TLR9 Activation Is Triggered by the Excess of Stimulatory versus Inhibitory Motifs Present in Trypanosomatidae DNA

DNA sequences purified from distinct organisms, e.g. non vertebrate versus vertebrate ones, were shown to differ in their TLR9 signalling properties especially when either mouse bone marrow-derived- or human dendritic cells (DCs) are probed as target cells. Here we found that the DC-targeting immunostimulatory property of Leishmania major DNA is shared by other Trypanosomatidae DNA, suggesting that this is a general trait of these eukaryotic single-celled parasites. We first documented, in vitro, that the low level of immunostimulatory activity by vertebrate DNA is not due to its limited access to DCs'' TLR9. In addition, vertebrate DNA inhibits the activation induced by the parasite DNA. This inhibition could result from the presence of competing elements for TLR9 activation and suggests that DNA from different species can be discriminated by mouse and human DCs. Second, using computational analysis of genomic DNA sequences, it was possible to detect the presence of over-represented inhibitory and under-represented stimulatory sequences in the vertebrate genomes, whereas L. major genome displays the opposite trend. Interestingly, this contrasting features between L. major and vertebrate genomes in the frequency of these motifs are shared by other Trypanosomatidae genomes (Trypanosoma cruzi, brucei and vivax). We also addressed the possibility that proteins expressed in DCs could interact with DNA and promote TLR9 activation. We found that TLR9 is specifically activated with L. major HMGB1-bound DNA and that HMGB1 preferentially binds to L. major compared to mouse DNA. Our results highlight that both DNA sequence and vertebrate DNA-binding proteins, such as the mouse HMGB1, allow the TLR9-signaling to be initiated and achieved by Trypanosomatidae DNA.     

Spatio-Temporal Factors Associated with Meningococcal Meningitis Annual Incidence at the Health Centre Level in Niger, 2004–2010

Background

Epidemics of meningococcal meningitis (MM) recurrently strike the African Meningitis Belt. This study aimed at investigating factors, still poorly understood, that influence annual incidence of MM serogroup A, the main etiologic agent over 2004–2010, at a fine spatial scale in Niger.

Methodology/Principal Findings

To take into account data dependencies over space and time and control for unobserved confounding factors, we developed an explanatory Bayesian hierarchical model over 2004–2010 at the health centre catchment area (HCCA) level. The multivariate model revealed that both climatic and non-climatic factors were important for explaining spatio-temporal variations in incidence: mean relative humidity during November–June over the study region (posterior mean Incidence Rate Ratio (IRR) = 0.656, 95% Credible Interval (CI) 0.405–0.949) and occurrence of early rains in March in a HCCA (IRR = 0.353, 95% CI 0.239–0.502) were protective factors; a higher risk was associated with the percentage of neighbouring HCCAs having at least one MM A case during the same year (IRR = 2.365, 95% CI 2.078–2.695), the presence of a road crossing the HCCA (IRR = 1.743, 95% CI 1.173–2.474) and the occurrence of cases before 31 December in a HCCA (IRR = 6.801, 95% CI 4.004–10.910). At the study region level, higher annual incidence correlated with greater geographic spread and, to a lesser extent, with higher intensity of localized outbreaks.

Conclusions

Based on these findings, we hypothesize that spatio-temporal variability of MM A incidence between years and HCCAs result from variations in the intensity or duration of the dry season climatic effects on disease risk, and is further impacted by factors of spatial contacts, representing facilitated pathogen transmission. Additional unexplained factors may contribute to the observed incidence patterns and should be further investigated.     

Modulation of Phosphorylation of Tocopherol and Phosphatidylinositol by hTAP1/SEC14L2-Mediated Lipid Exchange

The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-³²P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, βT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains.     

A serpin regulates dorsal-ventral axis formation in the Drosophila embryo

Extracellular serine protease cascades have evolved in vertebrates and invertebrates to mediate rapid, local reactions to physiological or pathological cues. The serine protease cascade that triggers the Toll signaling pathway in Drosophila embryogenesis shares several organizational characteristics with those involved in mammalian complement and blood clotting. One of the hallmarks of such cascades is their regulation by serine protease inhibitors (serpins). Serpins act as suicide substrates and are cleaved by their target protease, forming an essentially irreversible 1:1 complex. The biological importance of serpins is highlighted by serpin dysfunction diseases, such as thrombosis caused by a deficiency in antithrombin. Here, we describe how a serpin controls the serine protease cascade, leading to Toll pathway activation. Female flies deficient in Serpin-27A produce embryos that lack dorsal-ventral polarity and show uniform high levels of Toll signaling. Since this serpin has been recently shown to restrain an immune reaction in the blood of Drosophila, it demonstrates that proteolysis can be regulated by the same serpin in different biological contexts.     

Role of CD4 hinge region in GP120 utilization by immunoglobulin domain 1

Immunoglobulin-like domain 1 of CD4 (D1-CD4) promotes HIV infection by binding the envelope glycoprotein (ENV) and exposing its coreceptor-binding site. To study CD4-ENV-coreceptor interactions, we characterized hybrid receptors having domains 1 and 2 of CD4 (D1D2-CD4) joined to the N-terminus of chemokine receptors CCR5, CXCR4, CXCR2, and DARC. Hybrid receptors showed conserved ENV-coreceptor specificity in cell-cell fusion assays. Although D1D2-CD4-CCR5 was sufficient to permit ENV-mediated fusion, D1-CD4-CCR5 and human D1/mouse D2-CD4-CCR5 lacked CD4 function and binding to a neutralizing antibody mapped to D1-CD4. Chimeric D1D2-CD4 joined to CCR5 revealed that the C-terminal 20 residues of human D2-CD4 are required for efficient ENV-mediated fusion. Mutagenesis of hybrid receptors showed the importance of residues forming D1-D2 CD4 interdomain contacts and hinge region proximal residues. Mutagenesis of WT human CD4 confirmed that residues forming D1-D2 interdomain contacts and hinge-region proximal residues contribute positively to CD4 activity in the full-length receptor.     

Role of proteomics to differentiate between benign and potentially malignant pancreatic cysts

Pancreatic cystic neoplasms represent 10-15% of primary cystic masses of the pancreas. While pancreatic cysts are detected with an increasing frequency due to the use of advanced imaging modalities in clinical practice, the diagnosis of pancreatic cystic neoplasms remains unsatisfactory because available diagnostic techniques proved not sensitive enough so far. This study was designed to characterize the proteomic pattern of pancreatic cyst fluids obtained from various cystic lesions. Cyst fluids were collected by direct puncture during open surgery to avoid any possible contamination from other tissues. CEA, CA-19-9, and amylase concentrations were measured using specific immunoassays. After immunodepletion and fractionation by SDS-PAGE, proteins were digested and analyzed by LC-MS/MS. Specific histological lesions were found to be associated with distinct protein patterns. Interestingly, some of these proteins have been proposed as biomarkers of pancreatic cancer. Immunoblots allowed for verifying the differential expression in specific cyst fluids of two selected proteins, olfactomedin-4 and mucin-18. Finally, immunohistochemistry was performed to correlate these data with the expression pattern of olfactomedin-4 and mucin-18 in pancreatic cyst tissues. Results from this study indicate that proteomic analysis of cyst fluid could provide reliable candidates for developing new biomarkers for the preoperative management of malignant and premalignant pancreatic cysts.     

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.     

Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH

The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.     

Proteomics reveals potential biomarkers of seed vigor in sugarbeet

To unravel biomarkers of seed vigor, an important trait conditioning crop yield, a comparative proteomic study was conducted with sugarbeet seed samples of varying vigor as generated by an invigoration treatment called hydropriming and an aging treatment called controlled deterioration. Comparative proteomics revealed proteins exhibiting contrasting behavior between seed samples. Thus, 18 proteins were up-regulated during priming and down-regulated during aging and further displayed an up-regulation upon priming of the aged seeds, meaning that down-regulation of these spot volumes during aging was reversible upon subsequent priming. Also, 11 proteins exhibited the converse behavior characterized by a decrease and an increase of the spot volumes during priming and aging of the control seeds, respectively, and a decrease in the spot volumes upon priming of the aged seeds. The results underpinned the role in seed vigor of several metabolic pathways involved in lipid and starch mobilization, protein synthesis or the methyl cycle. They also corroborate previous studies suggesting that the glyoxylate enzyme isocitrate lyase, the capacity of protein synthesis and components of abscisic acid signaling pathways are likely contributors of seed vigor.