Tree shape‐based approaches for the comparative study of cophylogeny

Cophylogeny is the congruence of phylogenetic relationships between two different groups of organisms due to their long‐term interaction. We investigated the use of tree shape distance measures to quantify the degree of cophylogeny. We implemented a reverse‐time simulation model of pathogen phylogenies within a fixed host tree, given cospeciation probability, host switching, and pathogen speciation rates. We used this model to evaluate 18 distance measures between host and pathogen trees including two kernel distances that we developed for labeled and unlabeled trees, which use branch lengths and accommodate different size trees. Finally, we used these measures to revisit published cophylogenetic studies, where authors described the observed associations as representing a high or low degree of cophylogeny. Our simulations demonstrated that some measures are more informative than others with respect to specific coevolution parameters especially when these did not assume extreme values. For real datasets, trees’ associations projection revealed clustering of high concordance studies suggesting that investigators are describing it in a consistent way. Our results support the hypothesis that measures can be useful for quantifying cophylogeny. This motivates their usage in the field of coevolution and supports the development of simulation‐based methods, i.e., approximate Bayesian computation, to estimate the underlying coevolutionary parameters.     

Genomic Evidence for Island Population Conversion Resolves Conflicting Theories of Polar Bear Evolution

Despite extensive genetic analysis, the evolutionary relationship between polar bears (Ursus maritimus) and brown bears (U. arctos) remains unclear. The two most recent comprehensive reports indicate a recent divergence with little subsequent admixture or a much more ancient divergence followed by extensive admixture. At the center of this controversy are the Alaskan ABC Islands brown bears that show evidence of shared ancestry with polar bears. We present an analysis of genome-wide sequence data for seven polar bears, one ABC Islands brown bear, one mainland Alaskan brown bear, and a black bear (U. americanus), plus recently published datasets from other bears. Surprisingly, we find clear evidence for gene flow from polar bears into ABC Islands brown bears but no evidence of gene flow from brown bears into polar bears. Importantly, while polar bears contributed <1% of the autosomal genome of the ABC Islands brown bear, they contributed 6.5% of the X chromosome. The magnitude of sex-biased polar bear ancestry and the clear direction of gene flow suggest a model wherein the enigmatic ABC Island brown bears are the descendants of a polar bear population that was gradually converted into brown bears via male-dominated brown bear admixture. We present a model that reconciles heretofore conflicting genetic observations. We posit that the enigmatic ABC Islands brown bears derive from a population of polar bears likely stranded by the receding ice at the end of the last glacial period. Since then, male brown bear migration onto the island has gradually converted these bears into an admixed population whose phenotype and genotype are principally brown bear, except at mtDNA and X-linked loci. This process of genome erosion and conversion may be a common outcome when climate change or other forces cause a population to become isolated and then overrun by species with which it can hybridize.     

Changing Dietary Calcium-Phosphorus Level and Cereal Source Selectively Alters Abundance of Bacteria and Metabolites in the Upper Gastrointestinal Tracts of Weaned Pigs

Several dietary ingredients may affect the bacterial community structure and metabolism in the porcine gut and may therefore influence animals'' health and performance. This study investigated the effects of cereal source and calcium-phosphorus (CaP) level in the diet on bacterial microbiota and metabolites, nutrient intake, and gut environment in weaned pigs. Pigs (n = 8/treatment) were fed wheat-barley- or corn-based diets with an adequate or high CaP level for 14 days. Effects on microbiota in the stomach, ileum, and midcolon were assessed using quantitative PCR. Data showed that Enterobacteriaceae, Campylobacter spp., and Helicobacter spp., which all contain highly immune reactive lipopolysaccharide (LPS), were abundant at all gut sites. Diet effects on bacteria and metabolites were moderate and occurred mainly in the upper gut, whereas no effects on bacteria, fermentation products, and LPS could be observed in the colon. Differences in carbohydrate intake with corn versus wheat-barley diets selectively stimulated Bifidobacterium in the stomach and ileum. There was a growth advantage for a few bacterial groups in the stomach and ileum of pigs fed the high versus adequate CaP level (i.e., gastric Enterobacteriaceae and ileal Enterococcus, Bacteroides-Prevotella-Porphyromonas, and Campylobacter). Interestingly, gastrointestinal pH was not affected by dietary CaP level. The present findings demonstrate the stability of the bacterial community and gut environment toward dietary changes even in young pigs. The results on stimulation of gastric and ileal Bifidobacterium by corn diets may be employed in nutritional strategies to support gut health after weaning.     

Interference between Proteins Hap46 and Hop/p60, Which Bind to Different Domains of the Molecular Chaperone hsp70/hsc70

Several structurally divergent proteins associate with molecular chaperones of the 70-kDa heat shock protein (hsp70) family and modulate their activities. We investigated the cofactors Hap46 and Hop/p60 and the effects of their binding to mammalian hsp70 and the cognate form hsc70. Hap46 associates with the amino-terminal ATP binding domain and stimulates ATP binding two- to threefold but inhibits binding of misfolded protein substrate to hsc70 and reactivation of thermally denatured luciferase in an hsc70-dependent refolding system. By contrast, Hop/p60 interacts with a portion of the carboxy-terminal domain of hsp70s, which is distinct from that involved in the binding of misfolded proteins. Thus, Hop/p60 and substrate proteins can form ternary complexes with hsc70. Hop/p60 exerts no effect on ATP and substrate binding but nevertheless interferes with protein refolding. Even though there is no direct interaction between these accessory proteins, Hap46 inhibits the binding of Hop/p60 to hsc70 but Hop/p60 does not inhibit the binding of Hap46 to hsc70. As judged from respective deletions, the amino-terminal portions of Hap46 and Hop/p60 are involved in this interference. These data suggest steric hindrance between Hap46 and Hop/p60 during interaction with distantly located binding sites on hsp70s. Thus, not only do the major domains of hsp70 chaperones communicate with each other, but cofactors interacting with these domains affect each other as well.     

Identification, replication, and fine-mapping of Loci associated with adult height in individuals of african ancestry

Adult height is a classic polygenic trait of high heritability (h ² ∼0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain ∼10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10⁻¹² and 2p14-rs4315565, P = 1.2×10⁻⁸). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10⁻⁴ for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits.     

Classification of protein profiles from antibody microarrays using heat and detergent treatment

Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.     

Subretinal delivery of adeno‐associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice

Background

Adeno‐associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long‐term therapy is the development of immune responses against the vector or transgene product.

Methods

We evaluated cellular and humoural responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression.

Results

Following subretinal administration of vector, splenocytes and T‐cells from draining lymph nodes showed minimal activation following stimulation by co‐culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low‐dose AAV.hRPE65.hRPE65 to both eyes of RPE65^?/? mice resulted in transgene expression and functional rescue, but re‐administration of high‐dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye.

Conclusions

These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose‐dependent. Low‐dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration. Copyright © 2009 John Wiley & Sons, Ltd.

     

Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase‐mediated effect on post‐stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488–4496). In the present study, the global function of ClpC in physiology, metabolism, and late‐stationary phase survival was examined using DNA microarrays and 2‐D PAGE followed by MALDI‐TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose‐phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell‐associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late‐stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.     

The MalF P2 Loop of the ATP-Binding Cassette Transporter MalFGK2 from Escherichia coli and Salmonella enterica Serovar Typhimurium Interacts with Maltose Binding Protein (MalE) throughout the Catalytic Cycle

We have investigated the interaction of the uncommonly large periplasmic P2 loop of the MalF subunit of the maltose ATP-binding cassette transporter (MalFGK₂) from Escherichia coli and Salmonella enterica serovar Typhimurium with maltose binding protein (MalE) by site-specific chemical cross-linking in the assembled transport complex. We focused on possible distance changes between two pairs of residues of the P2 loop and MalE during the transport cycle. The distance between MalF(S205C) and MalE(T80C) (∼5 Å) remained unchanged under all conditions tested. Cross-linking did not affect the ATPase activity of the complex. The distance between MalF(T177C) and MalE(T31C) changed from ∼10 Å to ∼5 Å upon binding of ATP (or maltose, with a less pronounced result) and was reset to ∼10 Å after hydrolysis of one ATP. A cross-link (∼25 Å) between MalF(S205C) and MalE(T31C) was observed only when the transporter resided in a transition state-like conformation, as was the case after vanadate trapping or in a binding protein-independent mutant, both of which are characterized by tight binding of unliganded MalE to the transporter. Thus, we propose that the observed cross-link is indicative of catalytic intermediates of the transporter. Together, our results strengthen the notion that the MalF P2 loop plays an important role in intersubunit communication. In particular, this loop is involved in keeping MalE in close contact with the transporter. The data are discussed with respect to a crystal structure and current transport models.ATP-binding cassette (ABC) transporters utilize the free energy of ATP hydrolysis to translocate substrates across biological membranes and can function as import or export systems (17). ABC transporters are generally composed of two hydrophobic, pore-forming transmembrane subunits and transmembrane domains (TMDs) and two hydrophilic nucleotide-binding (or ABC) subunits and nucleotide-binding domains (NBDs) that hydrolyze ATP (9). The crystal structures of isolated NBDs (6, 23, 34, 43) revealed that NBDs can be divided into a RecA-like subdomain comprising both the Walker A and the Walker B motifs, which are involved in nucleotide binding, and a helical subdomain harboring the unique LSGGQ motif (35). Furthermore, in the physiologically relevant NBD dimer, the nucleotide is complexed between the Walker A and B sites of one monomer and the LSGGQ motif of the opposing monomer. Both subdomains are joined by the “Q loop” containing a conserved glutamine residue that binds to the Mg²⁺ ion and attacking water and is likely to be involved in communicating ATP binding to the TMDs (10, 20, 29). ATP-dependent closing of the NBD dimer is thought to provide one possibility of the power stroke of ABC transporters (38).ABC importers that are confined to prokaryotes mediate the uptake of a large variety of solutes, including inorganic ions, amino acids, sugars, vitamins, oligopeptides, and polyamines (5). They require an additional protein, the extracytoplasmic solute binding protein (SBP), in order to capture the substrate and to deliver it to the cognate ABC transporter (37). SBPs typically consist of two lobes that are connected by a linker region. The interface between the two lobes forms the substrate binding site. Upon binding of the ligand, the proteins undergo a conformational change from an open toward a closed state (33) which, by interaction with extracytoplasmic peptide regions of TMDs of the cognate ABC transporter, initiates the transport process (31). The molecular events by which binding of ATP to the NBDs and interaction of liganded binding proteins with the TMDs are communicated to eventually trigger substrate translocation are still poorly understood.The maltose ABC transporter of Escherichia coli and Salmonella enterica serovar Typhimurium is one of the best-characterized transporters and thus serves as a model system for studying the mechanism by which ABC transporters exert their functions in general (15). The transporter is composed of the extracytoplasmic (periplasmic) maltose binding protein (MalE), the membrane-spanning subunits MalF and MalG, and two copies of the ATP-hydrolyzing subunit (MalK) (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.(A) Structure of the catalytic intermediate of the maltose transporter [MalFGK(E159Q)₂-E]. The complex is shown in a ribbon diagram. White horizontal bars mark the boundaries of the membrane. Color code: yellow, MalE; cyan, MalF; red, MalG; green and magenta, MalK dimer. (B) Close-up view of the contact site between MalF P2 and the N-terminal lobe of MalE. The color code is the same as that for panel A. Residues from regions I and II that were replaced by cysteines are indicated in pink (MalF) and green (MalE). Residue MalE-K179, which was used as a control, is shown in green. The figure was drawn with DS ViewerPro 6.0 (Accelrys, Cambridge, United Kingdom), using the coordinates from entry 2R6G in the Brookhaven Protein Data Bank.Recently, suppressor mutational analysis provided a first hint that substrate availability is communicated from MalE to the MalK dimer via periplasmic loop regions of MalFG (11). Moreover, by site-directed cross-linking based on previous genetic evidence (19, 40), we demonstrated a close proximity of MalE G13 to Pro-78 in the first periplasmic loop (P1) of MalG, independently of cofactors such as maltose or ATP. Interaction of both residues was also observed in intact cells (11). These findings led us to propose that a copy of MalE is permanently associated with the transporter throughout the catalytic cycle. Furthermore, we have found that a region of the large, periplasmic P2 loop of MalF around Ser-205 (Fig. ​(Fig.1)1) is in cross-linking distance from MalE in the presence of maltose and MgATP only or when the transporter resides in the vanadate-trapped transition state. These results were perfectly confirmed by the subsequently published crystal structure of the MalFGK(E159Q)₂-E complex, which represents a transport intermediate (32). Here, the MalK dimer is complexed with two ATP molecules, and MalE is tightly associated with MalFG, but maltose has already been released into a binding pocket formed by MalF only. In particular, the N-terminal lobe of MalE is in close contact with the P2 loop of MalF (Fig. ​(Fig.1A1A).In this communication, we have taken advantage of this structural information to gain further insight into the MalF P2-MalE interaction during the transport cycle. We demonstrate ATP- and maltose-dependent distance changes between selected pairs of residues of the loop and MalE in the assembled complex by site-specific cross-linking. Our data demonstrate for the first time that the MalF P2 loop is in close contact to MalE throughout the catalytic cycle.     

Characterization of the amino acids involved in substrate specificity of methionine sulfoxide reductase A

Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide (MetSO) back to methionine. In vivo, Msrs are essential in protecting cells against oxidative damages on proteins and in the virulence of some bacteria. There exists two structurally unrelated classes of Msrs. MsrAs are stereo-specific toward the S epimer on the sulfur of the sulfoxide, whereas MsrBs are specific toward the R isomer. Both classes of Msrs display a similar catalytic mechanism of sulfoxide reduction by thiols via the sulfenic acid chemistry and a better affinity for protein-bound MetSO than for free MetSO. Recently, the role of the amino acids implicated in the catalysis of the reductase step of Neisseria meningitidis MsrA was determined. In the present study, the invariant amino acids potentially involved in substrate binding, i.e. Phe-52, Trp-53, Asp-129, His-186, Tyr-189, and Tyr-197, were substituted. The catalytic parameters under steady-state conditions and of the reductase step of the mutated MsrAs were determined and compared with those of the wild type. Altogether, the results support the presence of at least two binding subsites. The first one, whose contribution is major in the efficiency of the reductase step and in which the epsilon-methyl group of MetSO binds, is the hydrophobic pocket formed by Phe-52 and Trp-53, the position of the indole ring being stabilized by interactions with His-186 and Tyr-189. The second subsite composed of Asp-129 and Tyr-197 contributes to the binding of the main chain of the substrate but to a lesser extent.