Charakterisierung von Genen des Zellwandstoffwechsels (Rv1500, hddA) und der Nitratassimilation (glnR) von Mycobacterium tuberculosis

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Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼10$^{2}$ colony forming units (CFUs) and ∼10$^{3}$ CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity

Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases

Tuotannonohjausjärjestelmän uudistaminen

Tämä toiminnallinen opinnäytetyö tehtiin toimeksiantona KavoKerr Finland oy:lle. Tavoitteena oli saada valitulle tuotantolinjalle käyttöön kokonaan uusi tuotannonohjausjärjestelmä ja samalla muokata linjaa siten, että sen toiminta olisi mahdollisimman tehokasta uuden järjestelmän kanssa. Linjan nykyisessä käytössä oleva järjestelmä on Microsoft Access pohjainen tietokanta, joka ei vastaa tämän päivän vaatimuksia nopeassa tahdissa toimivalle tuotantolinjalle. Se ei myöskään sisällä riittävää tuotteen seurattavuutta eri tuotantovaiheiden aikana. Teoriaosassa esitellään yleisesti tuotannonohjausjärjestelmien toimintatapaa ja sitä, miten niiden käyttäminen edistää tuotannon toimivuutta. Lisäksi käydään läpi kohdeyrityksessä käytössä olevaa lean-filosofiaa. Tähän liittyen erityisesti standardoidut työtavat sekä virheiden estämiseen suunnattu poka-yoke ovat työn keskiössä. Toiminnallisen osan aiheena on varsinaisen suunnitelman tekemistä ja siihen liittyvien osa-alueiden läpikäyntiä. Suunnitelmaan sisältyy varsinaisen tuotantolinjan muokkaamista koskevat toimet, sekä käyttöönotettavan tuotannonohjausjärjestelmän konfigurointiin ja ohjelmointiin liittyviä asioita. Lopputuloksena syntyvät suunnitelmat mahdollistavat tuotantolinjan siirtämisen uuteen järjestelmään

Enolase of Streptococcus pneumoniae Binds Human Complement Inhibitor C4b-Binding Protein and Contributes to Complement Evasion.

Streptococcus pneumoniae (pneumococcus) is a pathogen that causes severe local and life-threatening invasive diseases, which are associated with high mortality rates. Pneumococci have evolved several strategies to evade the host immune system, including complement to disseminate and to survive in various host niches. Thus, pneumococci bind complement inhibitors such as C4b-binding protein (C4BP) and factor H via pneumococcal surface protein C, thereby inhibiting the classical and alternative complement pathways. In this study, we identified the pneumococcal glycolytic enzyme enolase, a nonclassical cell surface and plasminogen-binding protein, as an additional pneumococcal C4BP-binding protein. Furthermore, we demonstrated that human, but not mouse, C4BP bound pneumococci. Recombinant enolase bound in a dose-dependent manner C4BP purified from plasma, and the interaction was reduced by increasing ionic strength. Enolase recruited C4BP and plasminogen, but not factor H, from human serum. Moreover, C4BP and plasminogen bound to different domains of enolase as they did not compete for the interaction with enolase. In direct binding assays with recombinant C4BP mutants lacking individual domains, two binding sites for enolase were identified on the complement control protein (CCP) domain 1/CCP2 and CCP8 of the C4BP α-chains. C4BP bound to the enolase retained its cofactor activity as determined by C4b degradation. Furthermore, in the presence of exogenously added enolase, an increased C4BP binding to and subsequently decreased C3b deposition on pneumococci was observed. Taken together, pneumococci specifically interact with human C4BP via enolase, which represents an additional mechanism of human complement control by this versatile pathogen