Diversification, evolution and sub-functionalization of 70kDa heat-shock proteins in two sister species of Antarctic krill: differences in thermal habitats, responses and implications under climate change

A comparative thermal tolerance study was undertaken on two sister species of Euphausiids (Antarctic krills) Euphausia superba and Euphausia crystallorophias. Both are essential components of the Southern Ocean ecosystem, but occupy distinct environmental geographical locations with slightly different temperature regimes. They therefore provide a useful model system for the investigation of adaptations to thermal tolerance. Methodology/Principal Finding Initial CTmax studies showed that E. superba was slightly more thermotolerant than E. crystallorophias. Five Hsp70 mRNAs were characterized from the RNAseq data of both species and subsequent expression kinetics studies revealed notable differences in induction of each of the 5 orthologues between the two species, with E. crystallorophias reacting more rapidly than E. superba. Furthermore, analyses conducted to estimate the evolutionary rates and selection strengths acting on each gene tended to support the hypothesis that diversifying selection has contributed to the diversification of this gene family, and led to the selective relaxation on the inducible C form with its possible loss of function in the two krill species. Conclusions The sensitivity of the epipelagic species E. crystallorophias to temperature variations and/or its adaptation to cold is enhanced when compared with its sister species, E. superba. These results indicate that ice krill could be the first of the two species to be impacted by the warming of coastal waters of the Austral ocean in the coming years due to climate change

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Molecular evolution of two antimicrobial peptides encoding genes characterized from extremophile annelids : the hydrothermal worm Alvinella pompejana and the coastal annelid Capitella spp

Les peptides antimicrobiens (PAMs) font partie intégrante du système immunitaire inné de la plupart des organismes en constituant une première ligne de défense contre un large éventail d'agents pathogènes et peuvent également être impliqués dans le contrôle et/ou le confinement de la microflore symbiotique. Le but de cette thèse était d'étudier l'évolution moléculaire de deux gènes codant pour deux précurseurs protéiques de PAMs (preprocapitellacine et preproalvinellacine) caractérisés chez deux annélides extrémophiles : le ver côtier Capitella spp (Cc) et le ver hydrothermal Alvinella pompejana (Ap). Ces précurseurs à partir desquels sont maturés les PAM présentent une structure typique des protéines à BRICHOS: un peptide signal, une propiece, un domaine chaperon BRICHOS et un peptide en épingle à cheveux beta (ici le PAM). Les résultats ont montrés que le même type de mécanismes pourrait co-exister entre les deux taxons annélides étudiés pour promouvoir et maintenir la diversité génétique des deux effecteurs immunitaires dans les différents domaines des précurseurs protéiques (duplication, recombinaison, sélection positive, introgression). Une différence majeure peut être mise en évidence dans la région du PAM qui est monomorphe (sélection purifiante) pour Ap et polymorphe pour Cc. Ceci serait dû à l'absolue nécessité de cultiver une communauté épibiotique hautement spécialisée et obligatoire pour le ver hydrothermal malgré des conditions abiotiques très fluctuantes alors que les espèces côtières de Capitella spp évoluent dans un environnement pathogène dans lequel la diversification de l'arsenal immunitaire constitue un avantage pour renforcer leur potentiel défensif.Antimicrobial peptides (AMP) are integral components of the innate immune system of most organisms in which they provide an early and a first line of defense against a wide range of microbial and microeukaryotic agents. They are also known to shape and control the symbiotic microflora. The aim of this thesis was to study the molecular evolution of two antimicrobial peptides encoding the genes: preproalvinellacin and preprocapitellacin that have been characterized from two annelids: the coastal species Capitella spp (Cc) and the hydrothermal species Alvinella pompejana (Ap). These precursors from which are matured the AMPs alvinellacin and capitellacin, display an original structure of a BRICHOS chaperon: a signal peptide, a propiece, a BRICHOS domain and a beta hairpin peptide (here the AMP). Results show that the same kind of mechanisms might co-occur between the two distinct annelid taxa to promote and maintain genetic diversity for both immune effectors in the precursor domains (duplication, recombination, positive selection, introgression). One major difference can be highlighted in the AMP region that is strictly monomorphic for the Ap species (purifiying selection) and is highly polymorphic in the Cc species. This can be due to the absolute need of farming a highly specialized epibiotic community for the hydrothermal worm despite highly fluctuating abiotic conditions whereas the coastal species of Capitella spp evolved in a more pathogenic environment in which the immune arsenal diversification should be an advantage in enhancing their defensive potential

Antagonistic evolution of an antibiotic and its molecular chaperone: how to maintain a vital ectosymbiosis in a highly fluctuating habitat

International audienceEvolution of antimicrobial peptides (AMPs) has been shown to be driven by recurrent duplications and balancing/positive selection in response to new or altered bacterial pathogens. We use Alvinella pompejana, the most eurythermal animal known on Earth, to decipher the selection patterns acting on AMP in an ecological rather than controlled infection approach. The preproalvinellacin multigenic family presents the uniqueness to encode a molecular chaperone (BRICHOS) together with an AMP (alvinellacin) that controls the vital ectosymbiosis of Alvinella. In stark contrast to what is observed in the context of the Red queen paradigm, we demonstrate that exhibiting a vital and highly conserved ecto-symbiosis in the face of thermal fluctuations has led to a peculiar selective trend promoting the adaptive diversification of the molecular chaperone of the AMP, but not of the AMP itself. Because BRICHOS stabilizes beta-stranded peptides, this polymorphism likely represents an eurythermal adaptation to stabilize the structure of alvinellacin, thus hinting at its efficiency to select and control the epibiosis across the range of temperatures experienced by the worm; Our results fill some knowledge gaps concerning the function of BRICHOS in invertebrates and offer perspectives for studying immune genes in an evolutionary ecological framework

Alignment of five Hsp70 isoforms from <i>E</i>. <i>superba</i> and <i>E</i>. <i>crystallorophias</i>.

<p>In red: Hsp70 diagnostic motifs; in black: possible glycosylation sites; in blue: hydrophobic linker between Nucleotide Binding Domain and Substrate Binding Domain.</p

Immune failure reveals vulnerability of populations exposed to pollution in the bioindicator species Hediste diversicolor

International audienceHuman activities on the shoreline generate a growing pollution, creating deleterious habitats in coastal zones. Some species nevertheless succeed in such harsh milieus, raising the question of their tolerance to environmental stress. The annelid Hediste diversicolor lives buried in the sediments, directly exposed to contaminants trapped in the mud. After verifying the similarity of their genetic contexts, we compared reproductive output and individual immune resistance measures of populations living in polluted vs. ‘clean’ sediments, and related these assessments with measures of phthalates and metal pollution, and associated toxicity indices. Chemical analyses predicted no toxicity to the local infauna, and phenological studies evidenced no direct cost of living in noxious habitats. However, populations exposed to pollutants showed a significantly reduced survival upon infection with a local pathogen. Surprisingly, physiological studies evidenced a basal overinflammatory state in the most exposed populations. This over-activated baseline immune phenotype likely generates self-damage leading to enhanced immune cell death rate and immune failure. Monitoring the immune status of individual worms living in anthropic areas could thus be used as a reliable source of information regarding the actual health of wild populations

Non-synonymous (dN) tree of the 11 Hsp70 genes found in <i>E</i>. <i>superba</i> and <i>E</i>. <i>crystallorophias</i> obtained using the free-ratio (M1) branch model of CodeML according to the Goldman & Yang (1994) model of codon substitution.

<p>Above internal branches: dN/dS (ω) ratio, below internal branches: number of non-synonymous and synonymous mutations estimated from the ancestral sequence reconstruction. * indicates saturation at the synonymous sites. The green colour represents branches under putative selective relaxation and the red one, branches (mostly terminal) under positive selection. Next to external (terminal) branches also corresponds to the ω ratio and the number of non-synonymous and synonymous mutations obtained from the ancestral sequence reconstruction using the M_2A branch-site model of selection. If any, a list of amino-acid replacements that corresponds to positively selected sites with a BEB probability greater than 90% is provided together with these values.</p

Phylogeny of the HSP70 family in Eucrustacea based on Maximum Likelihood and Bayesian analyses of amino acid data set.

<p>Numbers above branches are posterior probabilities and bootstrap values (based on 100 replicated).</p

Number of synonymous and non-synonymous mutations between the two krill species.

<p>N.dN and S.dS estimates obtained between <i>E</i>. <i>superba</i> and <i>E</i>. <i>crystallorophias</i> for the 5 paralogous <i>Hsp70</i> genes using the Nei-Gojobori method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121642#pone.0121642.ref079" target="_blank">79</a>] to make evolutionary-rate comparisons under the assumption of a global molecular clock.</p><p>Number of synonymous and non-synonymous mutations between the two krill species.</p