Antigenic variation enables pathogens to avoid the host immune response by continual switching of surface proteins. The protozoan blood parasite Trypanosoma brucei causes human African trypanosomiasis ("sleeping sickness") across sub-Saharan Africa and is a model system for antigenic variation, surviving by periodically replacing a monolayer of variant surface glycoproteins (VSG) that covers its cell surface. We compared the genome of Trypanosoma brucei with two closely related parasites Trypanosoma congolense and Trypanosoma vivax, to reveal how the variant antigen repertoire has evolved and how it might affect contemporary antigenic diversity. We reconstruct VSG diversification showing that Trypanosoma congolense uses variant antigens derived from multiple ancestral VSG lineages, whereas in Trypanosoma brucei VSG have recent origins, and ancestral gene lineages have been repeatedly coopted to novel functions. These historical differences are reflected in fundamental differences between species in the scale and mechanism of recombination. Using phylogenetic incompatibility as a metric for genetic exchange, we show that the frequency of recombination is comparable between Trypanosoma congolense and Trypanosoma brucei but is much lower in Trypanosoma vivax. Furthermore, in showing that the C-terminal domain of Trypanosoma brucei VSG plays a crucial role in facilitating exchange, we reveal substantial species differences in the mechanism of VSG diversification. Our results demonstrate how past VSG evolution indirectly determines the ability of contemporary parasites to generate novel variant antigens through recombination and suggest that the current model for antigenic variation in Trypanosoma brucei is only one means by which these parasites maintain chronic infections.

Human long-lasting PerAF is characterized by heterogeneous and unstable patterns of activation including wavefronts, transient rotational circuits, and disorganized activity.

Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

No abstract

SUMMARY The transition from replication to non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenesis, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating populations is a priority for tuberculosis treatment, but few drug targets in non-replicating Mtb are currently known. Here, we directly measured the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication using activity-based proteomics. We predict SH activity for 78 proteins, including 27 proteins with unknown function, and identify 37 SHs that remain active in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with major shifts in SH activity. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation of SHs. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

Neuronal nicotinic acetylcholine receptor subunit a5 mRNA is widely expressed in the CNS. An a5 gene polymorphism has been implicated in behavioral differences between mouse strains, and a5-null mutation induces profound changes in mouse acute responses to nicotine. In this study, we have examined the distribution and prevalence of a5* nicotinic acetylcholine receptor in mouse brain, and quantified the effects of a5-null mutation on pre-synaptic nicotinic acetylcholine receptor function (measured using synaptosomal 86 Rb + efflux) and overall [ 125 I]epibatidine binding site expression. a5* nicotinic acetylcholine receptor expression was found in nine of fifteen regions examined, although < 20% of the total nicotinic acetylcholine receptor population in any region contained a5. Deletion of the a5 subunit gene resulted in localized loss of function (thalamus, striatum), which was itself confined to the DHbE-sensitive receptor population. No changes in receptor expression were seen. Consequently, functional changes must occur as a result of altered function per unit of receptor. The selective depletion of high agonist activation affinity sites results in overall nicotinic function being reduced, and increases the overall agonist activation affinity. Together, these results describe the receptor-level changes underlying altered behavioral responses to nicotine in nicotinic acetylcholine receptor a5 subunit-null mutants.

Chronic nicotine treatment elicits a brain region-selective increase in the number of high-affinity agonist binding sites, a phenomenon termed up-regulation. Nicotine-induced up-regulation of ␣4␤2-nicotinic acetylcholine receptors (nAChRs) in cell cultures results from increased assembly and/or decreased degradation of nAChRs, leading to increased nAChR protein levels. To evaluate whether the increased binding in mouse brain results from an increase in nAChR subunit proteins, C57BL/6 mice were treated with nicotine by chronic intravenous infusion. 125 I]A85380, perhaps because the small ligand penetrated deeply into the sections, whereas the much larger mAbs encountered permeability barriers. Immunoprecipitation of [125 I]epibatidine binding sites with mAb 270 in select regions of nicotine-treated mice was nearly quantitative, although somewhat less so with mAb 299, confirming that the mAbs effectively recognize their targets. The patterns of change measured using immunoprecipitation were comparable with those determined autoradiographically. Thus, increases in ␣4␤2*-nAChR binding sites after chronic nicotine treatment reflect increased nAChR protein.

Immunolabeling of beta2 and alpha4 subunits was quantitated in brain sections (14 mum) using [(125)I]mAb 270 and [(125)I]mAb 299, respectively. Specificity was demonstrated by signal loss in beta2(-/-) and alpha4(-/-) brain sections, respectively. Even mild paraformaldehyde fixation severely affected immunolabeling, so this study used unfixed sections. Immunolabeling autoradiography was used to map and quantitate the effects of beta2 and alpha4 subunit-null mutations on their putative partner subunits' protein expression. [(125)I]mAb 299 labeling was nearly eliminated in beta2(-/-) sections, although dorsal interpeduncular nucleus (IPN) retained a faint signal. Therefore, alpha4 subunit expression is almost universally beta2-dependent. In contrast, alpha4-null mutation effects on [(125)I]mAb 270 immunolabeling varied widely among brain regions. In corticothalamic regions, [(125)I]mAb 270 labeling was eliminated. However, in habenulopeduncular regions, alpha4 genotype had no effect. Other (predominantly dopaminergic and optic tract) nuclei also retained reduced [(125)I]mAb 270 labeling in alpha4(-/-) sections. Thus, although most beta2 subunit protein expression is alpha4-dependent, this dependence is not universal. Presumably, residual beta2 subunits are found in non-alpha4* subtypes. Together, these results show that immunolabeling is applicable to reliable, quantitative investigations of neuronal nAChRs, and that subunit-null mutants can be appropriate controls for such experiments. In situ mRNA hybridization was also performed to determine if altered mRNA transcription mediated the interdependence of alpha4 and beta2 subunit expression. alpha4-Null mutation did not affect beta2 mRNA expression, nor did beta2 genotype affect alpha4 mRNA expression. Consequently, it seems that the two subunits' effects on each other's expression are mediated at the protein, rather than gene expression level.