Full engagement of liganded maltose‐binding protein stabilizes a semi‐open ATP‐binding cassette dimer in the maltose transporter

MalFGK₂ is an ATP‐binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose‐binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide‐binding subunits (MalK dimer). This MBP‐stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose‐bound MBP unto MalFGK₂ is facilitated by nucleotides and stabilizes a semi‐open MalK dimer. Maltose‐bound MBP promotes the transition to the semi‐open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi‐open MalK₂ conformation by maltose‐bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi‐open conformation, from which it can proceed to hydrolyze ATP.     

Aberrant IgM signaling promotes survival of transitional T1 B cells and prevents tolerance induction in lupus-prone New Zealand black mice

New Zealand Black (NZB) mice develop a lupus-like syndrome. Although the precise immune defects leading to autoantibody production in these mice have not been characterized, they possess a number of immunologic abnormalities suggesting that B cell tolerance may be defective. In the bone marrow, immature self-reactive B cells that have failed to edit their receptors undergo apoptosis as a consequence of Ig receptor engagement. Splenic transitional T1 B cells are recent bone marrow emigrants that retain these signaling properties, ensuring that B cells recognizing self-Ags expressed only in the periphery are deleted from the naive B cell repertoire. In this study we report that this mechanism of tolerance is defective in NZB mice. We show that NZB T1 B cells are resistant to apoptosis after IgM cross-linking in vitro. Although extensive IgM cross-linking usually leads to deletion of T1 B cells, in NZB T1 B cells we found that it prevents mitochondrial membrane damage, inhibits activation of caspase-3, and promotes cell survival. Increased survival of NZB T1 B cells was associated with aberrant up-regulation of Bcl-2 after Ig receptor engagement. We also show that there is a markedly increased proportion of NZB T1 B cells that express elevated levels of Bcl-2 in vivo and provide evidence that up-regulation of Bcl-2 follows encounter with self-Ag in vivo. Thus, we propose that aberrant cell signaling in NZB T1 B cells leads to the survival of autoreactive B cells, which predisposes NZB mice to the development of autoimmunity.     

Vector Choice Determines Immunogenicity and Potency of Genetic Vaccines against Angola Marburg Virus in Nonhuman Primates

The immunogenicity and durability of genetic vaccines are influenced by the composition of gene inserts and choice of delivery vector. DNA vectors are a promising vaccine approach showing efficacy when combined in prime-boost regimens with recombinant protein or viral vectors, but they have shown limited comparative efficacy as a stand-alone platform in primates, due possibly to suboptimal gene expression or cell targeting. Here, regimens using DNA plasmids modified for optimal antigen expression and recombinant adenovirus (rAd) vectors, all encoding the glycoprotein (GP) gene from Angola Marburg virus (MARV), were compared for their ability to provide immune protection against lethal MARV Angola infection. Heterologous DNA-GP/rAd5-GP prime-boost and single-modality rAd5-GP, as well as the DNA-GP-only vaccine, prevented death in all vaccinated subjects after challenge with a lethal dose of MARV Angola. The DNA/DNA vaccine induced humoral responses comparable to those induced by a single inoculation with rAd5-GP, as well as CD4⁺ and CD8⁺ cellular immune responses, with skewing toward CD4⁺ T-cell activity against MARV GP. Vaccine regimens containing rAd-GP, alone or as a boost, exhibited cellular responses with CD8⁺ T-cell dominance. Across vaccine groups, CD8⁺ T-cell subset dominance comprising cells exhibiting a tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) double-positive functional phenotype was associated with an absence or low frequency of clinical symptoms, suggesting that both the magnitude and functional phenotype of CD8⁺ T cells may determine vaccine efficacy against infection by MARV Angola.The filoviruses Marburgvirus (MARV) and Ebolavirus (EBOV) are endemic primarily to central Africa and cause a severe form of viral hemorrhagic fever. Of all the filovirus strains or species, the Angola strain of MARV is associated with the highest mortality rate (90%) in humans observed to date (26). An increase in natural filovirus outbreak frequency over the past decade and the potential for use to cause deliberate human mortality have focused attention on the need for therapeutics and vaccines against filoviruses. While regulatory pathways have been proposed to facilitate licensing of a preventive vaccine against potently lethal pathogens such as these, there is as yet no licensed vaccine for use in humans, and efforts remain targeted to the optimization of vaccine performance in nonhuman primates (NHP) since this animal model recapitulates many aspects of disease pathogenesis observed in humans.Genetic vaccines are a promising approach for immunization against pathogens that are rapidly changing due to natural evolution, cross-species transmission, or intentional modification. Gene-based vaccines are produced rapidly and can be delivered by a variety of vectors. DNA vectors are advantageous because they are inherently safe and stable and can be used repeatedly without inducing antivector immune responses. However, while filovirus DNA vaccines have demonstrated efficacy in small animal models, efforts to induce protective immunity by injection of plasmid DNA alone into NHP have yielded less encouraging results. EBOV DNA vectors generate immune protection in mice and guinea pigs, but this has not been demonstrated in NHP unless DNA immunization is boosted with a viral vector vaccine (23). MARV DNA fully protects mice and guinea pigs but provides only partial protection in NHP (17). The discordant results between rodent and primate species may be due to the use of slightly modified infectious challenge viruses in rodent models or may reflect underlying differences in vaccine performance and the mechanisms of immune protection between rodents and NHP.In the current study, we examined whether DNA plasmid-based vaccines could be improved to increase potency in NHP and compared immunogenicity of this vaccine modality with those of viral vector and prime-boost approaches. DNA-vectored vaccines were modified by codon optimizing gene target inserts for enhanced expression in primates. These vectors induced antigen-specific cellular and humoral immune responses similar to immunization using a recombinant adenoviral vector and provided protection after lethal challenge with MARV Angola. However, macaques vaccinated with DNA vectors exhibited clinical symptoms associated with MARV hemorrhagic fever (MHF) that were absent in NHP receiving a single inoculation with recombinant adenovirus (rAd) vectors, suggesting qualitative differences in the immune responses elicited by the different modalities.     

Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A)

The prototypical form of the Ser/Thr phosphatase PP2A is a heterotrimeric complex consisting of catalytic subunit (C), and A and B regulatory subunits. C-terminal methylation of PP2A-C influences holoenzyme assembly. Using late gestation development in the rat as an in vivo model of liver growth, we found that PP2A-C protein and activity levels were higher in fetal compared to adult liver extracts. However, unmethylated PP2A-C was much higher in the adult extracts. In MonoQ fractionation, unmethylated C eluted separately from methylated C, which was present predominantly in ABC heterotrimers. Gel filtration chromatography revealed that some unmethylated C was present as free catalytic subunit in adult liver. In addition, a significant proportion of PP2A was in inactive forms that may involve novel regulatory subunits. Our results indicate that methylation of PP2A-C appears to be a primary determinant for the biogenesis of PP2A heterotrimers.     

Pneumococcal Carriage in Children under Five Years in Uganda-Will Present Pneumococcal Conjugate Vaccines Be Appropriate?

BackgroundPneumonia is the major cause of death in children globally, with more than 900,000 deaths annually in children under five years of age. Streptococcus pneumoniae causes most deaths, most often in the form of community acquired pneumonia. Pneumococcal conjugate vaccines (PCVs) are currently being implemented in many low-income countries. PCVs decrease vaccine-type pneumococcal carriage, a prerequisite for invasive pneumococcal disease, and thereby affects pneumococcal disease and transmission. In Uganda, PCV was launched in 2014, but baseline data is lacking for pneumococcal serotypes in carriage.ObjectivesTo study pneumococcal nasopharyngeal carriage and serotype distribution in children under 5 years of age prior to PCV introduction in UgandaMethodsThree cross-sectional pneumococcal carriage surveys were conducted in 2008, 2009 and 2011, comprising respectively 150, 587 and 1024 randomly selected children aged less than five years from the Iganga/Mayuge Health and Demographic Surveillance Site. The caretakers were interviewed about illness history of the child and 1723 nasopharyngeal specimens were collected. From these, 927 isolates of S. pneumoniae were serotyped.ResultsOverall, the carriage rate of S. pneumoniae was 56% (957/1723). Pneumococcal carriage was associated with illness on the day of the interview (OR = 1.50, p = 0.04). The most common pneumococcal serotypes were in descending order 19F (16%), 23F (9%), 6A (8%), 29 (7%) and 6B (7%). One percent of the strains were non-typeable. The potential serotype coverage rate for PCV10 was 42% and 54% for PCV13.ConclusionAbout half of circulating pneumococcal serotypes in carriage in the Ugandan under-five population studied was covered by available PCVs.     

Defining the importance of landscape metrics for large branchiopod biodiversity and conservation: the case of the Iberian Peninsula and Balearic Islands

The deficiency in the distributional data of invertebrate taxa is one of the major impediments acting on the bias towards the low awareness of its conservation status. The present study sets a basic framework to understand the large branchiopods distribution in the Iberian Peninsula and Balearic Islands. Since the extensive surveys performed in the late 1980s, no more studies existed updating the information for the whole studied area. The present study fills the gap, gathering together all available information on large branchiopods distribution since 1995, and analysing the effect of human population density and several landscape characteristics on their distribution, taking into consideration different spatial scales (100 m, 1 km and 10 km). In overall, 28 large branchiopod taxa (17 anostracans, 7 notostracans and 4 spinicaudatans) are known to occur in the area. Approximately 30% of the sites hosted multiple species, with a maximum of 6 species. Significant positive co-occurring species pairs were found clustered together, forming 4 different associations of large branchiopod species. In general, species clustered in the same group showed similar responses to analysed landscape characteristics, usually showing a better fit at higher spatial scales.     

Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

A newClostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from theC. perfringens replicon pIP404 and theE. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants inE. coli. Both chloramphenicol and erythromycin resistance can be selected inC. perfringens andE. coli since pJIR418 carries theC. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes fromC. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Recruitment of C-terminal Src kinase by the leukocyte inhibitory receptor CD85j

The CD85j inhibitory receptor (also termed ILT2 or LIR-1) is a type-I transmembrane protein that belongs to the Ig superfamily and is expressed by different leukocyte lineages. The extracellular region of CD85j binds HLA class I molecules and its cytoplasmic domain displays four immunoreceptor tyrosine-based inhibition motifs (ITIM). Upon tyrosine phosphorylation CD85j recruits the SHP-1 tyrosine phosphatase, involved in negative signaling. In order to identify other molecules to which CD85j might interact with in a phosphotyrosine-dependent manner, a cDNA B-cell library was screened in a three-hybrid system in yeast using the CD85j cytoplasmic tail as bait in the presence of the Src-kinase c-fyn420, 531Y-F, 176R-Q mutant. In this system, the C-terminal Src kinase (Csk) was shown to interact with CD85j. Phosphorylation-dependent recruitment of Csk to the CD85j cytoplasmic tail was confirmed in CD85j-transfected mammalian cells by immunoprecipitation and Western blot analysis. Mutational analyses and phospho-peptide mapping suggested that the SH2 domain of Csk may preferentially bind to ITIM Y562 of CD85j; yet, mutation to phenylalanine of Y533, Y614, and Y644 also significantly reduced Csk recruitment by CD85j. Even though CD85j was detected in both anti-SHP1 and CSK immunoprecipitates, these two molecules did not co-precipitate together with CD85j. Our data support the possibility that Csk regulates the function of CD85j.