Jomaa, and M. Eberl, FEBS Lett. 561:99-104, 2004). Here we describe a targeted mutagenesis strategy to create a double pathway mutant in L. monocytogenes which cannot grow in the absence of exogenously provided mevalonate, confirming the requirement for at least one intact pathway for growth. In addition, murine studies revealed that mutants lacking the MEP pathway were impaired in virulence relative to the parent strain during intraperitoneal infection, while mutants lacking the classical mevalonate pathway were not impaired in virulence potential. In vivo bioluminescence imaging also confirmed in vivo expression of the gcpE gene (MEP pathway) during murine infection.
PMCID: PMC2573353  PMID: 18765739
8.  Fosmidomycin plus Clindamycin for Treatment of Pediatric Patients Aged 1 to 14 Years with Plasmodium falciparum Malaria 
Fosmidomycin plus clindamycin was shown to be efficacious in the treatment of uncomplicated Plasmodium falciparum malaria in a small cohort of pediatric patients aged 7 to 14 years, but more data, including data on younger children with less antiparasitic immunity, are needed to determine the potential value of this new antimalarial combination. We conducted a single-arm study to improve the precision of efficacy estimates for an oral 3-day fixed-ratio combination of fosmidomycin and clindamycin at 30 and 10 mg/kg of body weight, respectively, every 12 hours for the treatment of uncomplicated P. falciparum malaria in 51 pediatric outpatients aged 1 to 14 years. Fosmidomycin plus clindamycin was generally well tolerated, but relatively high rates of treatment-associated neutropenia (8/51 [16%]) and falls of hemoglobin concentrations of ≥2 g/dl (7/51 [14%]) are of concern. Asexual parasites and fever were cleared within median periods of 42 h and 38 h, respectively. All patients who could be evaluated were parasitologically and clinically cured by day 14 (49/49; 95% confidence interval [CI], 93 to 100%). The per-protocol, PCR-adjusted day 28 cure rate was 89% (42/47; 95% CI, 77 to 96%). Efficacy appeared to be significantly reduced in children aged 1 to 2 years, with a day 28 cure rate of only 62% for this small subgroup (5/8). The inadequate efficacy in children of <3 years highlights the need for continued systematic studies of the current dosing regimen, which should include randomized trial designs.
PMCID: PMC1538678  PMID: 16870763
9.  Short-Course Regimens of Artesunate-Fosmidomycin in Treatment of Uncomplicated Plasmodium falciparum Malaria 
Fosmidomycin is effective against malaria, but it needs to be given for ≥4 days when used alone. We conducted a study of 50 children with Plasmodium falciparum malaria to evaluate the safety and efficacy of consecutively shortened regimens of artesunate-fosmidomycin (1 to 2 mg/kg of body weight and 30 mg/kg of body weight, respectively; doses given every 12 hours). All dosing regimens were well tolerated. Artesunate-fosmidomycin acted rapidly, resulting in consolidated geometric mean parasite and fever clearance times of 24 h and 15 h, respectively. Treatment regimens of ≥2 days led to cure ratios of 100% by day 14 (39/39; 95% confidence interval [95% CI], 91% to 100%). Most importantly, the 3-day regimen achieved 100% cure on day 28 (10/10; 95% CI, 69% to 100%). Treatment with artesunate-fosmidomycin was associated with transient grade I or II neutropenia (absolute neutrophil counts of 750 to 1,200/μl and 400 to 749/μl, respectively) in six or two patients, respectively. Artesunate-fosmidomycin demonstrates the feasibility and potential value of short-course artemisinin-based combination chemotherapy with rapidly eliminated combination partners.
PMCID: PMC1195450  PMID: 16127049
10.  Fosmidomycin, a Novel Chemotherapeutic Agent for Malaria 
In previous studies, fosmidomycin has been shown to possess activity against Plasmodium falciparum in vitro and in the mouse model. It has a novel mode of action through inhibition of 1-deoxy-d-xylulose 5-phosphate reductoisomerase, an enzyme of the nonmevalonate pathway of isoprenoid biosynthesis, which is absent in humans. In this open-label, uncontrolled trial, the efficacy and safety of fosmidomycin, in an oral dose of 1,200 mg every 8 h for 7 days, were evaluated in the treatment of acute uncomplicated Plasmodium falciparum malaria in 20 adult subjects in Gabon and Thailand. Clinical assessments were performed and thick blood smears were evaluated every 8 h until parasite clearance and resolution of symptoms were achieved; assessments continued at weekly intervals thereafter for the duration of the 28-day followup period. All subjects were clinically and parasitologically cured on day 7 (primary end point). Parasite and fever clearance were rapid, with means of 44 and 41 h, respectively. On day 28, seven out of nine subjects (78%) were cured in Gabon and two out of nine subjects (22%) were cured in Thailand. The drug was well tolerated, although mild gastrointestinal side effects were recorded for five subjects. Analysis of hematological and biochemical parameters showed no clinically significant changes throughout the study. Fosmidomycin is an effective and safe antimalarial drug, although its use as a single agent is restricted by the occurrence of recrudescent infections. However, its role in combination therapy should be explored.
PMCID: PMC151759  PMID: 12543685
11.  In Vitro and In Vivo Synergy of Fosmidomycin, a Novel Antimalarial Drug, with Clindamycin 
Fosmidomycin acts through inhibition of 1-deoxy-d-xylulose 5-phosphate (DOXP) reductoisomerase, a key enzyme of the nonmevalonate pathway of isoprenoid biosynthesis. It possesses potent antimalarial activity in vitro and in murine malaria. In a recent clinical study, fosmidomycin was effective and well tolerated in the treatment of patients with acute uncomplicated Plasmodium falciparum malaria but resulted in an unacceptably high rate of recrudescence. In order to identify a potential combination partner, the interaction of fosmidomycin with a number of antimalarial drugs in current use was investigated in a series of in vitro experiments. Synergy was observed between fosmidomycin and the lincosamides, lincomycin and clindamycin. The efficacy of a combination of fosmidomycin and clindamycin was subsequently demonstrated in the Plasmodium vinckei mouse model.
PMCID: PMC127394  PMID: 12183243
12.  GcpE Is Involved in the 2-C-Methyl-d-Erythritol 4-Phosphate Pathway of Isoprenoid Biosynthesis in Escherichia coli 
Journal of Bacteriology  2001;183(8):2411-2416.
In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.
PMCID: PMC95155  PMID: 11274098
13.  Differential Stimulation of the Na+/H+ Exchanger Determines Chloroquine Uptake in Plasmodium falciparum  
The Journal of Cell Biology  1998;140(2):335-345.
Here we describe the identification and characterization of a physiological marker that is associated with the chloroquine-resistant (CQR) phenotype in the human malarial parasite Plasmodium falciparum. Single cell in vivo pH measurements revealed that CQR parasites consistently have an elevated cytoplasmic pH compared to that of chloroquine-sensitive (CQS) parasites because of a constitutively activated Na+/H+ exchanger (NHE). Together, biochemical and physiological data suggest that chloroquine activates the plasmodial NHE of CQS parasites, resulting in a transitory phase of rapid sodium/hydrogen ion exchange during which chloroquine is taken up by this protein. The constitutively stimulated NHE of CQR parasites are capable of little or no further activation by chloroquine. We propose that the inability of chloroquine to stimulate its own uptake through the constitutively activated NHE of resistant parasites constitutes a minimal and necessary event in the generation of the chloroquine-resistant phenotype.
PMCID: PMC2132566  PMID: 9442109

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