Identification of Leishmania-specific protein phosphorylation sites by LC-ESI-MS/MS and comparative genomics analyses.

Human pathogenic protozoa of the genus Leishmania undergo various developmental transitions during the infectious cycle that are triggered by changes in the host environment. How these parasites sense, transduce, and respond to these signals is only poorly understood. Here we used phosphoproteomic approaches to monitor signaling events in L. donovani axenic amastigotes, which may be important for intracellular parasite survival. LC-ESI-MS/MS analysis of IMAC-enriched phosphoprotein extracts identified 445 putative phosphoproteins in two independent biological experiments. Functional enrichment analysis allowed us to gain insight into parasite pathways that are regulated by protein phosphorylation and revealed significant enrichment in our data set of proteins whose biological functions are associated with protein turn-over, stress response, and signal transduction. LC-ESI-MS/MS analysis of TiO(2)-enriched phosphopeptides confirmed these results and identified 157 unique phosphopeptides covering 181 unique phosphorylation sites in 126 distinct proteins. Investigation of phosphorylation site conservation across related trypanosomatids and higher eukaryotes by multiple sequence alignment and cluster analysis revealed L. donovani-specific phosphoresidues in highly conserved proteins that share significant sequence homology to orthologs of the human host. These unique phosphorylation sites reveal important differences between host and parasite biology and post-translational protein regulation, which may be exploited for the design of novel anti-parasitic interventions.

Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage.

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1(-/-) null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.

Sorting of Leishmania-bearing dendritic cells reveals subtle parasite-induced modulation of host-cell gene expression.

Once in the mouse skin, Leishmania (L) amazonensis amastigotes are hosted by professional mononuclear phagocytes such as dendritic cells (DCs). When monitored after parasite inoculation, the frequency of amastigote-hosting DCs is very low (<1%) in both the skin and skin-draining lymph nodes. Therefore, we designed and validated an efficient procedure to purify live amastigotes-hosting DCs with the objective to facilitate quantitative and qualitative analysis of such rare cells. To this end, a L. amazonensis transgenic parasite expressing DsRed2 fluorescent protein was generated and added to mouse bone marrow-derived DC cultures. Then, a high speed sorting procedure, performed in BSL-2 containment, was setup to pick out only DCs hosting live amastigotes. This study reveals, for the first time, a unique transcript pattern from sorted live amastigotes-hosting DCs that would have been undetectable in unsorted samples. It was indeed possible to highlight a significant and coordinated up-regulation of L-arginine transporter and arginase2 transcripts in Leishmania-hosting DCs compared to un-parasitized DCs. These results indicate that arginine catabolism for polyamine generation is dominating over L-arginine catabolism for NO generation. In conclusion, this approach provides a powerful method for further characterisation, of amastigote-hosting DCs in the skin and the skin-draining lymph nodes.

Early over expression of messenger RNA for multiple genes, including insulin, in the Pancreatic Lymph Nodes of NOD mice is associated with Islet Autoimmunity.

BACKGROUND: Autoimmune diabetes (T1D) onset is preceded by a long inflammatory process directed against the insulin-secreting beta cells of the pancreas. Deciphering the early autoimmune mechanisms represents a challenge due to the absence of clinical signs at early disease stages. The aim of this study was to identify genes implicated in the early steps of the autoimmune process, prior to inflammation, in T1D. We have previously established that insulin autoantibodies (E-IAA) predict early diabetes onset delineating an early phenotypic check point (window 1) in disease pathogenesis. We used this sub-phenotype and applied differential gene expression analysis in the pancreatic lymph nodes (PLN) of 5 weeks old Non Obese Diabetic (NOD) mice differing solely upon the presence or absence of E-IAA. Analysis of gene expression profiles has the potential to provide a global understanding of the disease and to generate novel hypothesis concerning the initiation of the autoimmune process. METHODS: Animals have been screened weekly for the presence of E-IAA between 3 and 5 weeks of age. E-IAA positive or negative NOD mice at least twice were selected and RNAs isolated from the PLN were used for microarray analysis. Comparison of transcriptional profiles between positive and negative animals and functional annotations of the resulting differentially expressed genes, using software together with manual literature data mining, have been performed. RESULTS: The expression of 165 genes was modulated between E-IAA positive and negative PLN. In particular, genes coding for insulin and for proteins known to be implicated in tissue remodelling and Th1 immunity have been found to be highly differentially expressed. Forty one genes showed over 5 fold differences between the two sets of samples and 30 code for extracellular proteins. This class of proteins represents potential diagnostic markers and drug targets for T1D. CONCLUSION: Our data strongly suggest that the immune related mechanisms taking place at this early age in the PLN, correlate with homeostatic changes influencing tissue integrity of the adjacent pancreatic tissue. Functional analysis of the identified genes suggested that similar mechanisms might be operating during pre-inflammatory processes deployed in tissues i) hosting parasitic microorganisms and ii) experiencing unrestricted invasion by tumour cells.

Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes.

BACKGROUND: Mammal macrophages (MPhi) display a wide range of functions which contribute to surveying and maintaining tissue integrity. One such function is phagocytosis, a process known to be subverted by parasites like Leishmania (L). Indeed, the intracellular development of L. amazonensis amastigote relies on the biogenesis and dynamic remodelling of a phagolysosome, termed the parasitophorous vacuole, primarily within dermal MPhi. RESULTS: Using BALB/c mouse bone marrow-derived MPhi loaded or not with amastigotes, we analyzed the transcriptional signatures of MPhi 24 h later, when the amastigote population was growing. Total RNA from MPhi cultures were processed and hybridized onto Affymetrix Mouse430_2 GeneChips, and some transcripts were also analyzed by Real-Time quantitative PCR (RTQPCR). A total of 1,248 probe-sets showed significant differential expression. Comparable fold-change values were obtained between the Affymetrix technology and the RTQPCR method. Ingenuity Pathway Analysis software pinpointed the up-regulation of the sterol biosynthesis pathway (p-value = 1.31e-02) involving several genes (1.95 to 4.30 fold change values), and the modulation of various genes involved in polyamine synthesis and in pro/counter-inflammatory signalling. CONCLUSION: Our findings suggest that the amastigote growth relies on early coordinated gene expression of the MPhi lipid and polyamine pathways. Moreover, these MPhi hosting multiplying L. amazonensis amastigotes display a transcriptional profile biased towards parasite-and host tissue-protective processes.

AffyGCQC: a web-based interface to detect outlying genechips with extreme studentized deviate tests.

Affymetrix GeneChip oligonucleotide arrays are dedicated to analyzing gene expression differences across distinct experimental conditions. Data production for such arrays is an elaborate process with many potential sources of variability unrelated to biologically relevant gene expression variations. Therefore, rigorous data quality assessment is fundamental throughout the process for downstream biologically meaningful analyses. We have developed a program named AffyGCQC, which is the acronym for a bioinformatics tool designed to perform Affymetrix GeneChip Quality Control. This program implements a graphical representation of QC metrics recommended by Affymetrix for GeneChip oligonucleotide array technology. Most importantly, it performs extreme studentized deviate statistical tests for the set of arrays being compared in a given experiment, thus providing an objective measure for outlier detection. AffyGCQC has been designed as an easy-to-use Web-based interface (online supplementary information: http://www.transcriptome.ens.fr/AffyGCQC/; contact: affygcqc@biologie.ens.fr).

Unveiling pathways used by Leishmania amazonensis amastigotes to subvert macrophage function.

This article provides a summary and discussion of properties of Leishmania amazonensis-loaded mouse macrophages. It illustrates how high-throughput analysis is expected to contribute to deciphering features displayed by macrophages when they are subverted as host cells for replicating Leishmania amastigotes. Firstly, we discuss features of mouse mononuclear phagocytes in steady-state conditions, including the phagocytosis of apoptotic cells. Secondly, we discuss results from ongoing investigations aimed at characterizing transcriptional signatures displayed by BALB/c mouse bone marrow-derived macrophages housing replicating L. amazonensis amastigotes. After a brief presentation on the feasibility of high-throughput microscopy relying on our robust culture system, we share some perspectives on the perpetuation of L. amazonensis in their hosts. Within this latter context, a novel question is formulated and its relevance is discussed: do the Leishmania amastigotes that persist within the mammalian dermis reach a non-replicating developmental stage? If so, is this developmental stage the only one displaying the features required for further development as promastigotes within the sand fly gut lumen?

Dense genome-wide linkage analysis of rheumatoid arthritis, including covariates.

OBJECTIVE: Rheumatoid arthritis (RA) is a heterogeneous disease that exhibits a complex genetic component. Previous RA genome scans confirmed the involvement of the HLA region and generated data on suggestive signals at non-HLA regions, albeit with few overlaps in findings between studies. The present study was undertaken to detect potential RA gene regions and to estimate the number of true RA gene regions, taking into account the heterogeneity of RA, through performance of a dense genome scan. METHODS: In a study of 88 French Caucasian families (105 RA sibpairs), 1,088 microsatellite markers were genotyped (3.3-cM genome scan), and a multipoint model-free linkage analysis was performed. The statistical assessment of the results relied on 10,000 computer simulations. A covariate-based multipoint model-free linkage analysis was performed on the locations of regions with suggestive evidence for linkage. RESULTS: Involvement of the HLA region was strongly confirmed (P = 6 x 10(-5)), and 19 non-HLA regions showed suggestive evidence for linkage (P < 0.05); 9 of these overlapped with regions suggested in other published RA genome scans. A routine 12-cM genome scan with the same families would have detected only 7 of the 19 regions, including only 4 of the 9 overlapping regions. From the 10,000 computer simulations, we estimated that 8 +/- 4 regions (mean +/- SD) were true-positives. RA covariate-based analysis provided additional linkage evidence for 3 regions, with age at disease onset, erosions, and HLA-DRB1 shared epitope as covariates. CONCLUSION: The results of this study provide evidence of 19 non-HLA RA gene regions, with an estimate of 8 +/- 4 as true-positives, and provide additional evidence for 3 regions from covariate-based analysis.