Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A

Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1–411), middle (residues 296–701), and C-terminal portions (residues 524–911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels of hemagglutinin (HA) activity, with the exception of the F1 fragment, which demonstrates no HA activity; and (iii) in the presence of alum, all fragments elicit various levels of anti-toxin A-neutralizing antibody responses, but those neutralizing antibodies elicited by F2 did not protect mice against a TcdA challenge. Because TcdA rRBD, F1 and F3 formulated with alum can elicit immune protective responses against the cytotoxicity of TcdA, they represent potential components of future candidate vaccines against C. difficile-associated diseases.     

Male brood provisioning rates provide evidence for inter‐age competition for mates in female Cooper's Hawks Accipiter cooperii

Life history theory predicts that individuals should maximize lifetime reproductive success (LRS) by breeding as soon as they reach sexual maturity, yet many species delay breeding, either because there are insufficient available mates or breeding sites, or because delayed breeding yields higher LRS. Accipitriform species, such as Cooper's Hawk Accipiter cooperii, exhibit both delayed breeding and delayed plumage maturation. However, in certain circumstances, first‐year females in non‐definitive plumage do breed and apparently compete with older females for high‐quality breeding territories. We predicted that these young females are at a competitive disadvantage compared with older females and that older females would have both higher reproductive success and be able to acquire higher quality nesting territories. We conducted brood counts and measured prey delivery rates by male Cooper's Hawks in an expanding urban population located in Albuquerque, New Mexico (USA), to assess our prediction. We found that older females had higher reproductive success, fledging 1.6 more offspring than younger females, and that they occupied territories where males provisioned at higher rates of 0.37 more prey items per 2‐h period. Our results showed that older females fared better than first‐year females but it is unclear if this is the result of passive or active competition. Older females initiated nesting 14.3 days sooner than first‐year females and thus may have filled vacant, high‐quality territories before first‐year females began seeking mates. Additionally, first‐year females were never observed persistently to confront older females for breeding territories, but they did actively compete against each other. First‐year females may defer to older females who, in a direct competitive interaction, would be most likely to prevail. Thus, delayed plumage maturation in Cooper's Hawks may serve to focus competition for nesting territories within age classes.     

Exploiting whole genome sequence data to fine map and characterize candidate genes within a quantitative trait loci region affecting androstenone on porcine chromosome 5

Male piglets are routinely castrated to eliminate boar taint. However, this treatment is undesirable, and alternative approaches, including genetic strategies to reduce boar taint, are demanded. Androstenone is one of the causative agents of boar taint, and a QTL region affecting this pheromone has previously been reported on SSC5: 22.6–24.8 Mb in Duroc. The QTL region is one of the few reported for androstenone that does not simultaneously affect levels of other sex steroids. The main objective of this study was to fine map this QTL. Whole genome sequence data from 23 Norwegian Duroc boars were analyzed to detect new polymorphisms within the QTL region. A subset of 161 SNPs was genotyped in 834 Duroc sires and analyzed for association with androstenone in adipose tissue and testosterone, estrone sulphate and 17β‐estradiol in blood plasma. Our results revealed 100 SNPs significantly associated with androstenone levels in fat (P < 0.001) with 94 of the SNPs being in strong linkage disequilibrium in the region 23.03–24.27 Mb. This haplotype block contains at least four positional candidate genes (HSD17B6, SDR9C7, RDH16 and STAT6) involved in androstenone biosynthesis. No significant associations were found between any of the SNPs and levels of testosterone and estrogens, confirming previous findings. The amount of phenotypic variance explained by single SNPs within the haplotype block was as high as 5.4%. As the SNPs in this region significantly affect levels of androstenone without affecting levels of other sex steroids, they are especially interesting as genetic markers for selection against boar taint.     

α–E-catenin binds to dynamitin and regulates dynactin-mediated intracellular traffic

α–Epithelial catenin (E-catenin) is an important cell–cell adhesion protein. In this study, we show that α–E-catenin also regulates intracellular traffic by binding to the dynactin complex component dynamitin. Dynactin-mediated organelle trafficking is increased in α–E-catenin^−/− keratinocytes, an effect that is reversed by expression of exogenous α–E-catenin. Disruption of adherens junctions in low-calcium media does not affect dynactin-mediated traffic, indicating that α–E-catenin regulates traffic independently from its function in cell–cell adhesion. Although neither the integrity of dynactin–dynein complexes nor their association with vesicles is affected by α–E-catenin, α–E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton. Because the actin-binding domain of α–E-catenin is necessary for this regulation, we hypothesize that α–E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.     

Comparable length at weaning in cetaceans

Weaning represents the transition of mammalian life from maternal dependence to independence in both energetics and behavior. The length at weaning (Lw) is determined by the maternal investment during gestation and lactation. It affects calf survival and impacts on the long‐term persistence of species, but the measurement is not readily obtainable for many cetaceans. A general linear model and residual correlation were used to explore the correlations between the published Lw and five other life history traits of cetaceans, including female asymptotic length (Lx), length at birth (Lb), lactation period (LP), gestation period (GP), and calving interval (CI). Lx was a significant confounding parameter on the other five traits. By eliminating the confounding Lx, ANCOVA revealed that baleen whales (Mysticeti) and toothed cetaceans (Odontoceti) had comparable Lw. By residual correlation, which factors the confounding Lx, Lw was significantly correlated with Lb but not with GP, LP, and CI. After Lb was further eliminated, convergent Lw in cetaceans could still be observed. Therefore, we proposed a generic expression Lw= 1.239Lx^0.877, which allows us to further estimate Lw of undocumented cetacean species.     

Production of chicken anemia virus (CAV) VP1 and VP2 protein expressed by recombinant Escherichia coli

Chicken anemia virus (CAV) is an anemia agent of breeder and young chicks. This virus is the cause of economic losses across the chicken industry worldwide as a consequence of severe anemia and immunodeficiency among the birds. Two genes of CAV encoding the VP1 and VP2 proteins were cloned and expressed in Escherichia coli BL21 (DE3). A Western blot assay using His-tag antiserum was used to assess the expression level of the CAV viral proteins in E. coli. The results demonstrated that only full-length VP2 can be successfully expressed in E. coli, but not full-length VP1. A serial of N-terminus deletions of the VP1 protein, VP1Nd30, VP1Nd60 and VP1 Nd129, were created using PCR in order to improve VP1 expression. The results demonstrated that all three of these recombinant VP1 mutant proteins can be expressed in E. coli. VP1Nd129 protein demonstrates the highest expression level compared to the other two proteins. The specificity of Nd129-VP1 and VP2 protein were confirmed by mass spectrometry. By comparing the expression level of VP1Nd129 and VP2 protein after the addition of IPTG, the results indicated that the VP1Nd129 protein gave a higher level of protein expression than VP2. The highest yields of VP1Nd129 and VP2 were 26.2 and 15.5 mg/L, respectively, after IPTG induction with 0.1 mM IPTG for 6 h, respectively. The identification of the optimized conditions for production of the CAV viral proteins VP1 and VP2 will allow them to be used in the future as an antigen for the development of vaccines and diagnostic tests.     

Male hosts are responsible for the transmission of a trophically transmitted parasite, Pterygodermatites peromysci, to the intermediate host in the absence of sex-biased infection

Field studies have identified that male-biased infection can lead to increased rates of transmission, so we examined the relative importance of host sex on the transmission of a trophically transmitted parasite (Pterygodermatites peromysci) where there is no sex-biased infection. We experimentally reduced infection levels in either male or female white-footed mice (Peromyscus leucopus) on independent trapping grids with an anthelmintic and recorded subsequent infection levels in the intermediate host, the camel cricket (Ceuthophilus pallidipes). We found that anthelmintic treatment significantly reduced the prevalence of infection among crickets in both treatment groups compared with the control, and at a rate proportional to the number of mice de-wormed, indicating prevalence was not affected by the sex of the shedding definitive host. In contrast, parasite abundance in crickets was higher on the grids where females were treated compared with the grids where males were treated. These findings indicate that male hosts contribute disproportionately more infective stages to the environment and may therefore be responsible for the majority of parasite transmission even when there is no discernable sex-biased infection. We also investigated whether variation in nematode length between male and female hosts could account for this male-biased infectivity, but found no evidence to support that hypothesis.     

The L7Ae protein binds to two kink-turns in the Pyrococcus furiosus RNase P RNA

The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg²⁺-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR''s catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.