Characterization of the Regions Involved in the Calcium-Induced Folding of the Intrinsically Disordered RTX Motifs from the Bordetella pertussis Adenylate Cyclase Toxin

Repeat in toxin (RTX) motifs are nonapeptide sequences found among numerous virulence factors of Gram-negative bacteria. In the presence of calcium, these RTX motifs are able to fold into an idiosyncratic structure called the parallel β-roll. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the best-characterized RTX cytolysins. CyaA contains a C-terminal receptor domain (RD) that mediates toxin binding to the eukaryotic cell receptor. The receptor-binding domain is composed of about forty RTX motifs organized in five successive blocks (I to V). The RTX blocks are separated by non-RTX flanking regions of variable lengths. It has been shown that block V with its N- and C-terminal flanking regions constitutes an autonomous subdomain required for the toxicity of CyaA. Here, we investigated the calcium-induced biophysical changes of this subdomain to identify the respective contributions of the flanking regions to the folding process of the RTX motifs. We showed that the RTX polypeptides, in the absence of calcium, exhibited the hallmarks of intrinsically disordered proteins and that the C-terminal flanking region was critical for the calcium-dependent folding of the RTX polypeptides, while the N-terminal flanking region was not involved. Furthermore, the secondary and tertiary structures were acquired concomitantly upon cooperative binding of several calcium ions. This suggests that the RTX polypeptide folding is a two-state reaction, from a calcium-free unfolded state to a folded and compact conformation, in which the calcium-bound RTX motifs adopt a β-roll structure. The relevance of these results to the toxin physiology, in particular to its secretion, is discussed.     

Historical Biogeography of endemic seed plant genera in the Caribbean: Did GAARlandia play a role?

The Caribbean archipelago is a region with an extremely complex geological history and an outstanding plant diversity with high levels of endemism. The aim of this study was to better understand the historical assembly and evolution of endemic seed plant genera in the Caribbean, by first determining divergence times of endemic genera to test whether the hypothesized Greater Antilles and Aves Ridge (GAARlandia) land bridge played a role in the archipelago colonization and second by testing South America as the main colonization source as expected by the position of landmasses and recent evidence of an asymmetrical biotic interchange. We reconstructed a dated molecular phylogenetic tree for 625 seed plants including 32 Caribbean endemic genera using Bayesian inference and ten calibrations. To estimate the geographic range of the ancestors of endemic genera, we performed a model selection between a null and two complex biogeographic models that included timeframes based on geological information, dispersal probabilities, and directionality among regions. Crown ages for endemic genera ranged from Early Eocene (53.1 Ma) to Late Pliocene (3.4 Ma). Confidence intervals for divergence times (crown and/or stem ages) of 22 endemic genera occurred within the GAARlandia time frame. Contrary to expectations, the Antilles appears as the main ancestral area for endemic seed plant genera and only five genera had a South American origin. In contrast to patterns shown for vertebrates and other organisms and based on our sampling, we conclude that GAARlandia did not act as a colonization route for plants between South America and the Antilles. Further studies on Caribbean plant dispersal at the species and population levels will be required to reveal finer‐scale biogeographic patterns and mechanisms.     

Infection with Trypanosoma cruzi restricts the repertoire of parasite-specific CD8+ T cells leading to immunodominance

Interference or competition between CD8(+) T cells restricted by distinct MHC-I molecules can be a powerful means to establish an immunodominant response. However, its importance during infections is still questionable. In this study, we describe that following infection of mice with the human pathogen Trypanosoma cruzi, an immunodominant CD8(+) T cell immune response is developed directed to an H-2K(b)-restricted epitope expressed by members of the trans-sialidase family of surface proteins. To determine whether this immunodominance was exerted over other non-H-2K(b)-restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2K(b)-, H-2K(k)-, or H-2K(d)-restricted CD8(+) T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2K(b)-restricted immunodominant epitope. In contrast, H-2K(k)- or H-2K(d)-restricted immune responses were significantly impaired in heterozygote infected mice when compared with homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi Ags. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8(+) T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.     

Developmental arrest during embryonic development of the common chameleon (Chamaeleo chamaeleon) in Spain

Embryonic development of the common chameleon, Chamaeleo chamaeleon, was monitored from oviposition to hatching at a field site in southwestern Spain and in the laboratory under five experimental temperature regimes. Embryos were diapausing gastrulae at the time of oviposition; developmental arrest in the field continued as cold torpor during winter. Postarrest development in the field commenced in April, and hatching occurred in August, for a total incubation period of 10.5 mo. In the laboratory, one group of eggs was incubated at a constant warm (26 degrees C) temperature. The remaining treatments simulated field conditions and consisted of initial periods of warm temperature of 0, 27, 46, and 71 d, a subsequent 4-mo period of cold winter (16 degrees C) temperature, and a final period of warm (26 degrees C) temperature. Embryos in the constant warm temperature treatment were in diapause an average of 3 mo, with clutch means ranging from 2 to 4 mo. Hatching among clutches occurred over 2 mo. In contrast, for field and experimental eggs that experienced cold winter conditions, hatching within treatments occurred over 2-14 d; "winter" conditions synchronized development. The length of time between the end of cold conditions and hatching did not differ among treatments; development thus resumed as soon as temperature was suitable regardless of the initial period of warm temperature. Diapause in nature thus insures that embryos remain gastrulae after oviposition despite nest temperatures that may be warm enough to support development.     

Long-term calorie restriction has minimal impact on brain metabolite and fatty acid profiles in aged rats on a Western-style diet

The effect of long-term calorie restriction (CR) on metabolites, fatty acid profiles and energy substrate transporter expression in the brain was assessed in aged rats. Three groups of male Sprague–Dawley rats were studied: (i) a 2 month old ad libitum-fed (2AL group), (ii) a 19 month old ad libitum-fed (19AL group), and (iii) a 19 month old group subjected to 40% CR from the age of 7.5 to 19 months (19CR group). The diet contained high sucrose and low n-3 polyunsaturated fatty acids (PUFA) so as to imitate a Western-style diet. High resolution magic angle spinning-¹H NMR showed an effect of aging on brain cortex metabolites compared to 2AL rats, the largest differences being for myo-inositol (+251% and +181%), lactate (+203% and +188%), β-hydroxybutyrate (+176% and +618%) and choline (+148% and +120%), in 19AL and 19 CR rats, respectively. However, brain metabolites did not differ between the 19AL and 19CR groups. Cortex fatty acid profiles showed that n-3 PUFA were 35–47% lower but monounsaturated fatty acids were 40–52% higher in 19AL and 19CR rats compared to 2AL rats. Brain microvessel glucose transporter (GLUT1) was 68% higher in 19AL rats than in 2AL rats, while the monocarboxylate transporter, MCT1, was 61% lower in 19CR rats compared to 19AL rats. We conclude that on a high-sucrose, low n-3 PUFA diet, the brain of aged AL rats had higher metabolites and microvessel GLUT1 expression compared to 2AL rats. However, long-term CR in aged rats did not markedly change brain metabolite or fatty acid profile, but did reduce brain microvessel MCT1 expression.     

Niche conservatism and the potential for the crayfish Procambarus clarkii to invade South America

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