The use of the periplasmic alkaline phosphatase (PhoA) reporter protein from E. coli has been critical for definition of the topology of transmembrane proteins of multiple bacterial species. This report demonstrates development of a PhoA reporter system in B. burgdorferi. Codon usage of the E. coli phoA in B. burgdorferi was analyzed and an optimized version of the gene was obtained. In order to assess the differential activity of the reporter system, two optimized PhoA-fusion construct using B. burgdorferi proteins were engineered: one using the periplasmic protein OppAIV and one using the cytoplasmic protein PncA. The activity of PhoA requires periplasmic localization. The periplasmic OppAIV-PhoA fusion as well as the cytoplasmic PncA-PhoA fusion produced detectable PhoA protein in E. coli and in B. burgdorferi. The periplasmic fusion construct, but not the cytoplasmic fusion construct, resulted in functional alkaline phosphatase (AP) activity in E. coli, as observed by blue colonies on agar plates containing a chromogenic substrate for AP. In contrast, both of the fusion constructs produced limited detectable levels of functional alkaline phosphatase activity in B. burgdorferi, as observed by yellow color change in liquid protein lysate containing a chromogenic substrate for AP. Development of a PhoA fusion reporter system for use in B. burgdorferi will provide a new molecular genetics tool for analyzing the topology of B. burgdorferi transmembrane proteins. These types of studies are critical for understanding the function of B. burgdorferi transport systems and may identify novel molecular approaches for the treatment of Lyme disease.

Biologiska läkemedel är stora molekyler som har renats eller producerats från ett biologiskt ursprung. De används idag i stor utsträckning runt om i världen på grund av deras effektivitet att minska symtomen som kan uppstå vid olika mycket svåra sjukdomar. Detta arbete fokuserar på biologiska läkemedel som hämmar cytokinet tumörnekrotisk faktor (TNF). Läkemedlen används bland annat vid svåra autoimmuna sjukdomar. Vid autoimmuna sjukdomar aktiveras cellerna i immunsystemet vilket leder till utsöndring av olika cytokiner till exempel interleukin 1 och TNF. Dessa cytokiner påverkar kroppen på olika sätt genom att aktivera eller inhibera vissa produktionsprocesser i kroppen. Syftet med projektet var att verifiera en cellbaserad metod som används för koncentrationsmätning av det biologiska läkemedlet Infliximab (en TNF antagonist) i patientserum. Metoden som används i detta projekt kallas för reporter gene assay och den baseras på användning av en iLite cellinje. iLite är humana erytroleukemiceller K562 som är transfekterade med nukleär faktor kappa-B. Denna faktor aktiveras när TNF binder sig till TNF receptorer på cellytan. Aktiveringen av denna faktor leder till produktion av enzymet Eldflugeluciferas. Produktionen av enzymet är indirekt proportionell mot koncentrationen av TNF antagonist i serum. Dessutom innehåller cellerna en reporter gen för Renilla luciferas som uttrycks konstant. Renilla luciferas används som en internkontroll för att normalisera variationer som kan orsakas av provhantering samt transfektionseffektivitet. Luminescensen som utvecklas på grund av inverkan av Eldflugeluciferas på luciferas substrat samt inverkan av Renilla luciferas på stopp substrat mäts med hjälp av en luminometer. Dag-till-dag variationen för fem analyserade prov som analyserades vid fyra olika tillfällen var mellan 5 - 28 %, lot-till-lot variationen för fyra prov som analyserades fyra gånger vid fyra olika tillfällen var mellan 14 - 21 %. Alla normala serumprover från friska frivilliga blodgivare var negativa med en koncentration av Infliximab mindre än 0,65 µg/ ml. Resultaten visar att metoden är stabil och känslig jämfört med Biomonitors resultat. Dessutom visar resultaten att medelvärden av de olika körningarna från Euro Diagnostica (ett laboratorium som tillverkar immunologiska kit, Malmö) korrelerar väl med resultaten från Biomonitors laboratorium i Köpenhamn. Korrelationen för 19 analyserade prov var 0,99. / Biological drugs are large molecules that have been purified from or produced from a biological origin. They are used in great extent around the whole world because of their efficiency in reducing the symptoms of very severe diseases. The present work focus on biological drugs that inhibit tumor necrosis factor (TNF). Biological drugs are used for treatment of different autoimmune diseases. In autoimmune diseases activated cells of the immune system leads to secretion of various cytokines such as interleukin 1 and TNF. These cytokines affect the body in different ways by activating or inhibiting certain processes in the body. The purpose of this project was to verify a cell-based method which used for concentration measurement of the biological drug Infliximab (a TNF antagonist) in patient serum. The method which used in this project is called the reporter gene assay, and it is based on the use of an iLite cell line. iLite are human erythroleukemia K562 cells which are transfected with the nuclear factor kappa-B. This factor is activated when the tumor necrosis factor binds to its receptors on the cell surface. The activation of this factor leads to the production of the enzyme Firefly luciferase. Production of the enzyme is indirectly proportional to the concentration of TNF antagonist in the serum. Moreover, the cells contain a reporter gene for Renilla luciferase which is under constant expression. Renilla luciferase is used as an internal control to normalize the variations that may be caused by sample handling and transfection efficiency. The luminescence developed due to the influence of Firefly luciferase on luciferase substrate and Renilla luciferase on stop substrate is measured by using a luminometer. Day-to-day variations for five samples were analyzed at four different times were between 5 - 28 % while variations of lot-to-lot of 4 samples were analyzed at four different times were 14 - 21 %. The concentration of Infliximab in all the serum samples from healthy volunteer blood donors were less than 0.65 µg / ml. The results show that the method is robust and sensitive as compared to Biomonitors results. Furthermore, the results show that the mean value of the different runs from Euro Diagnostica (a laboratory that produces immunological kits, Malmö) for day-to-day and lot-to-lot variation agrees quite well with those from Biomonitors laboratory in Copenhagen. The correlation for the nineteen analyzed samples was 0.99.

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According to the Centers for Disease Control and Prevention (CDC), 8 million people each year are infected with Mycobacterium tuberculosis (Mtb) leading to 1.5 million deaths annually. This staggering number calls for advancements in understanding this bacterium so progress can be made in treating and preventing the disease. It is particularly important to understand mechanisms by which TB survives inside hostile host immune cells known as macrophages and within hypoxic granuloma lesions of the lung. Preliminary microarray data has shown that a TB gene known as Rv2633c is induced upon macrophage invasion. Bioinformatic analysis of Rv2633c coding sequence shows the product of Rv2633c has homology with hemerythrin-like proteins. Hemerythrins are a class of proteins commonly used to bind oxygen and sense nitric oxide and iron, leading us to hypothesize a role for Rv2633c in surviving hypoxic or nitrosative stress encountered within macrophages and granulomas. My first aim will be to generate a reporter strain of Mycobacterium smegmatis (Msm) expressing the mCherry fluorescent protein driven by the Rv2633c promoter. This tool will allow us to determine the stress conditions (i.e. hypoxia, nitric oxide treatment, acid pH) that activate expression of this gene by measuring the change in fluorescence. Linking the regulation of Rv2633c to specific environmental cues relevant to infections in vivo will provide insight into the role of this unique protein. Secondly, a knockout mutant of Rv2633c in the attenuated M. bovis BCG will be constructed and characterized to determine the importance and function of this protein during TB infections.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a growing problem worldwide due to the emergence of multi-drug resistant and extensively-drug resistant strains of the bacteria. A key to combatting the spread of these strains lies in the understanding of gene expression occurring in Mtb. This study focuses on the development and optimization of a luciferase-based bioluminescent transcriptional reporter that can be used to monitor gene expression in Mtb. The luminescent signal emitted from the reporter can be measured and correlated with the level of transcription of certain genes. This study focuses specifically on a gene called whiB7 which encodes a transcription factor known to contribute to the drug resistance of Mtb. The drug-inducible whiB7 promoter was cloned into various locations in the luciferase plasmid in order to determine the ideal configuration of the reporter for maximum luminescence. The optimized luciferase reporter was then compared with a fluorescent transcriptional reporter, mCherry, also under control of the whiB7 promoter. Fluorescent reporters present some disadvantages including delayed kinetics and inability to accurately reflect gene downregulation due to long half-life of reporter proteins. It was hypothesized that the luciferase reporter would solve these problems by offering a more sensitive and dynamic tool to monitor gene expression. Quantitative real-time PCR was used to measure whiB7 mRNA present in cultures containing either the luciferase or mCherry reporters. The luminescent and fluorescent signal given from these reporters was then compared to actual mRNA expression. It was observed that the signal from the luciferase reporter more closely matched mRNA expression at each timepoint, indicating that the luciferase reporter is a better gauge of actual gene expression levels than the mCherry reporter.

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