Borrelia burgdorferi bb0426 encodes a 2′-deoxyribosyltransferase that plays a central role in purine salvage

Borrelia burgdorferi is an obligate parasite with a limited genome that severely narrows its metabolic and biosynthetic capabilities. Thus survival of this spirochaete in an arthropod vector and mammalian host requires that it can scavenge amino acids, fatty acids and nucleosides from a blood meal or various host tissues. Additionally, the utilization of ribonucleotides for DNA synthesis is further complicated by the lack of a ribonucleotide reductase for the conversion of nucleoside-5′-diphosphates to deoxynucleosides-5′-diphosphates. The data presented here demonstrate that B. burgdorferi must rely on host-derived sources of purine bases, deoxypurines and deoxypyrimidines for the synthesis of DNA. However, if deoxyguanosine (dGuo) is limited in host tissue, the enzymatic activities of a 2′-deoxyribosyltransferase (DRTase, encoded by bb0426), IMP dehydrogenase (GuaB) and GMP synthase (GuaA) catalyse the multistep conversion of hypoxanthine (Hyp) to dGMP for DNA synthesis. This pathway provides additional biochemical flexibility for B. burgdorferi when it colonizes and infects different host tissues

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric “RCR-complex”. We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called “R78C”, combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial

Bacterial enzymes in thymidylate synthesis

Deoxyribonucletides (dNTPs) are synthesized via the de novo and salvage pathways. The de novo synthesis involves initial synthesis of ribonucleotides whereas the salvage pathway uses deoxyribonucleosides. Deoxynucleoside kinases (dNKs) initiate the salvage pathway and this step is often considered as rate limiting. The two pathways eventually coincide to one where nucleoside monophosphate kinases are the shared enzymes. In this thesis two thymidine kinases (TKs) and a thymidylate kinase (TMPK) are cloned, expressed and characterized and they originate from Ureaplasma urealyticum (Uu) and Bacillus anthracis (Ba). Uu causes urethritis and is associated with complications in pregnancy, e.g. premature births, infertility, spontaneous abortions and chronic lung disease in the infants. Ba causes anthrax; cutaneous, inhalational and gastrointestinal, where the two latter have a high mortality rate. Ba is also considered as a potent bioweapon because of its spore forming ability. TK from Uu (Uu-TK) was strictly pyrimidine specific and used all nucleoside triphosphates as phosphate donors, except dTTP that was a feedback inhibitor. Further studies discovered that analogs with halogen substitutions at the 5-position gave the highest activity. Analogs with modifications at the N3- or 3’-position showed good to moderate activities while 2’-substituents were not substrates. A fluorine substitution was tolerated in the 2’-arabinosyl position. These results correlated well with the active site structure of Uu-TK. The Uu-TK structure contained a unique domain, the lasso domain with a structural zinc ion, and belonged structurally to another enzyme family than the other dNKs. TK and TMPK from Ba (Ba-TK and Ba-TMPK) were strictly pyrimidine and thymidylate specific, respectively. Ba-TK used all nucleoside triphosphates as phosphate donors, except dTTP, and phosphorylated several nucleoside analogs. The analog activities of Ba-TK were similar to that of Uu-TK. Ba-TMPK used ATP and dATP as phosphate donors and a number of analogs as substrates. FMAUMP (1-(2-deoxy-2-fluoro-D-arabinofuranosyl)-5-methyluracil-5’-monophosphate) was the best substrate and its nucleoside form was a potent inhibitor. Enzymes in thymidylate synthesis are potential targets for antibacterial therapy and the studies conducted in this thesis have discovered several potential leads, which will contribute to future design of antibiotics

Marginal zone B cells are naturally reactive to collagen type II and are involved in the initiation of the immune response in collagen-induced arthritis

Antibodies against type II collagen (CII) are essential for development of collagen-induced arthritis (CIA), but how and where the B-cell response to CII is initiated is not fully known. We show here that naive DBA/1 mice display naturally reactive IgM and IgG anti-CII producing B cells prior to immunization. The CII-reactive B cells were observed in the spleen and recognized as marginal zone (MZ) B cells. After CII immunization, CII-specific B cells expanded rapidly in the spleen, in contrast to the lymph nodes, with the initial response derived from MZ B cells and later by follicular (FO) B cells. This was evident despite that the MZ B cells were subject to stringent tolerance mechanisms by having a greater Fc gamma receptor IIb expression than the FO B cells. Further, the MZ B cells migrated to the FO areas upon immunization, possibly providing antigen and activating FO T cells and subsequently FO B cells. Thus, around CIA onset increased numbers of IgG anti-CII producing FO B cells was seen in the spleen, which was dominated by IgG2a- and IgG2b-positive cells. These data demonstrate that CII-reactive MZ B cells are present before and expand after CII immunization, suggesting an initiating role of MZ B cells in the development of CIA.Manuscript original title: Marginal zone B cells are naturally reactive to collagen type II and initiate the immune response in collagen-induced arthriti

Metacognition and Discourse Processing

Darbā tiek aplūkots psiholingvistikas pielietojums diskursa uztverē. Diskusija ir balstīta uz metakognitīvu stratēăiju izpēti ‘domāšanas artikulācijas’ protokolos, kur tiek atspoguļotas kognitīvas un metakognitīvas darbības teksta izpratnes laikā. Darba mērķis ir pierādīt, ka diskursa apstrādes laikā aktīvais monitorings noved pie aktīva metakognitītvo stratēģiju, kuras tieši iespaido sapratnes procesu, pielietojuma kognitīvo procesu kontrolei. Darbā ir izmantotas šādas metodes: atbilstošas literatūras analīze;‘domāšanas artikulācijas’ protokolu un diskursa analīzes kvalitatīvās metodes, balstītas uz Tīrnija un Pērsona (1983) un Flauera un Haisa (1981) izstrādātām struktūrām; un kvantitatīvās statistiskas metode. Gan teorētiskajā, gan praktiskajā daļā iegūtie dati ir apkopoti darba nobeigumā.The present paper deals with the application of psycholinguistic approach to the analysis of discourse processing. The discussion is based on researching metacognitive strategies in think aloud protocols reflecting cognitive and metacognitive activities during the session of text comprehension. The research is aimed to prove that during discourse processing active monitoring leads to control over cognitive processes through active use of metacognitive strategies, which have a direct impact on the process of comprehension. The research methods applied are: the analysis of relevant literature, the qualitative methods of think-aloud protocol and discourse analysis, based on Tierney and Pearson’s (1983) and Flower and Hayes’s (1981) frameworks, and the quantitative statistical method. All the findings of both theoretical and practical research are summarized in the concluding part of the research

Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase

Bacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis (Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5′-monophosphate (dTMP) efficiently with K m and V max values of 33 μM and 48 μmol mg−1 min−1, respectively. The efficiency of deoxyuridine-5′-monophosphate phosphorylation was ∼10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2′-fluoroarabinosyl-5-methyldeoxyuridine-5′-monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but l-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5′-monophosphate (5FdUMP) and 2′,3′-dideoxy-2′,3′-didehydrothymidine-5′-monophosphate (d4TMP). No activity could be detected with 3′-azidothymidine-5′-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and d-FMAU was a strong inhibitor with an IC50 value of 10 μM, while other nucleosides—l-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2′-arabinosylthymidine—were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents

Protection against collagen-induced arthritis in mice afforded by the parasitic worm product, ES-62, is associated with restoration of the levels of IL-10-producing B cells and reduced plasma cell infiltration of the joints

We have previously reported that ES-62, a molecule secreted by the parasitic filarial nematode Acanthocheilonema viteae, protects mice from developing collagen-induced arthritis (CIA). Together with increasing evidence that worm infection may protect against autoimmune conditions, this raises the possibility that ES-62 may have therapeutic potential in Rheumatoid Arthritis and hence, it is important to fully understand its mechanism of action. Towards this, we have established to date that ES-62 protection in CIA is associated with suppressed Th1/Th17 responses, reduced collagen-specific IgG2a antibodies and increased IL-10 production by splenocytes. IL-10-producing regulatory B cells (Bregs) have been proposed to suppress pathogenic Th1/Th17 responses in CIA: interestingly therefore, whilst the levels of IL-10-producing B cells were decreased in the spleens of mice with CIA, ES-62 was found to restore these to the levels found in naive mice. In addition, exposure to ES-62 decreased effector B cell, particularly plasma cell, infiltration of the joints, and such infiltrating B cells showed dramatically reduced levels of TLR4 and the activation markers, CD80 and CD86. Collectively, this induction of hyporesponsiveness of effector B cell responses, in the context of the resetting of the levels of IL-10-producing B cells, is suggestive of a modulation of the balance between effector and regulatory B cell responses that may contribute to ES-62-mediated suppression of CIA-associated inflammation and inhibition of production of pathogenic collagen-specific IgG2a antibodies