The Leishmania donovani Lipophosphoglycan Excludes the Vesicular Proton-ATPase from Phagosomes by Impairing the Recruitment of Synaptotagmin V

We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galβ1,4Manα1-PO4 units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification

Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum

Real-time PCR (qPCR) is the principal technique for the quantification of pathogen biomass in host tissue, yet no generic methods exist for the determination of the limit of quantification (LOQ) and the limit of detection (LOD) in qPCR. We suggest using the Youden index in the context of the receiver operating characteristic (ROC) curve analysis for this purpose. The LOQ was defined as the amount of target DNA that maximizes the sum of sensitivity and specificity. The LOD was defined as the lowest amount of target DNA that was amplified with a false-negative rate below a given threshold. We applied this concept to qPCR assays for Fusarium verticillioides and Fusarium proliferatum DNA in maize kernels. Spiked matrix and field samples characterized by melting curve analysis of PCR products were used as the source of true positives and true negatives. On the basis of the analysis of sensitivity and specificity of the assays, we estimated the LOQ values as 0.11 pg of DNA for spiked matrix and 0.62 pg of DNA for field samples for F. verticillioides. The LOQ values for F. proliferatum were 0.03 pg for spiked matrix and 0.24 pg for field samples. The mean LOQ values correspond to approximately eight genomes for F. verticillioides and three genomes for F. proliferatum. We demonstrated that the ROC analysis concept, developed for qualitative diagnostics, can be used for the determination of performance parameters of quantitative PCR

Failure of artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria in southern Cambodia

<p>Abstract</p> <p>Background</p> <p>Resistance to anti-malarial drugs hampers control efforts and increases the risk of morbidity and mortality from malaria. The efficacy of standard therapies for uncomplicated <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>malaria was assessed in Chumkiri, Kampot Province, Cambodia.</p> <p>Methods</p> <p>One hundred fifty-one subjects with uncomplicated falciparum malaria received directly observed therapy with 12 mg/kg artesunate (over three days) and 25 mg/kg mefloquine, up to a maximum dose of 600 mg artesunate/1,000 mg mefloquine. One hundred nine subjects with uncomplicated vivax malaria received a total of 25 mg/kg chloroquine, up to a maximum dose of 1,500 mg, over three days. Subjects were followed for 42 days or until recurrent parasitaemia was observed. For <it>P. falciparum </it>infected subjects, PCR genotyping of <it>msp1</it>, <it>msp2</it>, and <it>glurp </it>was used to distinguish treatment failures from new infections. Treatment failure rates at days 28 and 42 were analyzed using both per protocol and Kaplan-Meier survival analysis. Real Time PCR was used to measure the copy number of the <it>pfmdr1 </it>gene and standard 48-hour isotopic hypoxanthine incorporation assays were used to measure IC₅₀for anti-malarial drugs.</p> <p>Results</p> <p>Among <it>P. falciparum </it>infected subjects, 47.0% were still parasitemic on day 2 and 11.3% on day 3. The PCR corrected treatment failure rates determined by survival analysis at 28 and 42 days were 13.1% and 18.8%, respectively. Treatment failure was associated with increased <it>pfmdr1 </it>copy number, higher initial parasitaemia, higher mefloquine IC₅₀, and longer time to parasite clearance. One <it>P. falciparum </it>isolate, from a treatment failure, had markedly elevated IC₅₀for both mefloquine (130 nM) and artesunate (6.7 nM). Among <it>P. vivax </it>infected subjects, 42.1% suffered recurrent <it>P. vivax </it>parasitaemia. None acquired new <it>P. falciparum </it>infection.</p> <p>Conclusion</p> <p>The results suggest that artesunate-mefloquine combination therapy is beginning to fail in southern Cambodia and that resistance is not confined to the provinces at the Thai-Cambodian border. It is unclear whether the treatment failures are due solely to mefloquine resistance or to artesunate resistance as well. The findings of delayed clearance times and elevated artesunate IC₅₀suggest that artesunate resistance may be emerging on a background of mefloquine resistance.</p

Stage-Specific Pathways of Leishmania infantum chagasi Entry and Phagosome Maturation in Macrophages

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival

Effects of Point Mutations in Plasmodium falciparum Dihydrofolate Reductase and Dihydropterate Synthase Genes on Clinical Outcomes and In Vitro Susceptibility to Sulfadoxine and Pyrimethamine

Sulfadoxine-pyrimethamine was a common first line drug therapy to treat uncomplicated falciparum malaria, but increasing therapeutic failures associated with the development of significant levels of resistance worldwide has prompted change to alternative treatment regimes in many national malaria control programs. METHODOLOGY AND FINDING: We conducted an in vivo therapeutic efficacy trial of sulfadoxine-pyrimethamine at two locations in the Peruvian Amazon enrolling 99 patients of which, 86 patients completed the protocol specified 28 day follow up. Our objective was to correlate the presence of polymorphisms in P. falciparum dihydrofolate reductase and dihydropteroate synthase to in vitro parasite susceptibility to sulfadoxine and pyrimethamine and to in vivo treatment outcomes. Inhibitory concentration 50 values of isolates increased with numbers of mutations (single [108N], sextuplet [BR/51I/108N/164L and 437G/581G]) and septuplet (BR/51I/108N/164L and 437G/540E/581G) with geometric means of 76 nM (35-166 nM), 582 nM (49-6890- nM) and 4909 (3575-6741 nM) nM for sulfadoxine and 33 nM (22-51 nM), 81 nM (19-345 nM), and 215 nM (176-262 nM) for pyrimethamine. A single mutation present in the isolate obtained at the time of enrollment from either dihydrofolate reductase (164L) or dihydropteroate synthase (540E) predicted treatment failure as well as any other single gene alone or in combination. Patients with the dihydrofolate reductase 164L mutation were 3.6 times as likely to be treatment failures [failures 85.4% (164L) vs 23.7% (I164); relative risk = 3.61; 95% CI: 2.14 - 6.64] while patients with the dihydropteroate synthase 540E were 2.6 times as likely to fail treatment (96.7% (540E) vs 37.5% (K540); relative risk = 2.58; 95% CI: 1.88 - 3.73). Patients with both dihydrofolate reductase 164L and dihydropteroate synthase 540E mutations were 4.1 times as likely to be treatment failures [96.7% vs 23.7%; RR = 4.08; 95% CI: 2.45 - 7.46] compared to patients having both wild forms (I164 and K540).In this part of the Amazon basin, it may be possible to predict treatment failure with sulfadoxine-pyrimethamine equally well by determination of either of the single mutations dihydrofolate reductase 164L or dihydropteroate synthase 540E.ClinicalTrials.gov NCT00951106

S100A1: A Multifaceted Therapeutic Target in Cardiovascular Disease

Cardiovascular disease is the leading cause of death worldwide, showing a dramatically growing prevalence. It is still associated with a poor clinical prognosis, indicating insufficient long-term treatment success of currently available therapeutic strategies. Investigations of the pathomechanisms underlying cardiovascular disorders uncovered the Ca2+ binding protein S100A1 as a critical regulator of both cardiac performance and vascular biology. In cardiomyocytes, S100A1 was found to interact with both the sarcoplasmic reticulum ATPase (SERCA2a) and the ryanodine receptor 2 (RyR2), resulting in substantially improved Ca2+ handling and contractile performance. Additionally, S100A1 has been described to target the cardiac sarcomere and mitochondria, leading to reduced pre-contractile passive tension as well as enhanced oxidative energy generation. In endothelial cells, molecular analyses revealed a stimulatory effect of S100A1 on endothelial NO production by increasing endothelial nitric oxide synthase activity. Emphasizing the pathophysiological relevance of S100A1, myocardial infarction in S100A1 knockout mice resulted in accelerated transition towards heart failure and excessive mortality in comparison with wild-type controls. Mice lacking S100A1 furthermore displayed significantly elevated blood pressure values with abrogated responsiveness to bradykinin. On the other hand, numerous studies in small and large animal heart failure models showed that S100A1 overexpression results in reversed maladaptive myocardial remodeling, long-term rescue of contractile performance, and superior survival in response to myocardial infarction, indicating the potential of S100A1-based therapeutic interventions. In summary, elaborate basic and translational research established S100A1 as a multifaceted therapeutic target in cardiovascular disease, providing a promising novel therapeutic strategy to future cardiologists

BRCA1 is an essential regulator of heart function and survival following myocardial infarction

The tumour suppressor BRCA1 is mutated in familial breast and ovarian cancer but its role in protecting other tissues from DNA damage has not been explored. Here we show a new role for BRCA1 as a gatekeeper of cardiac function and survival. In mice, loss of BRCA1 in cardiomyocytes results in adverse cardiac remodelling, poor ventricular function and higher mortality in response to ischaemic or genotoxic stress. Mechanistically, loss of cardiomyocyte BRCA1 results in impaired DNA double-strand break repair and activated p53-mediated pro-apoptotic signalling culminating in increased cardiomyocyte apoptosis, whereas deletion of the p53 gene rescues BRCA1-deficient mice from cardiac failure. In human adult and fetal cardiac tissues, ischaemia induces double-strand breaks and upregulates BRCA1 expression. These data reveal BRCA1 as a novel and essential adaptive response molecule shielding cardiomyocytes from DNA damage, apoptosis and heart dysfunction. BRCA1 mutation carriers, in addition to risk of breast and ovarian cancer, may be at a previously unrecognized risk of cardiac failure

A Putative Transcription Factor MYT2 Regulates Perithecium Size in the Ascomycete Gibberella zeae

The homothallic ascomycete fungus Gibberella zeae is a plant pathogen that is found worldwide, causing Fusarium head blight (FHB) in cereal crops and ear rot of maize. Ascospores formed in fruiting bodies (i.e., perithecia) are hypothesized to be the primary inocula for FHB disease. Perithecium development is a complex cellular differentiation process controlled by many developmentally regulated genes. In this study, we selected a previously reported putative transcription factor containing the Myb DNA-binding domain MYT2 for an in-depth study on sexual development. The deletion of MYT2 resulted in a larger perithecium, while its overexpression resulted in a smaller perithecium when compared to the wild-type strain. These data suggest that MYT2 regulates perithecium size differentiation. MYT2 overexpression affected pleiotropic phenotypes including vegetative growth, conidia production, virulence, and mycotoxin production. Nuclear localization of the MYT2 protein supports its role as a transcriptional regulator. Transcriptional analyses of trichothecene synthetic genes suggest that MYT2 additionally functions as a suppressor for trichothecene production. This is the first study characterizing a transcription factor required for perithecium size differentiation in G. zeae, and it provides a novel angle for understanding sexual development in filamentous fungi

A Phenome-Based Functional Analysis of Transcription Factors in the Cereal Head Blight Fungus, Fusarium graminearum

Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The fungus produces mycotoxins that are harmful to animal and human. In this study, a systematic analysis of 17 phenotypes of the mutants in 657 Fusarium graminearum genes encoding putative transcription factors (TFs) resulted in a database of over 11,000 phenotypes (phenome). This database provides comprehensive insights into how this cereal pathogen of global significance regulates traits important for growth, development, stress response, pathogenesis, and toxin production and how transcriptional regulations of these traits are interconnected. In-depth analysis of TFs involved in sexual development revealed that mutations causing defects in perithecia development frequently affect multiple other phenotypes, and the TFs associated with sexual development tend to be highly conserved in the fungal kingdom. Besides providing many new insights into understanding the function of F. graminearum TFs, this mutant library and phenome will be a valuable resource for characterizing the gene expression network in this fungus and serve as a reference for studying how different fungi have evolved to control various cellular processes at the transcriptional level