Bacterial modulation of host glycosylation - in infection, biotechnology, and therapy

A majority of the proteins of the immune system are glycosylated and the glycans of IgG are essential for its functionality. Bacteria display enzymes that modulate the glycans of the immune system to weaken the host defense and favor bacterial survival. In this thesis we aimed at exploring bacterial modulation of host glycosylation in infection and to evaluate the usefulness of bacterial enzymes in biotechnology and for therapeutic use. The role of IgG endoglycosidase EndoS in streptococcal virulence was evaluated in a murine model of invasive infection and we found significant contribution when heterologously expressed. We also discovered and characterized EndoS2, a novel enzyme specific and conserved in serotype M49 of streptococci, with enzymatic activity on the glycans of IgG and α1-acid glycoprotein. Enterococcal pathogenesis was studied, and we found that the endoglycosidase EndoE cleaved glycans of lactoferrin to reduce the antibacterial functions and to support bacterial growth. A glycoform specificity difference between EndoS and EndoS2 was observed, and we suggested a method for quantification of high-mannose glycans on therapeutic antibodies, a key quality attribute. Finally, we explored the importance of Fc glycosylation of IgE and showed that EndoS cleaved glycans of this immunoglobulin causing a reduction of the immune cell activation in vivo, a potential new therapeutic strategy for severe IgE mediated allergies. In this thesis we demonstrate that glycans are an integral part of the immune system, and that the study of bacterial effectors of glycosylation paves the way for a deeper understanding of infections, for novel tools supporting the biotech arena, and for new therapeutic strategies

Differences in somatic embryogenesis initiation rates between individual trees of Norway spruce

Gran (Picea abies) har länge varit ett viktigt trädslag för den svenska skogsnäringen. Skogsbruket står inför en oviss framtid och med ett alltmer ostadigt klimat behövs ett genetiskt material som kan klara dessa nya prövningar. Målet är ett stort utbud av granmaterial med egenskaper som god tillväxt, högklassig virkeskvalitet och den alltmer nödvändiga förmågan att stå emot skadeinsekter och patogena svampar. En del i detta arbete för granens framtid är somatisk embryogenes (SE) som är en form av vegetativ förökning. SE är i sig en kostsam och tidskrävande process vilket gör att behovet av att välja ut träd med god initieringsframgång är av högsta prioritet. I denna studie undersöktes individer tillhörande olika trädfamiljer, gällande deras kapacitet i SE-initieringsförmåga. Resultatet från studien kommer utgöra en grund för att i ett annat projekt undersöka om initieringsfrekvensen kan sammankopplas (associeras) med molekylära markörer i en ’Genome-Wide Association Study (GWAS)’. Studien ingår därmed i ett forskningsprojekt ’GWAS: Marker-based earlier selection for high somatic embryo-initiation in Norway spruce’ för den efterföljande associationsstudien. Projektet är finansierat av ’UPSC Komptenscentrumprojekt’ tillsammans med Umeå Plant Science Centre (UPSC), Sveriges lantbruksuniversitet, Skogforsk och SweTree Technologies. Materialet till projektet utgjordes av omogna embryon från grankottar från olika moderträd plockade i klonarkiv i Ekebo (Svalöv) samt Maltesholm (Tollarp). Försöken genomfördes i Skogsmästarskolans laboratorium i Skinnskatteberg sommaren 2022. Under åtta veckor testades 101 trädindivider gällande deras initieringsförmåga. Resultaten kontrollerades därefter var tredje och var sjätte vecka och all information sammanställdes i en databas. Utvecklingsstadiet hos de zygotiska embryona (fröembryon) var av betydelse för resultaten. Små och outvecklade embryon var svårhanterliga för den laborativa processen vilket försvårade extraktionsprocessen. Förvaringen av kottarna hade en avgörande betydelse och lagringsförhållandena bör förbättras inför framtida studier. Problem med mögel, luftfuktighet och angrepp av sjukdomar och larver gav ett delvis defekt arbetsmaterial vilket påverkade den totala mängden användbart datamaterial som kunde samlas in. Skillnader kunde observeras mellan trädindivider gällande initieringsfrekvens. Av studiens 101 träd visade 66 träd initiering. Av de 35 träd där initiering helt uteblev hade embryon från 13 träd dock testats på ett icke optimalt sätt. Knappt en tredjedel (30 procent) av de 66 träden uppvisade lyckad initiering på en nivå mellan 10 och 20 procent av testade embryon. Något mer än en tredjedel (36 procent) uppvisade en initieringsandel på 30 till 40 procent. För knappt en tredjedel (33 procent) av träden registrerades en initieringsnivå på över 40 procent av. Endast ett testat träd hade 100 procent lyckade initieringar.Norway spruce (Picea abies) is an important tree species in Swedish forestry. With the uncertain future that forestry faces in an increasingly unstable climate, there is a need for genetic material that can withstand these new challenges. The goal is to secure good supply of Norway spruce genetic material and further improve traits such as optimal growth, high timber quality, and ability to resist pathogens and insect attacks. One potentially important part of the work of securing the future of Norway spruce in commercial forestry is somatic embryogenesis (SE), a form of vegetative propagation. SE is still a costly and time-consuming process, making the need to select trees with good initiation capacity a top priority. This study examined the SE initiation capacity of different tree families. The results will be utilized in a different study investigating whether initiation ability could be associated with certain molecular markers in a Genome-Wide Association Study (GWAS). The study was performed as one component within the project ‘GWAS: Marker-based earlier selection for high somatic embryo initiation in Norway spruce’. The project was financed by ‘UPSC Conpetence Centre’ in collaboration with Umeå Plant Science Centre (UPSC), The Swedish University of Agricultural Sciences, Skogforsk (the Forestry Research Institute of Sweden), and SweTree Technologies. The investigated material consisted of immature embryos from seed of Norway spruce cones harvested from mother trees collected in the clone archives Ekebo (Svalöv) and Maltesholm (Tollarp). The experiment was carried out in the laboratory at the SLU School for Forest Management in Skinnskatteberg during the summer of 2022. During eight weeks 101 trees were tested regarding their initiation ability and thereafter the development was monitored every three and six weeks. The recorded information was compiled in a database. The stage of development of the zygotic embryos (seed embryos) had a major impact on the results; small and undeveloped embryos were difficult to handle in the laboratory process and made the embryo extraction process much more difficult. Storage conditions negatively affected parts of the cone material and should be further improved in future studies. Problems with mold, non-optimal humidity, and infestation of diseases and larvae resulted in partly defective sample material for a subset of trees, which affected the total amount of usable data obtained in this study. Differences were observed regarding initiation ability of different trees. Of the total 101 trees in the study, 66 trees showed initiation. However, of the 35 trees that failed to initiate at all, embryos from 13 trees had been tested in a sub-optimal way. Slightly less than one third of the trees (30 percent) showed initiation rates of 10 to 20 percent of tested embryos. More than one-third (36 percent) showed initiation rates of 30 to 40 percent. For one-third (33 percent) of the trees, initiation rates were above 40 percent. Only one tested tree had 100 percent successful initiations

Study of the IgG endoglycosidase EndoS in group A streptococcal phagocyte resistance and virulence

<p>Abstract</p> <p>Background</p> <p>The secreted enzyme EndoS, an endoglycosidase from <it>Streptococcus pyogenes</it>, hydrolyzes the <it>N</it>-linked glycan of the constant region of immunoglobulin G (IgG) heavy chain and renders the antibody unable to interact with Fc receptors and elicit effector functions. In this study we couple targeted allelic replacement mutagenesis and heterologous expression to elucidate the contribution of EndoS to group A <it>Streptococcus </it>(GAS) phagocyte resistance and pathogenicity <it>in vitro </it>and <it>in vivo</it>.</p> <p>Results</p> <p>Knocking out the EndoS gene in GAS M1T1 background revealed no significant differences in bacterial survival in immune cell killing assays or in a systemic mouse model of infection. However, exogenous addition and heterologous expression of EndoS was found to increase GAS resistance to killing by neutrophils and monocytes <it>in vitro</it>. Additionally, heterologous expression of EndoS in M49 GAS increased mouse virulence <it>in vivo</it>.</p> <p>Conclusions</p> <p>We conclude that in a highly virulent M1T1 background, EndoS has no significant impact on GAS phagocyte resistance and pathogenicity. However, local accumulation or high levels of expression of EndoS in certain GAS strains may contribute to virulence.</p

Turning universal O into rare Bombay type blood

Red blood cell antigens play critical roles in blood transfusion since donor incompatibilities can be lethal. Recipients with the rare total deficiency in H antigen, the Oh Bombay phenotype, can only be transfused with group Oh blood to avoid serious transfusion reactions. We discover FucOB from the mucin-degrading bacteria Akkermansia muciniphila as an -1,2-fucosidase able to hydrolyze Type I, Type II, Type III and Type V H antigens to obtain the afucosylated Bombay phenotype in vitro. X-ray crystal structures of FucOB show a three-domain architecture, including a GH95 glycoside hydrolase. The structural data together with site-directed mutagenesis, enzymatic activity and computational methods provide molecular insights into substrate specificity and catalysis. Furthermore, using agglutination tests and flow cytometry-based techniques, we demonstrate the ability of FucOB to convert universal O type into rare Bombay type blood, providing exciting possibilities to facilitate transfusion in recipients/patients with Bombay phenotype

Regulation of learning and memory by meningeal immunity: a key role for IL-4

Proinflammatory cytokines have been shown to impair cognition; consequently, immune activity in the central nervous system was considered detrimental to cognitive function. Unexpectedly, however, T cells were recently shown to support learning and memory, though the underlying mechanism was unclear. We show that one of the steps in the cascade of T cell–based support of learning and memory takes place in the meningeal spaces. Performance of cognitive tasks led to accumulation of IL-4–producing T cells in the meninges. Depletion of T cells from meningeal spaces skewed meningeal myeloid cells toward a proinflammatory phenotype. T cell–derived IL-4 was critical, as IL-4−/− mice exhibited a skewed proinflammatory meningeal myeloid cell phenotype and cognitive deficits. Transplantation of IL-4−/− bone marrow into irradiated wild-type recipients also resulted in cognitive impairment and proinflammatory skew. Moreover, adoptive transfer of T cells from wild-type into IL-4−/− mice reversed cognitive impairment and attenuated the proinflammatory character of meningeal myeloid cells. Our results point to a critical role for T cell–derived IL-4 in the regulation of cognitive function through meningeal myeloid cell phenotype and brain-derived neurotrophic factor expression. These findings might lead to the development of new immune-based therapies for cognitive impairment associated with immune decline

Genomewide meta-analysis identifies loci associated with IGF-I and IGFBP-3 levels with impact on age-related traits

The growth hormone/insulin-like growth factor (IGF) axis can be manipulated in animal models to promote longevity, and IGF-related proteins including IGF-I and IGF-binding protein-3 (IGFBP-3) have also been implicated in risk of human diseases including cardiovascular diseases, diabetes, and cancer. Throug

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Bacterial glycosidases in pathogenesis and glycoengineering.

ABSTRACT Glycosylation is a common post-translational protein modification and many key proteins of the immune system are glycosylated. As the true experts of our immune system, pathogenic bacteria produce enzymes that can modify the carbohydrates (glycans) of the defense mechanisms in order to favor bacterial survival and persistence. At the intersection between bacterial pathogenesis and glycobiology, there is an increased interest in studying the bacterial enzymes that modify the protein glycosylation of their colonized or infected hosts. This is of great importance in order to fully understand bacterial pathogenesis, but it also presents itself as a valuable source for glycoengineering and glycoanalysis tools. This article highlights the role of bacterial glycosidases during infections, introduces the use of such enzymes as glycoengineering tools and discusses the potential of further studies in this emerging field