Y chromosome lineage- and village-specific genes on chromosomes 1p22 and 6q27 control visceral leishmaniasis in Sudan

Familial clustering and ethnic differences suggest that visceral leishmaniasis caused by Leishmania donovani is under genetic control. A recent genome scan provided evidence for a major susceptibility gene on Chromosome 22q12 in the Aringa ethnic group in Sudan. We now report a genome-wide scan using 69 families with 173 affected relatives from two villages occupied by the related Masalit ethnic group. A primary ten-centimorgan scan followed by refined mapping provided evidence for major loci at 1p22 (LOD score 5.65; nominal p = 1.72 × 10⁻⁷; empirical p < 1 × 10⁻⁵; λ_S = 5.1) and 6q27 (LOD score 3.74; nominal p = 1.68 × 10⁻⁵; empirical p < 1 × 10⁻⁴; λ_S = 2.3) that were Y chromosome–lineage and village-specific. Neither village supported a visceral leishmaniasis susceptibility gene on 22q12. The results suggest strong lineage-specific genes due to founder effect and consanguinity in these recently immigrant populations. These chance events in ethnically uniform African populations provide a powerful resource in the search for genes and mechanisms that regulate this complex disease.     

Comparative analysis of two slc11 (Nramp) loci in Takifugu rubripes

To study the evolution of the solute carrier family 11 (slc11; formerly Nramp) protein, we isolated and characterized two paralogs from the pufferfish Takifugu rubripes (Fugu). These teleost genes, designated Fugu slc11a-a and Fugu slc11a-b, comprise open reading frames of 1743 nucleotides (581 amino acids) and 1662 nt (554 aa), respectively. The proteins are 81% similar, and both exhibit signature features of the slc11 family of proteins including 12 transmembrane domains, a conserved transport motif and a glycosylated loop. Both Fugu paralogs are more Slc11a2-like based on sequence homology and phylogenetic studies. Analysis of gene environment placed both in the proximity of multiple loci syntenic to human chromosome 12q13, that is, within a SLC11A2 gene environment. However, Fugu slc11a-a also gave one match with chromosome 2q35, where human SLC11A1 resides. Functional diversification was suggested by differences in tissue distribution and subcellular localization. Fugu slc11a-a exhibits a restricted expression profile and a complex subcellular localization, including LAMP1 positive late endosomes/lysosomes in transiently transfected mouse macrophages. Fugu slc11a-b is expressed ubiquitously and localizes solely to late endosomes/lysosomes. This comparative analysis extends our understanding of the evolution and function of this important family of divalent cation transporters. [Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AJ496547/8/9 and AJ496550.]     

Assignment of rainbow trout linkage groups to specific chromosomes

The rainbow trout genetic linkage groups have been assigned to specific chromosomes in the OSU (2N=60) strain using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group. There was a rough correlation between chromosome size and size of the genetic linkage map in centimorgans for the genetic maps based on recombination from the female parent. Chromosome size and structure have a major impact on the female:male recombination ratio, which is much higher (up to 10:1 near the centromeres) on the larger metacentric chromosomes compared to smaller acrocentric chromosomes. Eighty percent of the BAC clones containing duplicate genes mapped to a single chromosomal location, suggesting that diploidization resulted in substantial divergence of intergenic regions. The BAC clones that hybridized to both duplicate loci were usually located in the distal portion of the chromosome. Duplicate genes were almost always found at a similar location on the chromosome arm of two different chromosome pairs, suggesting that most of the chromosome rearrangements following tetraploidization were centric fusions and did not involve homeologous chromosomes. The set of BACs compiled for this research will be especially useful in construction of genome maps and identification of QTL for important traits in other salmonid fishes.     

Cytokine Responses to Novel Antigens in an Indian Population Living in an Area Endemic for Visceral Leishmaniasis

Background

There are no effective vaccines for visceral leishmaniasis (VL), a neglected parasitic disease second only to malaria in global mortality. We previously identified 14 protective candidates in a screen of 100 Leishmania antigens as DNA vaccines in mice. Here we employ whole blood assays to evaluate human cytokine responses to 11 of these antigens, in comparison to known defined and crude antigen preparations.

Methods

Whole blood assays were employed to measure IFN-γ, TNF-α and IL-10 responses to peptide pools of the novel antigens R71, Q51, L37, N52, L302.06, J89, M18, J41, M22, M63, M57, as well as to recombinant proteins of tryparedoxin peroxidase (TRYP), Leishmania homolog of the receptor for activated C kinase (LACK) and to crude soluble Leishmania antigen (SLA), in Indian patients with active (n = 8) or cured (n = 16) VL, and in modified Quantiferon positive (EHC^+ve, n = 20) or modified Quantiferon negative (EHC^−ve, n = 9) endemic healthy controls (EHC).

Results

Active VL, cured VL and EHC^+ve groups showed elevated SLA-specific IFN-γ, but only active VL patients produced IL-10 and EHC^+ve did not make TNF-α. IFN-γ to IL-10 and TNF-α to IL-10 ratios in response to TRYP and LACK antigens were higher in cured VL and EHC^+ve exposed individuals compared to active VL. Five of the eleven novel candidates (R71, L37, N52, J41, and M22) elicited IFN-γ and TNF-α, but not IL-10, responses in cured VL (55–87.5% responders) and EHC^+ve (40–65% responders) subjects.

Conclusions

Our results are consistent with an important balance between pro-inflammatory IFNγ and TNFγ cytokine responses and anti-inflammatory IL-10 in determining outcome of VL in India, as highlighted by response to both crude and defined protein antigens. Importantly, cured VL patients and endemic Quantiferon positive individuals recognise 5 novel vaccine candidate antigens, confirming our recent data for L. chagasi in Brazil, and their potential as cross-species vaccine candidates.     

Unravelling the Leishmania genome

The past few years have seen significant advances in our understanding of eukaryotic genomes. In the field of parasitology, this is best exemplified by the application of genome mapping techniques to the study of genome structure and function in the protozoan parasite, Leishmania. Although much is known about the organism and the diseases it causes, molecular genetics has only recently begun to play a major part in elucidating some of the unusual characteristics of this interesting parasite. Mapping of the small (35 Mb) genome and determination of the functional role of genes by the application of in vitro homologous gene targeting techniques are revealing novel avenues for the development of prophylactic measures.     

Polymorphism in NOD2, Crohn's disease, and susceptibility to pulmonary tuberculosis

The nucleotide oligomerization binding domain 2 gene (NOD2) encodes an intracellular receptor for bacterial components, which is expressed in monocytes and is associated with Crohn's Disease (CD). This finding, along with epidemiological evidence, supports a role for infection in the pathogenesis of CD. Speculation that mycobacteria are involved in CD led us to investigate NOD2 in susceptibility to tuberculosis (TB), a global public health problem caused by Mycobacterium tuberculosis. CD-associated NOD2 variants were absent in a case-control study of 640 Gambians, where CD is rare. Novel NOD2 promoter polymorphisms were identified but showed no association with TB in this African population sample.     

Classification and regression tree and spatial analyses reveal geographic heterogeneity in genome wide linkage study of Indian visceral leishmaniasis

Background

Genome wide linkage studies (GWLS) have provided evidence for loci controlling visceral leishmaniasis on Chromosomes 1p22, 6q27, 22q12 in Sudan and 6q27, 9p21, 17q11-q21 in Brazil. Genome wide studies from the major focus of disease in India have not previously been reported.

Methods and Findings

We undertook a GWLS in India in which a primary ∼10 cM (515 microsatellites) scan was carried out in 58 multicase pedigrees (74 nuclear families; 176 affected, 353 total individuals) and replication sought in 79 pedigrees (102 nuclear families; 218 affected, 473 total individuals). The primary scan provided evidence (≥2 adjacent markers allele-sharing LOD≥0.59; nominal P≤0.05) for linkage on Chromosomes 2, 5, 6, 7, 8, 10, 11, 20 and X, with peaks at 6p25.3-p24.3 and 8p23.1-p21.3 contributed to largely by 31 Hindu families and at Xq21.1-q26.1 by 27 Muslim families. Refined mapping confirmed linkage across all primary scan families at 2q12.2-q14.1 and 11q13.2-q23.3, but only 11q13.2-q23.3 replicated (combined LOD = 1.59; P = 0.0034). Linkage at 6p25.3-p24.3 and 8p23.1-p21.3, and at Xq21.1-q26.1, was confirmed by refined mapping for primary Hindu and Muslim families, respectively, but only Xq21.1-q26.1 replicated across all Muslim families (combined LOD 1.49; P = 0.0045). STRUCTURE and SMARTPCA did not identify population genetic substructure related to religious group. Classification and regression tree, and spatial interpolation, analyses confirm geographical heterogeneity for linkages at 6p25.3-p24.3, 8p23.1-p21.3 and Xq21.1-q26.1, with specific clusters of families contributing LOD scores of 2.13 (P = 0.0009), 1.75 (P = 0.002) and 1.84 (P = 0.001), respectively.

Conclusions

GWLS has identified novel loci that show geographical heterogeneity in their influence on susceptibility to VL in India.     

Biosynthesis of colitose: expression, purification, and mechanistic characterization of GDP-4-keto-6-deoxy-D-mannose-3-dehydrase (ColD) and GDP-L-colitose synthase (ColC)

L-Colitose is a 3,6-dideoxyhexose found in the O-antigen of Gram-negative lipopolysaccharides. To study the biosynthesis of this unusual sugar, we have cloned and sequenced the L-colitose biosynthetic gene cluster from Yersinia pseudotuberculosis VI. The colD and colC genes in this cluster have been overexpressed and each gene product has been purified and characterized. Our results showed that ColD functions as GDP-4-keto-6-deoxy-D-mannose-3-dehydrase responsible for C-3 deoxygenation of GDP-4-keto-6-deoxy-D-mannose. This enzyme is coenzyme B(6)-dependent and its catalysis is initiated by a transamination step in which pyridoxal 5'-phosphate (PLP) is converted to pyridoxamine 5'-phosphate (PMP) in the presene of L-glutamate. This coenzyme forms a Schiff base with the keto sugar substrate and the resulting adduct undergoes a PMP-mediated beta-dehydration reaction to give a sugar enamine intermediate, which after tautomerization and hydrolysis to release ammonia yields GDP-4-keto-3,6-dideoxy-D-mannose as the product. The combined transamination-deoxygenation activity places ColD in a class by itself. Our studies also established ColC as GDP-L-colitose synthase, which is a bifunctional enzyme catalyzing the C-5 epimerization of GDP-4-keto-3,6-dideoxy-D-mannose and the subsequent C-4 keto reduction of the resulting L-epimer to give GDP-L-colitose. Reported herein are the detailed accounts of the overexpression, purification, and characterization of ColD and ColC. Our studies show that their modes of action in the biosynthesis of GDP-L-colitose represent a new deoxygenation paradigm in deoxysugar biosynthesis.