Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors

Several 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.     

The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton

The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen Trypanosoma brucei, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes, T. brucei PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton.In eukaryotes, the ubiquitous second messenger cyclic AMP (cAMP) is generated from ATP by membrane-integral or by cytoplasmic, CO₂-regulated cyclases (35, 44). The cAMP signal is processed by a small group of receiver proteins, including the regulatory subunit of protein kinase A (28), cAMP-gated ion channels (4), and the guanine-nucleotide-exchange proteins EPAC1 and EPAC2 (39). The cAMP signal is terminated by the action of a family of cyclic nucleotide-specific phosphodiesterases (PDEs) (9). This paradigm is rather straightforward, involves a limited number of players, and is generally well understood, at least in mammalian cells. However, much less is known about how individual cAMP signals are temporally and spatially controlled. Since most eukaryotic adenylyl cyclases are integral membrane proteins, often restricted to specific membrane subdomains (10), cAMP signaling is usually initiated at the cell membrane (40). However, diffusion of cAMP away from its site of generation is rapid, with diffusion coefficients being about 400 μm²/s (8, 15, 29), translating into diffusion velocities of 30 to 40 μm/s. As a consequence, the signal would reach the center of the cell with a diameter of 3 μm within less than 50 ms and would rapidly saturate the entire cell. While regulation through fluctuating cellular levels of cAMP represents a valid paradigm of cAMP signaling, it has become clear that other, more localized modes of cAMP signaling must also exist. Several groups have shown that the cAMP response of a given cell can differ depending on what set of receptors activates the cyclase response (14, 30, 41, 42). Similarly, the cAMP response of endothelial cells depends on the subcellular site where the cAMP is produced. They tighten their barrier function when cAMP is produced by membrane-bound adenylyl cyclases but become more permeable when cAMP is produced in the cytoplasm (17, 45). The distinct subcellular localization of cAMP signals was experimentally demonstrated using an array of techniques (29, 40, 55, 56).Physically tethered PDEs might serve to confine newly synthesized cAMP to defined microdomains. Only cAMP-binding proteins that are localized within or extend into such microdomains would be able to receive the cAMP signal (17, 49). cAMP concentrations within such domains might rise and fall rapidly, reaching peak concentrations much more rapidly and locally far beyond the steady-state cAMP levels measured in whole-cell extracts. Such spatially organized, tethered PDEs can generate local sinks into which cAMP disappears (1, 23). This paradigm would allow the simultaneous presence of numerous local cAMP concentration gradients within a single cell, allowing great flexibility in signal generation and intracellular signal transmission. This concept is based on the distinct subcellular localization and physical association of PDEs with subcellular structures and on the existence of localized subcellular cAMP pools, for which there is extensive experimental support (3, 5, 13, 50, 52). Interestingly, PDEs localized in different subcellular regions may still be able to compensate for each other. Ablation of the cilium-specific PDE1C from the olfactory neurons in the mouse did not prolong response termination, as long as the cytoplasmic PDE4 in the cell body was still present (11).The unicellular eukaryote Trypanosoma brucei is the causative agent of human sleeping sickness in sub-Saharan Africa. It belongs to the large order of the kinetoplastida, which includes many medically and economically important pathogens of humans, their livestock, and their crops worldwide (27). Trypanosomes are very small cells (about 15 by 3 μm in diameter) that carry a single flagellum (10 by 0.5 μm). The volume of a procyclic trypanosome of strain 427 is (9.6 ± 0.8) × 10⁻¹⁴ liter (Markus Engstler, personal communication), with the flagellum representing about 15% of this. A signaling threshold concentration of 1 μM cAMP corresponds to just about 30,000 molecules of cAMP per cell. Given a diffusion coefficient of 400 μm²/s (29), unrestricted diffusion of cAMP would swamp the cell within 50 ms. Obviously, temporal and spatial control of cAMP signaling is crucial for T. brucei. Strategically located, physically tethered PDEs might thus play an important role in the architecture of the cAMP signaling pathways in T. brucei.The genomes of T. brucei and of other kinetoplastids, such as T. vivax, T. cruzi, Leishmania major, L. infantum, and L. braziliensis, all code for the same set of five cyclic nucleotide-specific PDEs (25, 53). In T. brucei, the genes for T. brucei PDEB1 (TbrPDEB1; subsequently termed PDEB1) and TbrPDEB2 (PDEB2) are tandemly arranged on chromosome 9 and code for two very similar cAMP-specific PDEs, each with two GAF (mammalian cyclic GMP-dependent PDEs, Anabaena adenylyl cyclases, Escherichia coli FhlA) domains (21) in their N-terminal regions (38, 57). These two PDEs were also studied experimentally in T. cruzi (12) and L. major (24, 52), and orthologues are present in all kinetoplastid genomes available so far. Despite their high overall sequence similarity, PDEB1 and PDEB2 exhibit distinct subcellular localizations (31). PDEB1 is predominantly found in the flagellum, where it is stably associated with cytoskeletal components that are resistant to detergent extraction. In contrast, PDEB2 is mostly localized in the cell body, from where it is fully extractable by nonionic detergents. However, a minor fraction of PDEB2 also associates with the flagellar skeleton in a Triton-resistant manner, most likely through interaction with PDEB1. Earlier work has shown that both PDEB1and PDEB2 are essential enzymes in bloodstream-form T. brucei (31), while TbPDEA, TbPDEC, and TbPDED play minor roles (20; S. Kunz, unpublished data).     

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors (Part 2)

Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action.     

Genetic control of resistance to Trypanosoma brucei brucei infection in mice

Background

Trypanosoma brucei brucei infects livestock, with severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to this parasite. However, no genes controlling these differences were mapped.

Methods

We studied the genetic control of survival after T. b. brucei infection using recombinant congenic (RC) strains, which have a high mapping power. Each RC strain of BALB/c-c-STS/A (CcS/Dem) series contains a different random subset of 12.5% genes from the parental “donor” strain STS/A and 87.5% genes from the “background” strain BALB/c. Although BALB/c and STS/A mice are similarly susceptible to T. b. brucei, the RC strain CcS-11 is more susceptible than either of them. We analyzed genetics of survival in T. b. brucei-infected F₂ hybrids between BALB/c and CcS-11. CcS-11 strain carries STS-derived segments on eight chromosomes. They were genotyped in the F₂ hybrid mice and their linkage with survival was tested by analysis of variance.

Results

We mapped four Tbbr (Trypanosoma brucei brucei response) loci that influence survival after T. b. brucei infection. Tbbr1 (chromosome 3) and Tbbr2 (chromosome 12) have effects on survival independent of inter-genic interactions (main effects). Tbbr3 (chromosome 7) influences survival in interaction with Tbbr4 (chromosome 19). Tbbr2 is located on a segment 2.15 Mb short that contains only 26 genes.

Conclusion

This study presents the first identification of chromosomal loci controlling susceptibility to T. b. brucei infection. While mapping in F₂ hybrids of inbred strains usually has a precision of 40–80 Mb, in RC strains we mapped Tbbr2 to a 2.15 Mb segment containing only 26 genes, which will enable an effective search for the candidate gene. Definition of susceptibility genes will improve the understanding of pathways and genetic diversity underlying the disease and may result in new strategies to overcome the active subversion of the immune system by T. b. brucei.     

The Genome Sequence of Trypanosoma brucei gambiense,Causative Agent of Chronic Human African Trypanosomiasis

Background

Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.

Methods and Findings

An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.

Conclusions

This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.     

Phosphorylation-dependent protein interaction with Trypanosoma brucei 14-3-3 proteins that display atypical target recognition

Background

The 14-3-3 proteins are structurally conserved throughout eukaryotes and participate in protein kinase signaling. All 14-3-3 proteins are known to bind to evolutionally conserved phosphoserine-containing motifs (modes 1 and/or 2) with high affinity. In Trypanosoma brucei, 14-3-3I and II play pivotal roles in motility, cytokinesis and the cell cycle. However, none of the T. brucei 14-3-3 binding proteins have previously been documented.

Methodology/Principal Findings

Initially we showed that T. brucei 14-3-3 proteins exhibit far lower affinity to those peptides containing RSxpSxP (mode 1) and RxY/FxpSxP (mode 2) (where x is any amino acid residue and pS is phosphoserine) than human 14-3-3 proteins, demonstrating the atypical target recognition by T. brucei 14-3-3 proteins. We found that the putative T. brucei protein phosphatase 2C (PP2c) binds to T. brucei 14-3-3 proteins utilizing its mode 3 motif (–pS/pTx_1-2-COOH, where x is not Pro). We constructed eight chimeric PP2c proteins replacing its authentic mode 3 motif with potential mode 3 sequences found in Trypanosoma brucei genome database, and tested their binding. As a result, T. brucei 14-3-3 proteins interacted with three out of eight chimeric proteins including two with high affinity. Importantly, T. brucei 14-3-3 proteins co-immunoprecipitated with an uncharacterized full-length protein containing identified high-affinity mode 3 motif, suggesting that both proteins form a complex in vivo. In addition, a synthetic peptide derived from this mode 3 motif binds to T. brucei 14-3-3 proteins with high affinity.

Conclusion/Significance

Because of the atypical target recognition of T. brucei 14-3-3 proteins, no 14-3-3-binding proteins have been successfully identified in T. brucei until now whereas over 200 human 14-3-3-binding proteins have been identified. This report describes the first discovery of the T. brucei 14-3-3-binding proteins and their binding motifs. The high-affinity phosphopeptide will be a powerful tool to identify novel T. brucei 14-3-3-binding proteins.     

Differences between Trypanosoma brucei gambiense groups 1 and 2 in their resistance to killing by trypanolytic factor 1

Background

The three sub-species of Trypanosoma brucei are important pathogens of sub-Saharan Africa. T. b. brucei is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. T. b. rhodesiense and T. b. gambiense are able to resist lysis by TLF. There are two distinct sub-groups of T. b. gambiense that differ genetically and by human serum resistance phenotypes. Group 1 T. b. gambiense have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 T. b. gambiense are resistant to TLF-1 due in-part to reduced uptake of TLF-1 mediated by reduced expression of the TLF-1 receptor (the haptoglobin-hemoglobin receptor (HpHbR)) gene. Here we investigate if this is also true in group 2 parasites.

Methodology

Isogenic resistant and sensitive group 2 T. b. gambiense were derived and compared to other T. brucei parasites. Both resistant and sensitive lines express the HpHbR gene at similar levels and internalized fluorescently labeled TLF-1 similar fashion to T. b. brucei. Both resistant and sensitive group 2, as well as group 1 T. b. gambiense, internalize recombinant APOL1, but only sensitive group 2 parasites are lysed.

Conclusions

Our data indicate that, despite group 1 T. b. gambiense avoiding TLF-1, it is resistant to the main lytic component, APOL1. Similarly group 2 T. b. gambiense is innately resistant to APOL1, which could be based on the same mechanism. However, group 2 T. b. gambiense variably displays this phenotype and expression does not appear to correlate with a change in expression site or expression of HpHbR. Thus there are differences in the mechanism of human serum resistance between T. b. gambiense groups 1 and 2.     

Synthesis and evaluation of analogs of the phenylpyridazinone NPD-001 as potent trypanosomal TbrPDEB1 phosphodiesterase inhibitors and in vitro trypanocidals

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. Knock down of both enzymes leads to cell cycle arrest and is lethal to the parasite. Recently, we reported the phenylpyridazinone, NPD-001, with low nanomolar IC₅₀ values on both TbrPDEB1 (IC₅₀: 4 nM) and TbrPDEB2 (IC₅₀: 3 nM) (J. Infect. Dis. 2012, 206, 229). In this study, we now report on the first structure activity relationships of a series of phenylpyridazinone analogs as TbrPDEB1 inhibitors. A selection of compounds was also shown to be anti-parasitic. Importantly, a good correlation between TbrPDEB1 IC₅₀ and EC₅₀ against the whole parasite was observed. Preliminary analysis of the SAR of selected compounds on TbrPDEB1 and human PDEs shows large differences which shows the potential for obtaining parasite selective PDE inhibitors. The results of these studies support the pharmacological validation of the Trypanosome PDEB family as novel therapeutic approach for HAT and provide as well valuable information for the design of potent TbrPDEB1 inhibitors that could be used for the treatment of this disease.     

A Trypanosoma brucei Kinesin Heavy Chain Promotes Parasite Growth by Triggering Host Arginase Activity

Background

In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells.

Methodology/Principal findings

By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time.

Conclusion

A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.     

Rana grylio Virus (RGV) 50L Is Associated with Viral Matrix and Exhibited Two Distribution Patterns

Background

The complete genome of Rana grylio virus (RGV) was sequenced and analyzed recently, which revealed that RGV 50L had homologues in many iridoviruses with different identities; however, the characteristics and functions of 50L have not been studied yet.

Methodology/Principal Findings

We cloned and characterized RGV50L, and revealed 50L functions in virus assembly and gene regulation. 50L encoded a 499-amino acid structural protein of about 85 kDa in molecular weight and contained a nuclear localization signal (NLS) and a helix- extension-helix motif. Drug inhibition assay demonstrated that 50L was an immediate-early (IE) gene. Immuno-fluorescence assay revealed that 50L appeared early and persisted in RGV-infected cells following two distribution patterns. One pattern was that 50L exhibited a cytoplasm-nucleus- viromatrix distribution pattern, and mutagenesis of the NLS motif revealed that localization of 50L in the nucleus was NLS-dependent; the other was that 50L co-localized with viral matrix which plays important roles in virus assembly and the life circle of viruses.

Conclusions/Significance

RGV 50L is a novel iridovirus IE gene encoded structural protein which plays important roles in virus assembly.     

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background

Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.

Methodology/Principal Findings

The T. rangeli haploid genome is ∼24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins.

Conclusions/Significance

Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.     

Crystal Structures of TbCatB and Rhodesain,Potential Chemotherapeutic Targets and Major Cysteine Proteases of Trypanosoma brucei

Background

Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei.

Methods and Findings

We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002.

Conclusions

The mature domain of our TbCat•CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat•CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain•K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.     

Highly Sensitive In Vivo Imaging of Trypanosoma brucei Expressing “Red-Shifted” Luciferase

Background

Human African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.

Methodology/Principal findings

Here, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5′-variant surface glycoprotein untranslated region (UTR) and a downstream 3′-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter.

Conclusions/Significance

The optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.     

In vitro culture of freshly isolated Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes

Most researchers who study unicellular eukaryotes work with an extremely limited number of laboratory-adapted isolates that were obtained from the field decades ago, but the effects of passage in laboratory rodents, and adaptation to in vitro culture, have been little studied. For example, the vast majority of studies of Trypanosoma brucei biology have concentrated on just two strains, Lister 427 and EATRO1125, which were taken from the field over half a century ago and have since have undergone innumerable passages in rodents and culture. We here describe two new Trypanosoma brucei brucei strains. MAK65 and MAK98, which have undergone only 3 rodent passages since isolation from Ugandan cattle. High-coverage sequencing revealed that adaptation of the parasites to culture was accompanied by changes in gene copy numbers. T. brucei has so far been considered to be uniformly diploid, but we also found trisomy of chromosome 5 not only in one Lister 427 culture, but also in the MAK98 field isolate. Trisomy of chromosome 6, and increased copies of other chromosome segments, were also seen in established cultured lines. The two new T. brucei strains should be useful to researchers interested in trypanosome differentiation and pathogenicity. Initial results suggested that the two strains have differing infection patterns in rodents. MAK65 is uniformly diploid and grew more reproducibly in bloodstream-form culture than MAK98.     

Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize

Background and Aims

Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation.

Methods

Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses.

Key Results

During the period 0–28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo.

Conclusions

The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.     

Human and Animal Trypanosomes in C?te d'Ivoire Form a Single Breeding Population

Background

Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and contributes to the related veterinary disease, Nagana. T. brucei is segregated into three subspecies based on host specificity, geography and pathology. T. b. brucei is limited to animals (excluding some primates) throughout sub-Saharan Africa and is non-infective to humans due to trypanolytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species. T. b. gambiense is the more prevalent human, causing over 97% of human cases. Study of T. b. gambiense is complicated in that there are two distinct groups delineated by genetics and phenotype. The relationships between the two groups and local T. b. brucei are unclear and may have a bearing on the evolution of the human infectivity traits.

Methodology/Principal Findings

A collection of sympatric T. brucei isolates from Côte d’Ivoire, consisting of T. b. brucei and both groups of T. b. gambiense have previously been categorized by isoenzymes, RFLPs and Blood Incubation Infectivity Tests. These samples were further characterized using the group 1 specific marker, TgSGP, and seven microsatellites. The relationships between the T. b. brucei and T. b. gambiense isolates were determined using principal components analysis, neighbor-joining phylogenetics, STRUCTURE, F_ST, Hardy-Weinberg equilibrium and linkage disequilibrium.

Conclusions/Significance

Group 1 T. b. gambiense form a clonal genetic group, distinct from group 2 and T. b. brucei, whereas group 2 T. b. gambiense are genetically indistinguishable from local T. b. brucei. There is strong evidence for mating within and between group 2 T. b. gambiense and T. b. brucei. We found no evidence to support the hypothesis that group 2 T. b. gambiense are hybrids of group 1 and T. b. brucei, suggesting that human infectivity has evolved independently in groups 1 and 2 T. b. gambiense.     

Comparative genomics study of multi-drug-resistance mechanisms in the antibiotic-resistant Streptococcus suis R61 strain

Background

Streptococcus suis infections are a serious problem for both humans and pigs worldwide. The emergence and increasing prevalence of antibiotic-resistant S. suis strains pose significant clinical and societal challenges.

Results

In our study, we sequenced one multi-drug-resistant S. suis strain, R61, and one S. suis strain, A7, which is fully sensitive to all tested antibiotics. Comparative genomic analysis revealed that the R61 strain is phylogenetically distinct from other S. suis strains, and the genome of R61 exhibits extreme levels of evolutionary plasticity with high levels of gene gain and loss. Our results indicate that the multi-drug-resistant strain R61 has evolved three main categories of resistance.

Conclusions

Comparative genomic analysis of S. suis strains with diverse drug-resistant phenotypes provided evidence that horizontal gene transfer is an important evolutionary force in shaping the genome of multi-drug-resistant strain R61. In this study, we discovered novel and previously unexamined mutations that are strong candidates for conferring drug resistance. We believe that these mutations will provide crucial clues for designing new drugs against this pathogen. In addition, our work provides a clear demonstration that the use of drugs has driven the emergence of the multi-drug-resistant strain R61.     

Duplicated paralogous genes subject to positive selection in the genome of Trypanosoma brucei

Background

Whole genome studies have highlighted duplicated genes as important substrates for adaptive evolution. We have investigated adaptive evolution in this class of genes in the human parasite Trypanosoma brucei, as indicated by the ratio of non-synonymous (amino-acid changing) to synonymous (amino acid retaining) nucleotide substitution rates.

Methodology/Principal Findings

We have identified duplicated genes that are most rapidly evolving in this important human parasite. This is the first attempt to investigate adaptive evolution in this species at the codon level. We identify 109 genes within 23 clusters of paralogous gene expansions to be subject to positive selection.

Conclusions/Significance

Genes identified include surface antigens in both the mammalian and insect host life cycle stage suggesting that competitive interaction is not solely with the adaptive immune system of the mammalian host. Also surface transporters related to drug resistance and genes related to developmental progression are detected. We discuss how adaptive evolution of these genes may highlight lineage specific processes essential for parasite survival. We also discuss the implications of adaptive evolution of these targets for parasite biology and control.