Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic acid (M) to α-l-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

Competition for bumblebee visitation between Melampyrum pratense and Viscaria vulgaris with healthy and Ustilago-infected flowers

Summary We studied bumblebee foraging on two sympatrically and simultaneously flowering species, Melampyrum pratense (Scrophulariaceae) and Viscaria vulgaris (Caryophyllaceae) during the flowering season of Viscaria in south-west Sweden. We distinguished between healthy and Ustilago-infected Viscaria plants. Both species shared the main insect visitor, queens of Bombus hortorum, which collected nectar from both species but pollen from Melampyrum only. The pattern of visitation changed over the season: bumblebees preferred Viscaria early on, but changed to Melampyrum later in the season, probably because of the higher sugar content of Melampyrum nectar and the possibility of collecting both nectar and pollen from the same flower. Pollen collecting is probably of increasing importance since the need of pollen for the developing larvae will increase with time. Flowers of Viscaria received fewer visits in plots with other species than in pure Viscaria plots during one year and received more visits early than late in the season during both years. Melampyrum flowers received similar amounts of visits in mixed and pure environments. They also received more visits early than late, although this was probably a result of pollinator satiation since Melampyrum became very abundant with time. Ustilago-infected plants received far fewer visits but because of its long flowering time the proportion of open flowers receiving visits was still high. Viscaria flowers received significantly more visits than flowers of other species when bumblebees made heterospecific flower visits from Ustilago-infected plants; thus Ustilago spores were probably effectively dispersed from infected to healthy plants by the pollinators. The mechanism behind competition for pollination in this system was competition through pollinator preference, since the visitation rate to Viscaria actually decreased, but also competition through improper pollen transfer (grains of both species were found on the bodies of bumblebees) since the incidence of switching between the two species increased, probably resulting in an increased misplacement of conspecific pollen grains with time.     

Phylogeography and cryptic variation within the Lacerta viridis complex (Lacertidae, Reptilia)

It is well known that the current genetic pattern of many European species has been highly influenced by climatic changes during the Pleistocene. While there are many well known vertebrate examples, knowledge about squamate reptiles is sparse. To obtain more data, a range‐wide sampling of Lacerta viridis was conducted and phylogenetic relations within the L. viridis complex were analysed using an mtDNA fragment encompassing part of cytochrome b, the adjacent tRNA genes and the noncoding control region. Most genetic divergence was found in the south of the distribution range. The Carpathian Basin and the regions north of the Carpathians and Alps are inhabited by the same mitochondrial lineage, corresponding to Lacerta viridis viridis. Three distinct lineages occurred in the south‐eastern Balkans — corresponding to L. v. viridis, L. v. meridionalis, L. v. guentherpetersi— as well as a fourth lineage for which no subspecies name is available. This distribution pattern suggests a rapid range expansion of L. v. viridis after the Holocene warming, leading to a colonization of the northern part of the species range. An unexpected finding was that a highly distinct genetic lineage occurs along the western Balkan coast. Phylogenetic analyses (Bayesian, maximum likelihood, maximum parsimony) suggested that this west Balkan lineage could represent the sister taxon of Lacerta bilineata. Due to the morphological similarity of taxa within the L. viridis complex this cryptic taxon was previously assigned to L. v. viridis. The distribution pattern of several parapatric, in part highly, distinct genetic lineages suggested the existence of several refuges in close proximity on the southern Balkans. Within L. bilineata sensu stricto a generally similar pattern emerged, with a high genetic diversity on the Apennine peninsula, arguing for two distinct refuges there, and a low genetic diversity in the northern part of the range. Close to the south‐eastern Alps, three distinct lineages (L. b. bilineata, L. v. viridis, west Balkan taxon) occurred within close proximity. We suggest that the west Balkan lineage represents an early offshoot of L. bilineata that was isolated during a previous Pleistocene glacial from the more western L. bilineata populations, which survived in refuges on the Apennine peninsula.     

Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota

Most mucosal surfaces of the mammalian body are colonized by microbial communities (“microbiota”). A high density of commensal microbiota inhabits the intestine and shields from infection (“colonization resistance”). The virulence strategies allowing enteropathogenic bacteria to successfully compete with the microbiota and overcome colonization resistance are poorly understood. Here, we investigated manipulation of the intestinal microbiota by the enteropathogenic bacterium Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in a mouse colitis model: we found that inflammatory host responses induced by S. Tm changed microbiota composition and suppressed its growth. In contrast to wild-type S. Tm, an avirulent invGsseD mutant failing to trigger colitis was outcompeted by the microbiota. This competitive defect was reverted if inflammation was provided concomitantly by mixed infection with wild-type S. Tm or in mice (IL10^−/−, VILLIN-HA^CL4-CD8) with inflammatory bowel disease. Thus, inflammation is necessary and sufficient for overcoming colonization resistance. This reveals a new concept in infectious disease: in contrast to current thinking, inflammation is not always detrimental for the pathogen. Triggering the host's immune defence can shift the balance between the protective microbiota and the pathogen in favour of the pathogen.     

U7 snRNAs: a computational survey

U7 small nuclear RNA (snRNA) sequences have been described only for a handful of animal species in the past. Here we describe a computational search for functional U7 snRNA genes throughout vertebrates including the upstream sequence elements characteristic for snRNAs transcribed by polymerase II. Based on the results of this search, we discuss the high variability of U7 snRNAs in both sequence and structure, and report on an attempt to find U7 snRNA sequences in basal deuterostomes and non-drosophilids insect genomes based on a combination of sequence, structure, and promoter features. Due to the extremely short sequence and the high variability in both sequence and structure, no unambiguous candidates were found. These results cast doubt on putative U7 homologs in even more distant organisms that are reported in the most recent release of the Rfam database.