Die Speicheldrüsen der Schmeißfliege Calliphora vicina produzieren bei Stimulierung mit dem Neurohormon Serotonin (5-Hydroxytryptamine, 5-HT) einen KCl-reichen Primärspeichel. Der transepitheliale K+-Transport wird durch eine apikal lokalisierte vakuoläre H+-ATPase (V-ATPase) energetisiert. Stimulierung der Speicheldrüsen mit 5-HT aktiviert die apikale V-ATPase, die Protonen aus der Zelle in das Drüsenlumen transportiert. Trotz des auswärts gerichteten Protonentransportes führt die 5-HT-Stimulierung kurioserweise zu einer intrazellulären Ansäuerung. Die Ursachen dieser 5-HT-induzierten Ansäuerung waren unzureichend untersucht. Deshalb war das Ziel dieser Arbeit die Identifikation aller Transporter, die an der intrazellulären pH-(pHi)-Regulation in unstimulierten Speicheldrüsen von Calliphora vicina beteiligt sind und an der Entstehung und Regulation der 5-HT-induzierten pHi-Änderungen mitwirken. Von besonderem Interesse war hierbei die funktionelle Mitwirkung der V-ATPase, deren Beteiligung an der pHi-Regulation in tierischen Zellen bisher wenig untersucht war. Wesentliche Ergebnisse dieser Arbeit waren: • Messungen des pHi-Wertes in der unstimulierten Drüse zeigten, dass vor allem die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter an der Aufrechterhaltung des Ruhe-pHi beteiligt sind. • Zur Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung (NH4Cl-Vorpuls) tragen ebenfalls im Wesentlichen die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter bei. Der Na+/H+-Antiporter hat in der unstimulierten Drüse keinen messbaren Einfluss auf den Ruhe-pHi. • Die Wiederherstellung des Ruhe-pHi nach einer intrazellulären Alkalisierung (Na-acetat-Vorpuls) ist Cl--abhängig, aber auch unter extremen Bedingungen waren die Zellen noch in der Lage sich vollständig von einer intrazellullären Alkalisierung zu erholen. Einen entscheidenden Anteil daran hat offenbar die hohe intrazelluläre Puerkapazität. • Ein Na+-abhängiger Glutamat-Transporter ist per se kein pHi-regulierender Transporter, seine Aktivität hat jedoch Einfluss auf den Ruhe-pHi in der unstimulierten Speicheldrüse von Calliphora vicina. • 10 nM 5-HT induzieren in den Calliphora Speicheldrüsen eine intrazelluläre Ansäuerung. An dieser Ansäuerung ist der Na+/H+-Antiporter entscheidend beteiligt. Auch eine klare Cl--Abhängigkeit der 5-HT-induzierten Ansäuerung konnte beobachtet werden. Wahrscheinlich ist eine gekoppelte Aktivität von Na+/H+-Antiporter und Cl-/HCO3--Antiporter. • Messungen mit einem O2-empndlichen Fluoreszenzfarbstoff zeigten, dass Stimulierung der Speicheldrüsen mit 5-HT die Zellatmung aktivierte. Der cAMP- und der IP3/Ca2+-Weg tragen auf komplexe Weise zu der 5-HT-induzierten Aktivierung der Zellatmung und damit auch zu den 5-HT-induzierten pHi-Änderungen bei. • Mit molekularbiologischen Untersuchungen ist es gelungen den Na+-abhängigen Glutamat-Transporter, den Na+/H+-Antiporter, die Carboanhydrase und die Untereinheit C der V-ATPase in den Calliphora Speicheldrüsen direkt nachzuweisen. Zudem konnte erstmals der direkte Nachweis für die Expression eines nH+/K+-Antiporters in den Speicheldrüsen von Calliphora vicina erbracht werden. Diese Arbeit trug ganz wesentlich zum Verständnis der pHi-Regulation in der unstimulierten und stimulierten Speicheldrüse von Calliphora vicina bei. Mechanismen die zur Aufrechterhaltung und Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung bzw. Alkalisierung beitragen, konnten mit pHi-Messungen und auch molekularbiologisch nachgewiesen werden. Die Mechanismen, welche die 5-HT-induzierte intrazelluläre Ansäuerung verursachen, konnten ebenfalls aufgeklärt werden. Zudem wurde an den Calliphora Speicheldrüsen eine neue optische Methode zur Messung des O2-Verbrauchs in tierischen Geweben etabliert. / The tubular salivary glands of the blowfly Calliphora vicina consist of a single layer of epithelial cells. Stimulation with the neurohormone serotonin (5-hydroxytryptamine,5-HT) induces the secretion of a KCl-rich primary saliva. Transepithelial K+-transport is energized by a vacuolar-type H+-ATPase (V-ATPase) which is located in the apical membrane. 5-HT stimulates the apical V-ATPase which transports protons out of the cells into the lumen of the glands. Despite this outward directed proton transport, 5-HT stimulation leads to an intracellular acidication. The causes of this intracellular acidication were poorly understood. Therefore the aim of this thesis was the identication of all pHi regulating transporters which are involved in pHi regulation in the unstimulated salivary glands of Calliphora vicina and which contribute to the 5-HT-induced pHi changes. Of special interest was the functional role of the V-ATPase,whose contribution to pHi regulation in animal cells is, as yet, not well studied. Key results were: • pHi measurements in unstimulated glands showed that mainly the V-ATPase and at least one Na+-dependent HCO3--transporter are involved in maintenance of resting pHi. • V-ATPase and at least one Na+-dependent HCO3--transporter are also necessary for the recovery from an intracellular acidication (NH4Cl prepulse). • Recovery from an intracellular alkali load (Na-acetate prepulse) is partially Cl--dependent. • A Na+ dependent gluatamate-transporter is present in Calliphora salivary glands and its activity aects the resting pHi. • 10 nM 5-HT induce an intracellular acidication. This acidication is Na+-dependent, EIPA-sensitive and also Cl--dependent. No DIDS-sensitivity was observed. A coupled activity of a Na+/H+-antiporter and a Cl-/HCO3- -antiporter was suggested. • Using O2-sensitive fluorescent microbeads I could show that 5-HT stimulation of the Calliphora salivary glands activates cellular respiration. The cAMP and Ca2+-signalling pathways contribute in a complex manner to the 5-HT-induced activation of cellular respiration and consequently, also to the 5-HT-induced intracellular acidication. • The expression of a Na+ dependent glutamate-transporter, a Na+/H+-antiporter, a carbonic anhydrase, subunit C of the V-ATPase and a nH+/K+-antiporter were determined on mRNA level by RT-PCR. This thesis contributes signicantly to the understanding of pHi regulation in unstimulated and stimulated salivary glands of Calliphora vicina. Mechanisms which contribute to the maintenance and recovery of resting pHi were identied by using pHi measurements and molecular biological techniques. Mechanisms which are responsible for the 5-HT-induced intracellular acidication were also clarified. Furthermore a new optical method for measuring O2 consumption in animals cells was established by using the Calliphora salivary glands as a model.

In order to establish colonisation of a human host, pathogenic Yersinia use a type III protein secretion system to directly intoxicate host immune cells. Activation of this system requires target cell contact and is a highly regulated process. Both the intoxication and regulation events depend on the lcrGVHyopBD translocon operon, which is highly conserved in many bacterial pathogens. In this study, the role of individual operon members was analysed and functional domains identified by using the highly homologous pcrGVHpopBD operon of P. aeruginosa as a comparative tool. Yersinia spp. and P. aeruginosa were shown to form translocation pores of a similar size that promoted equally efficient protein delivery. A strong dependency on interactions between native translocator(s) in protein delivery was revealed, suggesting that each pathogen has delicately fine-tuned this process to suit its own infection niche. In particular, the C-terminus of YopD was shown to possess functional specificity for effector delivery in Yersinia that could not be conferred by the comparable region in homologous PopD. Moreover, a role for LcrV and PcrV in substrate recognition during the protein delivery process was excluded. The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in Yersinia, while equivalent complexes with analogous proteins were not formed in P. aeruginosa. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to Yersinia. Finally, PcrH was identified as a new member of the translocator class of chaperones, being essential for assembly of a functional PopB/PopD mediated translocon in P. aeruginosa. However, in contrast to the other members of this family, PcrH was dispensable for type III regulation. Moreover, both LcrH and PcrH were shown to possess tetratricopeptide repeats crucial for their chaperone function. One tetratricopeptide repeat mutant in LcrH was even isolated that failed to secrete both YopB and YopD substrates, even though stability was maintained. This demonstrates for the first time that LcrH has a role in substrate secretion in addition to its critical role in promoting substrate stability.

Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca2+ concentration ([Ca2+]o) in the body. The secretion of this hormone is suppressed at high [Ca2+]o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca2+ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca2+]o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca2+]o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca2+]o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca2+]o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca2+-independent. The magnitude of neomycin-induced release of Ca2+ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca2+ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca2+]o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca2+ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.

Francisella tularensis is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. F. tularensis can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of F. tularensis: the highly virulent tularensis (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent holarctica (Type B) and mediasiatica, and the essentially avirulent subspecies F. novicida. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between F. tularensis subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of F. tularensis, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the Francisella pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in Neisseria gonorrhoeae. pilA was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to F. tularensis and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (pilA) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them in cis could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia.

The type III secretion system (T3SS) is a highly conserved secretion system among Gram negative bacteria that translocates anti-host proteins directly into the infected cells to overcome the host immune system and establish a bacterial infection. Yersinia pseudotuberculosis is one of three pathogenic Yersinia spp. that use a plasmid encoded T3SS to establish an infection. This complex multi-component Ysc-Yop system is tightly regulated in time and space. The T3SS is induced upon target cell contact and by growth in the absence of calcium. There are two kinds of substrates for the secretion apparatus, the translocator proteins that make up the pore in the eukaryotic target cell membrane, and the translocated effector proteins, that presumably pass through this pore en route to the eukaryotic cell interior. The essential YopD translocator protein is involved in several important steps during effector translocation, such as pore formation, effector translocation. Moreover, in complex with its cognate chaperone LcrH, it maintains regulatory control of yop gene expression. To understand the molecular mechanism of YopD function, we made sequential in-frame deletions throughout the entire protein and identified discrete functional domains that made it possible to separate the role of YopD in translocation from its role in pore formation and regulation, really supporting translocation to be a multi-step process. Further site-directed mutagenesis of the YopD C-terminus, a region important for these functions, revealed no function for amino acids in the coiled-coil domain, while hydrophobic residues within the alpha-helical amphipathic domain are functionally significant for regulation, pore formation and translocation of effectors. Unique to the T3SSs are the chaperones which are required for efficient type III protein secretion. The translocator-class chaperone LcrH binds two translocator proteins, YopB and YopD, which is necessary for their pre-secretory stabilization and their efficient secretion. We have shown that LcrH interacts with each translocator at a unique binding-site established by the folding of its three tandem tetratricopeptide repeats (TPRs). Beside the regulatory LcrH-YopD complex, LcrH complexes with YscY, a component of the Ysc-Yop T3SS, that is also essential for regulatory control. Interestingly the roles for LcrH do not end here, because it also appears to function in fine tuning the amount of effector translocation into target cells upon cell contact. Moreover, LcrH’s role in pre-secretory stability appears to be an in vitro phenomenon, since upon bacteria-host cell contact we found accumulated levels of YopB and YopD inside the bacteria in absence of a LcrH chaperone. This suggests the true function of LcrH is seen during target cell contact. In addition, these stable YopB and YopD are secreted in a Ysc-Yop independent manner in absence of a functional LcrH. We propose a role for LcrH in conferring substrate secretion pathway specificity, guiding its substrate to the cognate Ysc-Yop T3SS to secure subsequent effector translocation. Together, this work has sought to better understand the key functions of LcrH and YopD in Yersinia pathogenicity. Using an approach based heavily on recombinant DNA technology and tissue culture infections, the complex molecular cross-talk between chaperone and its substrate, and the effect this has on the Yersinia lifestyle, are now being discovered.

Bacterial genomes provide records of the molecular processes associated with emergence and evolution of different bacterial lifestyles. This thesis is based on whole-genome comparisons within the genus Bartonella, an excellent model system for studies of host- and vector-specificity and infection outcome in animal-associated bacteria. The louse-borne human specialist and trench fever agent Bartonella quintana was contrasted to the flea-borne generalist relatives Bartonella henselae and Bartonella grahamii, which cause asymptomatic infection in cat and mouse respectively. While B. henselae is commonly isolated from humans, and causes cat scratch disease, there is only one reported case of B. grahamii human infection. The gene complements of the three species are nested like Russian dolls with the smaller genome (B. quintana) being entirely contained in the medium sized (B. henselae), which in turned is contained in the largest (B. grahamii). Size differences reflect differences in the horizontally and vertically acquired gene content, and in the number of genus- and species- specific genes, owing to differential impact of bacteriophages and plasmids, and to different degrees of genome decay. These processes can be attributed to the three distinct lifestyles. Comparisons with other alpha-proteobacteria suggest that the Bartonella genus as a whole evolved from plant-associated species, and that horizontal transfer, in particular of genes involved in interaction with the host, played a key role in the transition to animal intracellular lifestyle. The long-term genome decay associated with this lifestyle is most advanced in the host-restricted B. quintana. The broad host-range species B. grahamii has the largest genome and the largest proportion of auxiliary DNA of the three, probably because it has access to a larger gene pool. In encodes all the known pathogenicity determinants found in the genomes of B. henselae and B. quintana, suggesting that these genes primarily evolved to facilitate colonization in the reservoir host.

cAMP and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting pancreatic β-cells. Glucose-stimulated insulin secretion from β-cells is pulsatile and driven by oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the kinetics of cAMP signaling and the mechanisms of cAMP action. Evanescent wave microscopy and fluorescent translocation biosensors were used to monitor plasma membrane-related signaling events in single MIN6-cells and primary mouse β-cells. Glucose stimulation of insulin secretion resulted in pronounced oscillations of the membrane phospholipid PIP3 caused by autocrine activation of insulin receptors. Glucose also triggered oscillations of the sub-plasma membrane cAMP concentration ([cAMP]pm). These oscillations were preceded and enhanced by elevations of [Ca2+]i, but conditions raising cytoplasmic ATP triggered [cAMP]pm elevations without accompanying changes in [Ca2+]i. The [cAMP]pm oscillations were also synchronized with PIP3 oscillations and both signals were suppressed after inhibition of adenylyl cyclases. Protein kinase A (PKA) was important for promoting concomitant initial elevations of [cAMP]pm and [Ca2+]i, and PKA inhibitors diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The glucose-induced PIP3 oscillations were markedly suppressed in cells treated with siRNA against the cAMP-dependent guanine nucleotide exchange factor Epac2. Pharmacological activation of Epac restored PIP3 responses after adenylyl cyclase or PKA inhibition. Glucose and other cAMP-elevating stimuli induced redistribution of fluorescence-tagged Epac2 from the cytoplasm to the plasma membrane. This translocation was modulated by [Ca2+]i and depended on intact cyclic nucleotide-binding and Ras-association domains. In conclusion, glucose generates cAMP oscillations in β-cells via a concerted action of Ca2+ and metabolically generated ATP. The oscillations are important for the magnitude and kinetics of insulin secretion. While both protein kinase A and Epac is required for initiation of insulin secretion the cAMP-dependence of established pulsatility is mediated by Epac2.

In order to establish an extra-cellular infection the gram-negative bacteria Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to translocate a set of anti-host effectors into eukaryotic cells. The toxins disrupt signalling pathways important for phagocytosis, cytokine production and cell survival. Secretion and translocation via this T3SS is strictly regulated on several levels. In this context, the function of YopE and LcrH during Yersinia infections has been analysed. YopE is an essential translocated effector that disrupts the actin cytoskeleton of infected eukaryotic cells, by inactivating small GTPases through its GTPase activating protein (GAP) activity. However, cytotoxicity can be uncoupled from in vitro GAP activity towards the RhoA, Rac1 and Cdc42 GTPases. Furthermore, in vivo studies of the YopE GAP activity revealed that only RhoA and Rac1 are targeted, but this is not a pre-requisite for Yersinia virulence. Hence, YopE must target one or more additional GTPases to cause disease in mice. YopE was the only Yersinia effector that blocks LDH release from infected cells. Moreover, translocated YopE could regulate the level of subsequent effector translocation by a mechanism that involved the YopE GAP function and another T3S component, YopK. Loss of translocation control elevated total T3S gene expression in the presence of eukaryotic cells. This indicated the existence of a regulatory loop for feedback control of T3S gene expression in the bacteria that originates from the interior of the eukaryotic cell after effector translocation is completed. This might represent the true virulence function of YopE. Exoenzyme S (ExoS) of Pseudomonas aeruginosa has a YopE-like GAP domain with similar activity towards RhoA, Rac1 and Cdc42. However, ExoS is unable to complement hyper-translocation resulting from loss of YopE. This indicates a unique function for YopE in translocation control in Yersinia that might be dependent on correct intracellular localisation. It follows that the Membrane Localisation Domain in YopE was important for translocation control, but dispensable for cytotoxicity and blockage of LDH release. YopD and its cognate chaperone LcrH are negative regulatory elements of the T3S regulon and together with YopB, are involved in the effector translocation process. Randomly generated point mutants in LcrH specifically effected stability and secretion of both the YopB and YopD substrates in vitro and prevented their apparent insertion as translocon pores in the membranes of infected cells. Yet, these mutants still produced stable substrates in the presence of eukaryotic cells and most could mediate at least partial effector translocation. Thus, only minimal amounts of the YopB and YopD translocator proteins are needed for translocation and the LcrH chaperone may regulate this process from inside the bacteria.

The increasing use of antibiotics has led to a huge problem for society, as some bacteria have developed resistance towards many of the antibiotics currently available. To help find solutions to this problem we studied small molecules that inhibit bacterial virulence, the ability to cause disease. The type III secretion system (T3SS) is a conserved virulence system found in several gram-negative bacteria, including human and plants pathogens, such as Yersinia spp., Pseudomonas aeruginosa, Chlamydia spp., Salmonella spp., Shigella spp, enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), and Erwinia spp. One class of virulence-blocking compounds is the salicylidene acylhydrazides. They were first identified in a screen towards the T3SS in Yersinia pseudotuberculosis and have since been shown to block the T3SS in a panel of gram-negative bacteria such as Chlamydia spp. Salmonella enterica, Shigella flexneri and EPEC. We designed and synthesized a library of 58 salicylidene acylhydrazides and evaluated their activity as virulence-blocking compounds in Y. pseudotuberculosis followed by calculations of quantitative structure activity relationships (QSARs). Four QSAR models were calculated, and when used in consensus they correctly classified between five out of eight compounds for Y. pseudotuberculosis as active or inactive and six out of eight compounds for C. trachomatis. Since the target and mode of action of the salicylidene acylhydrazides were unknown, we used solution and solid phase synthesis to synthesize three different affinity reagents. One of these affinity reagents was used in affinity chromatography experiments, where 19 putative target proteins from an E. coli O157 bacterial lysate were identified. We studied four of the proteins, Tpx, WrbA, FolX, and AdhE, in more detail in Y. pseudotuberculosis and E. coli O157. We believe that the salicylidene acylhydrazides act on multiple targets that together result in down-regulation of T3SS functions. A knockout of AdhE in E. coli O157 showed a similar phenotype as salicylidene acylhydrazide treated E. coli, suggesting that this protein may be particularly interesting as a drug target. Many of the antibiotics used today originate form natural sources. In contrast, most virulence-blocking compounds towards the T3SS are small synthetic organic molecules. Therefore, a prefractionated natural product library with marine and terrestrial biota samples was screened towards the T3SS in Y. pseudotuberculosis. Neohopeaphenol A was identified as a hit and shown to have micromolar activity towards Y. pseudotuberculosis and P. aeruginosa in cell-based infection models. / Det ökande användandet av antibiotika har lett till stora problem för samhället. Många bakterier har utvecklat resistens mot de antibiotika som finns tillgängliga. För att försöka hitta en möjlig lösning på detta problem, arbetar vi med en strategi där vi med hjälp av små organiska molekyler inhiberar bakteriernas virulenssystem, deras förmåga att orsaka sjukdom. Traditionella antibiotika är antingen, bakteriocida, avdödande eller bakteriostatiska, tillväxthämmande. Bakteriernas enda sätt för att överleva antibiotikabehandlingen är att utveckla resistens. Forskarvärlden tror att molekyler som inhiberar bakteriernas virulenssystem, leder till ett minskat tryck att utveckla resistens mot dessa molekyler, eftersom de inte dödar eller hämmar bakterietillväxten, utan bara avväpnar bakterierna. Typ III sekretionssystemet är ett virulenssystem som finns i många gram-negativa bakterier, t.ex., Yersinia spp., Pseudomonas aeruginosa, Chlamydia spp., Salmonella spp., Shigella spp, enteropatogena Escherichia coli (EPEC) och Erwinia spp. Salicylidenacylhydraziderna är en substansklass virulensblockare som inhiberar typ III sekretionssystemet i de ovan nämnda bakterierna. I denna avhandling har vi designat och syntetiserat ett bibliotek med 58 salicylidenacylhydrazider och utvärderat deras biologiska aktivitet som virulensblockare i Y. pseudotuberculosis. Vi relaterade den biologiska aktiviteten till de kemiska egenskaperna hos salicylidenacylhydraziderna i kvantitativa strukturaktivitetssamband. Med hjälp av dessa samband kunde vi prediktera och validera aktiviteten till aktiv eller inaktiv för fem av åtta nya salicylidenacylhydrazider i Y. pseudotuberculosis och sex av åtta i C. trachomatis. Eftersom verkningsmekanismen för salicylidenacylhydraziderna var okänd, så syntetiserade vi tre olika affinitetsmolekyler med kombinerad lösnings- och fastfas-syntes. En av affinitetsmolekylerna användes sedan för att ”fiska ut” och identifiera 19 potentiella målproteiner i ett bakterielysat från E. coli. Fyra av dessa proteiner, TpX, WrbA, FolX och AdhE har vi studerat vidare i Y. pseudotuberculosis och E. coli. Utifrån resultaten tror vi att salicylidenacylhydraziderna interagerar med flera proteiner som tillsammans resulterar i en nedreglering av type III sekretionssystemen. Vår samarbetspartner, Andrew Roe och hans forskargrupp (Universitetet i Glasgow), har studerat AdhE i E. coli.  De har visat att E. coli som saknar genen för proteinet AdhE, har samma fenotyp som E. coli behandlad med salicylidenacylhydraziderna, d.v.s. ett nedreglerat T3SS, vilket gör AdhE till ett speciellt intressant målprotein. I jämförelse med många av våra nuvarande antibiotika som har ett naturligt ursprung så är de flesta studerade virulensblockare små syntetiska organiska molekyler. Därför testades en stor kollektion av naturprodukter från marina och landlevande växter och invertebrater från Sydostasien, för att hitta nya inhibitorer mot typ III sekretionssystemet i Y. pseudotuberculosis. Neohopeaphenol A som kommer från barken på Hopea hainanensis, ett träd som växer i sydostasiens regnskogar, identifierades som en ny virulensblockare. Neohopeaphenol A visade sig vara en potent virulensblockare i in vitro infektionsförsök med Y. psudotuberkulosis eller Pseudomonas aeruginosa. Forskningen i denna avhandling visar att virulensblockare kan hjälpa oss att förstå hur bakterier orsakar sjukdom. Förhoppningsvis kan det i framtiden leda till nya typer av läkemedel mot infektionssjukdomar.

The cadA gene in Dictyostelium encodes a unique Ca2+-dependent cell adhesion molecule DdCAD-1. It is synthesized as a soluble protein in the cytoplasm and then transported to the plasma membrane by contractile vacuoles. The solution structures of Ca2+-free and Ca2+-bound DdCAD-1 reveals that it contains two β-sandwich domains, belonging to the βγ-crystallin and immunoglobulin fold classes, respectively. Whereas the N-terminal domain has a major role in homophilic binding, the C-terminal domain tethers the protein to the cell membrane. Although hydrophobic interactions constitute the major force for adhesion, electrostatic interactions may act as a ‘switch’ to regulate the homophilic binding by a change in electrostatic potential caused by the binding of Ca2+ to the three binding sites. To further investigate DdCAD-1 transport, DdCAD-1-GFP fusion proteins were expressed in cadA-null cells. Time-lapse microscopy revealed that DdCAD-1 was imported by invagination of the contractile vacuole membrane. The N-terminal, C-terminal domains, and two of the three Ca2+-binding site mutant forms of DdCAD-1 failed to enter the contractile vacuole, suggesting that Ca2+-binding and the integrity of DdCAD-1 are required for import. Indeed, proteins with altered conformation failed to enter the contractile vacuole, indicating that the import signal is integrated in the three-dimensional structure of DdCAD-1. Finally, we describe how the cadA gene acts as a single-gene green beard. In chimera experiments, cells expressing DdCAD-1 were more likely to form fruiting bodies than cadA-null cells on soil plates. Here cadA behaved as a single gene green beard. However, cadA exhibited anti-green beard behaviour on non-nutrient agar plates. Wild-type cells differentiated mostly into prestalk cells and eventually died, whereas the cadA-null cells survived as spores. DdCAD-1 was enriched in cell-cell contact regions of anterior cells, while it was mostly localized in the cytoplasm of posterior cells. The presence of DdCAD-1 on the cell surface of prestalk cells is crucial for cell sorting, which in turn explain the anti-green beard effect observed in chimeras containing cadA+ and cadA- cells. These observations demonstrate that DdCAD-1 plays a direct role in cell sorting through differential cell-cell adhesion which results from the differential distribution of DdCAD-1.