Pathological mutations differentially affect the self-assembly and polymerisation of the innate immune system signalling adaptor molecule MyD88

Background

Higher-order self-assembly of proteins, or “prion-like” polymerisation, is now emerging as a simple and robust mechanism for signal amplification, in particular within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns needs to trigger a strong, binary response within cells. MyD88, an important adaptor protein downstream of TLRs, is one of the most recent candidates for involvement in signalling by higher order self-assembly. In this new light, we set out to re-interpret the role of polymerisation in MyD88-related diseases and study the impact of disease-associated point mutations L93P, R196C, and L252P/L265P at the molecular level.

Results

We first developed new in vitro strategies to characterise the behaviour of polymerising, full-length MyD88 at physiological levels. To this end, we used single-molecule fluorescence fluctuation spectroscopy coupled to a eukaryotic cell-free protein expression system. We were then able to explore the polymerisation propensity of full-length MyD88, at low protein concentration and without purification, and compare it to the behaviours of the isolated TIR domain and death domain that have been shown to have self-assembly properties on their own. These experiments demonstrate that the presence of both domains is required to cooperatively lead to efficient polymerisation of the protein. We then characterised three pathological mutants of MyD88.

Conclusion

We discovered that all mutations block the ability of MyD88 to polymerise fully. Interestingly, we show that, in contrast to L93P and R196C, L252P is a gain-of-function mutation, which allows the MyD88 mutant to form extremely stable oligomers, even at low nanomolar concentrations. Thus, our results shed new light on the digital “all-or-none” responses by the myddosomes and the behaviour of the oncogenic mutations of MyD88.

     

Muon spin relaxation studies of iron(II) spin crossover complexes

A series of model iron(II) spin crossover complexes have been investigated by temperature dependent muon spin relaxation (μSR) techniques at ISIS, UK. The thermally induced spin crossover in these materials could be monitored by following the initial asymmetry parameter, a₀, in zero-field. We established that the behavior of a₀ correlates well with the shape of the spin crossover curve derived from magnetic susceptibility measurements, whether hysteretic, smooth, or abrupt. In addition, the longitudinal field dependence of a₀ not only provides information on the nature of the muonic species but also on their interactions and respective localization in the crystal lattice. Useful insights to the electronic structure and dynamic phenomena of these model spin crossover complexes can be derived.     

Direct Correlation between Motile Behavior and Protein Abundance in Single Cells

Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage.     

Comparison of French and Worldwide Bacillus anthracis Strains Favors a Recent,Post-Columbian Origin of the Predominant North-American Clade

Background

Bacillus anthracis, the highly dangerous zoonotic bacterial pathogen species is currently composed of three genetic groups, called A, B and C. Group A is represented worldwide whereas group B is present essentially in Western Europe and Southern Africa. Only three strains from group C have been reported. This knowledge is derived from the genotyping of more than 2000 strains collected worldwide. Strains from both group A and group B are present in France. Previous investigations showed that the majority of sporadic French strains belong to the so-called A.Br.011/009 group A clade and define a very remarkable polytomy with six branches. Here we explore the significance of this polytomy by comparing the French B. anthracis lineages to worldwide lineages. We take advantage of whole genome sequence data previously determined for 122 French strains and 45 strains of various origins.

Results

A total of 6690 SNPs was identified among the available dataset and used to draw the phylogeny. The phylogeny of the French B group strains which belongs to B.Br.CNEVA indicates an expansion from the south-east part of France (the Alps) towards the south-west (Massif-Central and Pyrenees). The relatively small group A strains belonging to A.Br.001/002 results from at least two independent introductions. Strikingly, the data clearly demonstrates that the currently predominant B. anthracis lineage in North America, called WNA for Western North American, is derived from one branch of the A.Br.011/009 polytomy predominant in France.

Conclusions/Significance

The present work extends the range of observed substitution rate heterogeneity within B. anthracis, in agreement with its ecology and in contrast with some other pathogens. The population structure of the six branches A.Br.011/009 polytomy identified in France, diversity of branch length, and comparison with the WNA lineage, suggests that WNA is of post-Columbian and west European origin, with France as a likely source. Furthermore, it is tempting to speculate that the polytomy’s most recent common ancestor -MRCA- dates back to the Hundred Years'' war between France and England started in the mid-fourteenth century. These events were associated in France with deadly epidemics and major economic and social changes.     

Dynamic species co‐occurrence networks require dynamic biodiversity surrogates

In conservation it is inevitable that surrogates be selected to represent the occurrence of hard‐to‐find species and find priority locations for management. However, species co‐occurrence can vary over time. Here we demonstrate how temporal dynamics in species co‐occurrence influence the ability of managers to choose the best surrogate species. We develop an efficient optimisation formulation that selects the optimal set of complementary surrogate species from any co‐occurrence network. We apply it to two Australian datasets on successional bird responses to disturbances of revegetation and fire. We discover that a surprisingly small number of species are required to represent the majority of species co‐occurrences at any one time. Because co‐occurrence patterns are temporally dynamic, the optimal set of surrogates, and the number of surrogates required to achieve a desired surrogacy power, depend on sampling effort and the successional state of a system. Overlap in optimal sets of surrogates for representing 70% of co‐occurring species ranges from zero to 57% depending on when the surrogacy decision is made. Surrogate sets representing early successional communities over‐estimate the power of surrogacy decisions at later times. Our results show that in dynamic systems, optimal surrogates might be selected in different ways: 1) use short‐term monitoring to choose a larger number of static less‐informative surrogates; 2) use long‐term monitoring to choose a smaller number of static high‐power surrogates that may poorly represent early successional co‐occurrence; 3) develop adaptive surrogate selection frameworks with high short‐term and long‐term surrogacy power that update surrogate sets and capture temporal dynamics in species co‐occurrence. Our results suggest vigilance is needed when selecting surrogates for other co‐occurring species in dynamic landscapes, as selected surrogates from one time may have reduced effectiveness at a different time. Ultimately, decisions that fail to acknowledge dynamic species co‐occurrence will lead to uninformative or redundant surrogates.     

Morphological differentiation of Icelandic Redpolls,Acanthis flammea islandica

Capsule The analyses support the grouping of the three Acanthis species, although a large split is observed between the A. hornemanni subspecies.

Aims To investigate the morphological variation in A. f. islandica among different periods of the year and its morphological differentiation from the other subspecies A. f. flammea and A. f. rostrata, and also from the redpoll species, A. cabaret and the two subspecies of A. hornemanni, exilipes and hornemanni.

Methods The subspecies status of the Icelandic population was evaluated with Amadon's rule, by comparing its variation of traits to the distribution of the traits in different species/subspecies of the group.

Results A. f. islandica is characterized by intermediate wing, bill and tail lengths. Based on the 75% rule, wing length and bill depth can be used to discriminate A. f. islandica from both extreme morphs of redpolls (currently classified as different species); A. hornemanni and A. cabaret, and tail and wing length can distinguish A. f. islandica from its conspecifics A. f. flammea. The overall morphological divergence within the redpoll complex is not supported by association to the studied nuclear markers.

Conclusion The taxonomic status of the three redpoll species is supported by Amadon's rule, however the subspecies status of the Icelandic Redpoll remains unclear.     

Morpho morphometrics: Shared ancestry and selection drive the evolution of wing size and shape in Morpho butterflies

Butterfly wings harbor highly diverse phenotypes and are involved in many functions. Wing size and shape result from interactions between adaptive processes, phylogenetic history, and developmental constraints, which are complex to disentangle. Here, we focus on the genus Morpho (Nymphalidae: Satyrinae, 30 species), which presents a high diversity of sizes, shapes, and color patterns. First, we generate a comprehensive molecular phylogeny of these 30 species. Next, using 911 collection specimens, we quantify the variation of wing size and shape across species, to assess the importance of shared ancestry, microhabitat use, and sexual selection in the evolution of the wings. While accounting for phylogenetic and allometric effects, we detect a significant difference in wing shape but not size among microhabitats. Fore and hindwings covary at the individual and species levels, and the covariation differs among microhabitats. However, the microhabitat structure in covariation disappears when phylogenetic relationships are taken into account. Our results demonstrate that microhabitat has driven wing shape evolution, although it has not strongly affected forewing and hindwing integration. We also found that sexual dimorphism of forewing shape and color pattern are coupled, suggesting a common selective force.     

A new piperidinol derivative targeting mycolic acid transport in Mycobacterium abscessus

The natural resistance of Mycobacterium abscessus to most commonly available antibiotics seriously limits chemotherapeutic treatment options, which is particularly challenging for cystic fibrosis patients infected with this rapid‐growing mycobacterium. New drugs with novel molecular targets are urgently needed against this emerging pathogen. However, the discovery of such new chemotypes has not been appropriately performed. Here, we demonstrate the utility of a phenotypic screen for bactericidal compounds against M. abscessus using a library of compounds previously validated for activity against M. tuberculosis. We identified a new piperidinol‐based molecule, PIPD1, exhibiting potent activity against clinical M. abscessus strains in vitro and in infected macrophages. Treatment of infected zebrafish with PIPD1 correlated with increased embryo survival and decreased bacterial burden. Whole genome analysis of M. abscessus strains resistant to PIPD1 identified several mutations in MAB_4508, encoding a protein homologous to MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis was unaffected, PIPD1 strongly inhibited the transport of trehalose monomycolate, thereby abrogating mycolylation of arabinogalactan. Mapping the mutations conferring resistance to PIPD1 on a MAB_4508 tridimensional homology model defined a potential PIPD1‐binding pocket. Our data emphasize a yet unexploited chemical structure class against M. abscessus infections with promising translational development possibilities.     

Functional characterization of terpene synthases and chemotypic variation in three lavender species of section Stoechas

Lavandula pedunculata (Mill.) Cav. subsp. lusitanica, Lavandula stoechas L. subsp. stoechas and Lavandula viridis l'Hér. are three lavender taxa that belong to the botanical section Stoechas and are widely used as aromatherapy, culinary herb or folk medicine in many Mediterranean regions. The analysis of their bioactive volatile constituents revealed the presence of 124 substances, the most abundant being the bicyclic monoterpenes fenchone, camphor and 1,8‐cineole that give these three species their respective chemotypes. Most noteworthy was fenchone which, with its reduced form fenchol, made 48% of the total volatile constituents of L. pedunculata while present at 2.9% in L. stoechas and undetectable in L. viridis. In order to provide a molecular explanation to the differences in volatile compounds of these three species, two monoterpene synthases (monoTPS) and one sesquiterpene synthase (sesquiTPS) were cloned in L. pedunculata and functionally characterized as fenchol synthase (LpFENS), α‐pinene synthase (LpPINS) and germacrene A synthase (LpGEAS). The two other lavender species contained a single orthologous gene for each of these three classes of TPS with similar enzyme product specificities. Expression profiles of FENS and PINS genes matched the accumulation profile of the enzyme products unlike GEAS. This study provides one of the rare documented cases of chemotype modification during plant speciation via changes in the level of plant TPS gene expression, and not functionality.