[show abstract][hide abstract] ABSTRACT: Whole exome sequencing was performed on HLA-matched stem cell donors and
transplant recipients to measure sequence variation contributing to minor
histocompatibility antigen differences between the two. A large number of
nonsynonymous single nucleotide polymorphisms were identified in each of the
nine unique donor-recipient pairs tested. This variation was greater in
magnitude in unrelated donors as compared with matched related donors.
Knowledge of the magnitude of exome variation between stem cell transplant
recipients and donors may allow more accurate titration of immunosuppressive
therapy following stem cell transplantation.
[show abstract][hide abstract] ABSTRACT: Trypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont's contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here.
Analyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:18.104.22.168) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes.
We have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways.
[show abstract][hide abstract] ABSTRACT: Clostridium scindens ATCC 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11β-hydroxyandrost-4-ene-3,17-dione (11β-OHA) by high-resolution mass spectrometry, 1H and 13C NMR spectroscopy and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (~1000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative "transketolase" (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side-chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic "rheostat" controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine-dehydrogenase. The physiological effect of 11β-OHAD on the host or other gut microbes is currently unknown
The Journal of Lipid Research 06/2013; · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: CBA macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. Employing a transcriptomic approach, we previously showed the up-regulation of the genes involved in the classical pathway of macrophage activation in resistant mice. However, microarray analyses do not evaluate changes in gene expression that occur after translation. To circumvent this analytical limitation, we employed a proteomics approach to increase our understanding of the modulations that occur during infection and identify novel targets for the control of Leishmania infection. To identify proteins whose expression changes in CBA macrophages infected with L. major or L. amazonensis, protein extracts were obtained and digested and the peptides were characterized using multi-dimensional liquid chromatography coupled with tandem mass spectrometry analyses. A total of 162 proteins were selected as potentially modulated. Using biological network analyses, these proteins were classified as primarily involved in cellular metabolism and grouped into cellular development biological networks. This study is the first to use a proteomics approach to describe the protein modulations involved in cellular metabolism during the initial events of Leishmania-macrophage interaction. Based on these findings, we hypothesize that these differentially expressed proteins likely play a pivotal role in determining the course of infection.
Microbes and Infection 04/2013; · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: It has been long known that insect-infecting trypanosomatid flagellates from the genera Angomonas and Strigomonas harbor bacterial endosymbionts (Candidatus Kinetoplastibacterium or TPE - Trypanosomatid Proteobacterial Endosymbiont) that supplement the host metabolism. Based on previous analyses of other bacterial endosymbiont genomes from other lineages, a stereotypical path of genome evolution in such bacteria over the duration of their association with the eukaryotic host has been characterized. In this work, we sequence and analyze the genomes of five TPEs, perform their metabolic reconstruction, do an extensive phylogenomic analyses with all available Betaproteobacteria, and compare the TPEs with their nearest betaproteobacterial relatives. We also identify a number of housekeeping and central metabolism genes that seem to have undergone positive selection.Our genome structure analyses show total synteny among the five TPEs in spite of millions of years of divergence, and that this lineage follows the common path of genome evolution observed in other endosymbionts of diverse ancestries. As previously suggested by cell biology and biochemistry experiments, Ca. Kinetoplastibacterium spp. preferentially maintain those genes necessary for the biosynthesis of compounds needed by their hosts. We have also shown that metabolic and informational genes related to the cooperation with the host are overrepresented amongst genes shown to be under positive selection. Finally, our phylogenomic analysis shows that, while being in the Alcaligenaceae family of Betaproteobacteria, the closest relatives of these endosymbionts are not in the genus Bordetella as previously reported, but more likely in the Taylorella genus.
Genome Biology and Evolution 01/2013; · 4.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Until recently, the apicomplexan parasites, Cryptosporidium hominis and C. parvum, were considered the same species. However, the two parasites, now considered distinct species, exhibit significant differences in host range, infectivity, and pathogenicity, and their sequenced genomes exhibit only 95-97% identity. The availability of the complete genome sequences of these organisms provides the potential to identify the genetic variations that are responsible for the phenotypic differences between the two parasites. We compared the genome organization and structure, gene composition, the metabolic and other pathways, and the local sequence identity between the genes of these two Cryptosporidium species. Our observations show that the phenotypic differences between C. hominis and C. parvum are not due to gross genome rearrangements, structural alterations, gene deletions or insertions, metabolic capabilities, or other obvious genomic alterations. Rather, the results indicate that these genomes exhibit a remarkable structural and compositional conservation and suggest that the phenotypic differences observed are due to subtle variations in the sequences of proteins that act at the interface between the parasite and its host.
International journal of genomics. 01/2013; 2013:832756.
[show abstract][hide abstract] ABSTRACT: Some non-pathogenic trypanosomatids maintain a mutualistic relationship with a betaproteobacterium of the Alcaligenaceae family. Intensive nutritional exchanges have been reported between the two partners, indicating that these protozoa are excellent biological models to study metabolic co-evolution. We previously sequenced and herein investigate the entire genomes of five trypanosomatids which harbor a symbiotic bacterium (SHTs for Symbiont-Haboring Trypanosomatids) and the respective bacteria (TPEs for Trypanosomatid Proteobacterial Endosymbiont), as well as two trypanosomatids without symbionts (RTs for Regular Trypanosomatids), for the presence of genes of the classical pathways for vitamin biosynthesis. Our data show that genes for the biosynthetic pathways of thiamine, biotin, and nicotinic acid are absent from all trypanosomatid genomes. This is in agreement with the absolute growth requirement for these vitamins in all protozoa of the family. Also absent from the genomes of RTs are the genes for the synthesis of pantothenic acid, folic acid, riboflavin, and vitamin B6. This is also in agreement with the available data showing that RTs are auxotrophic for these essential vitamins. On the other hand, SHTs are autotrophic for such vitamins. Indeed, all the genes of the corresponding biosynthetic pathways were identified, most of them in the symbiont genomes, while a few genes, mostly of eukaryotic origin, were found in the host genomes. The only exceptions to the latter are: the gene coding for the enzyme ketopantoate reductase (EC:22.214.171.124) which is related instead to the Firmicutes bacteria; and two other genes, one involved in the salvage pathway of pantothenic acid and the other in the synthesis of ubiquinone, that are related to Gammaproteobacteria. Their presence in trypanosomatids may result from lateral gene transfer. Taken together, our results reinforce the idea that the low nutritional requirement of SHTs is associated with the presence of the symbiotic bacterium, which contains most genes for vitamin production.
PLoS ONE 01/2013; 8(11):e79786. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cryptosporidium is a protozoan parasite associated with acute and persistent diarrhea that, even in asymptomatic persons, can impair normal growth and potentially cognitive and physical development in young children. The recent availability of the complete gene sequence for Cryptosporidium hominis antigen Cp15 allows examination of innovative vaccine regimens involving intra-nasal antigen priming with live bacterial vectors applicable to human populations. We used a recently described weaned mouse model of cryptosporidiosis, where nourished and malnourished vaccinated mice receive the Cp15 antigen recombinant with cytolysinA on a Salmonella serovar Typhi CVD 908-htr A vector, followed by parenteral exposure to antigen with adjuvant. After challenge with Cryptosporidium oocysts via gavage, parameters of infection and disease (stool shedding of parasites, growth rates) were quantified, and serum/lymphoid tissue harvested to elucidate the Cp15-specific adaptive immune response. In vaccinated nourished mice, the regimen was highly immunogenic, with strong antigen-specific IL-6 and IFN-γ secretion and robust Cp15-specific immunoglobulin titers. In vaccinated malnourished mice, secretion of cytokines, particularly IFN-γ, and antigen-specific humoral immunity were generally undiminished despite protein deprivation and stunted growth. In contrast, after natural (oral) challenge with an identical inoculum of Cryptosporidium oocysts, cytokine and humoral responses to Cp15 were less than one-fourth those in vaccinated mice. Nevertheless, vaccination resulted in only transient reduction in stool shedding of parasites and was not otherwise protective against disease. Overall, immunogenicity for a C. hominis antigen was documented in mice, even in the setting of prolonged malnutrition, using an innovative vaccine regimen involving intra-nasal antigen priming with a live enteric bacterial vector, that has potential applicability to vulnerable human populations irrespective of nutritional status.