Browsing by Subject "gramnegatiivinen bakteeri"

Bacteria can communicate with each other using phenomenon called quorum sensing (QS). In QS the bacteria produce and release small signaling molecules which they use to communicate. Bacteria use QS in situations where it is beneficial to act on population level. QS has an important role e.g. in the formation of virulence factors and biofilms. There are several different QS systems. Gram-negative bacteria use i.a AI-1, AI-2, AI-3, and CAI-1 systems to communicate. All QS systems are based on the accumulation of signaling molecules when the bacterial concentration increases. When the concentration of signal molecules reaches the threshold level, the system activates. The activation of the signaling system then activates the expression of the genes controlled by the QS system. AI-2 signaling is assumed to be universal. That means that bacteria can use AI-2 signaling system in interspecies communication. In AI-2 signaling bacteria produce and release 4,5-dihydroxy-2,3-pentanedione (DPD) which works as a signaling molecule in the AI-2 system. Escherichia coli and Salmonella typhimurium use an ATP binding cassette ABC-type transporter to transport DPD molecules into the cell where LsrK kinase phosphorylates the DPD molecules. The phosphorylated DPD molecules bind to the LsrR regulator protein which acts as a suppressor of the lsr operon. The binding of the phosphorylated DPD molecules releases the LsrR from the lsr promoter region enabling the expression of the lsr genes. In Vibrio harveyi the surface proteins LuxP and LuxQ form a protein complex that recognizes DPD molecules. When the DPD concentration increases, the LuxPQ complex transform from kinase to phosphatase and the reaction chain, where LuxU phosphate transfer protein transfers a phosphate group from LuxO regulator protein, activates. The dephosphorylation of of LuxO releases the LuxR transcription factor and activates the expression of QS controlled genes. The aim of this thesis was to optimize two assays which can be used to screen for compounds that disrupt AI-2 signaling. The first assay was a bioreporter based assay where V. harveyi BB120 bioreporter strain was used. The second assay was protein based LsrK assay where the LsrK activity was monitored using assay kit which measures the concentration of ATP or ADP. The concentrations of bacteria, LsrK, and DPD used in the assays were optimized. The dimethyl sulfoxide (DMSO) tolerance of both assays were tested, the stability of the kits used in the LsrK assays was tested and the reaction buffer for the LsrK assay was selected from the two tested buffer options. The selected bacterial concentration for the V. harveyi BB120 assay was 100000 CFU/ml and DPD concentration 1 µM. The selected enzyme concentration for the LsrK assay was 300 nM and DPD concentration 300 µM. The tested DMSO concentrations had no effect on the kit measuring ATP but the highest concentrations tested had a small effect on the kit measuring ADP. A buffer containing triethanolamine, magnesium chloride, and bovine serum albumin was selected as the reaction buffer for the LsrK assay. Using the optimized LsrK assay, a screening was performed for a synthesized compound library. None of the compounds showed any LsrK inhibiting activity. The optimized assay was also used to make dose-response experiment to one LsrK inhibiting compound, named FIMM000642, which was found in a separate screening. The FIMM000642 dose-response as-say was also done against glycerol kinase to see if the compound would inhibit another enzyme from the same protein family or if the compound was a specific inhibitor to LsrK. FIMM000642 inhibited also the activity of glycerol kinase.