<p>In order to establish colonisation of a human host, pathogenic <i>Yersinia</i> 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 <i>lcrGVHyopBD </i>transloco<i>n</i> 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 <i>pcrGVHpopBD</i> operon of <i>P. aeruginosa</i> as a comparative tool. </p><p><i>Yersinia</i> spp. and<i> P. aeruginosa </i>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 <i>Yersinia</i> 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. </p><p>The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in <i>Yersinia</i>, while equivalent complexes with analogous proteins were not formed in <i>P. aeruginosa</i>. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to <i>Yersinia</i>. </p><p>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 <i>P. aeruginosa</i>. 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.</p>

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.

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.

Simple gel formulations may be applied to enhance the systemic and local exposure of potential compounds. The aim of this thesis is the development and characterization of controlled release formulations based on thermo-reversible poloxamer gels, which are suitable for novel drug delivery applications.  In particular co-solvents (DMSO, ethanol), mucoadhesive polymers (chitosan, alginate) and salts (sodium tripolyphosphate, CaCl2) have been used to enhance the applications of poloxamer 407 (P407) formulations in preclinical animal studies. The impact of these additives on the micellization and gelation properties of P407 aqueous solutions was studied by calorimetric methods, nuclear magnetic resonance spectroscopy (NMR) and “tube inversion” experiments. The drug release behavior of hydrophobic and hydrophilic drugs was characterized by using a membrane/membrane-free experimental setup. Finally, preliminary pharmacokinetic studies using a mouse model were conducted for screening of selected inhibitors of bacterial type III secretion and for evaluation of different formulations including P407 gel. All additives, used here, reduced the CMTs (critical micelle temperature) of dilute P407 solutions, with the exception of ethanol. The gelation temperature of concentrated P407 solutions was lowered in the presence of CaCl2, DMSO, TPP and alginate. 1H MAS (Magic Angle Spinning) NMR studies revealed that DMSO influences the hydrophobicity of the PPO segment of P407 polymers. Low concentrations of DMSO did not show any major effect on the drug release from P407 gels and may be used to improve the exposure of lead compounds in poloxamer gels. A newly developed in situ ionotropic gelation of chitosan in combination with TPP in P407 gels showed an enhanced resistance to water and reduced the release rates of model drugs. From preliminary pharmacokinetic studies in mice it was revealed that poloxamer formulations resulted in an increased plasma half-life of the lead compound.

The type III secretion system (T3SS) of pathogenic Yersinia pseudotuberculosis is involved in virulence. The syringe-like secretion system spans both bacterial membranes and is responsible for the ability of Yersinia to transfer toxic proteins (Yop proteins) into the eukaryotic target cell. The T3SS is believed to have evolved from the flagellum and regulation of the T3SS is a complex event that involves a series of regulatory proteins, whereby two are YscP and YscU. In a regulatory model, called the substrate specificity switch, both proteins act together to ensure proper T3SS structure and function by regulating a stop in YscF needle protein export with a shift to Yop effector secretion. YscU undergoes autoproteolysis at a conserved motif consisting of amino acids Asparagine-Proline-Threonine-Histidine (NPTH). Processing generates a C-terminal 10 kDa peptide, YscUCC. Processing is crucial for proper T3SS regulation and function both in vitro and in vivo. Full-length YscU does not support Yop secretion and after cleavage, YscUCC remains attached to the rest of YscU and acts as a negative block on T3S. Relief of this negative block is suggested to occur through displacement of YscUCC from the rest of YscU. Thorough control of many different cellular processes is brought by the heat shock proteins (HSPs) DnaK and DnaJ. Due to their multiple regulatory functions, mutations in the hsp-genes lead to pleiotropic effects. DnaK and DnaJ are essential for proper flagellum driven motion of bacteria, but more so; they ensure proper Yersinia T3SS function in vivo. Furthermore, DnaJ interacts with YscU and may be directly involved in T3SS regulation. Virulence of Yersinia is regulated on many levels. A previously identified virulence associated protein, VagH, is now characterized as an S-adenosyl-methionine dependent methyltransferase. The targets of the methylation activity of VagH are release factors 1 and 2 (RF1 and RF2), that are important for translation termination. The enzymatic activity of VagH is important for Yop secretion and a vagH mutant up-regulates a T3SS negative regulatory protein, YopD. Furthermore, a vagH mutant is avirulent in a mouse infection model, but is not affected in macrophage intracellular survival. The importance of VagH in vivo makes it a possible target for novel antimicrobial therapy.

Shigella flexneri attacks cells of the intestinal tract, causing over 1 million deaths annually from bacterial dysentery. A type III secretion system (T3SS) initiates the host-pathogen interaction and transports virulence factors directly into host cells via a needle complex (NC) comprising an extracellular needle and membrane-spanning basal body. Rings formed by the single-pass membrane proteins MxiG and MxiJ are arranged concentrically within the inner membrane ring (IMR) of the NC. The Neterminal domain of MxiG (MxiG-N) is the predominant IMR cytoplasmic structure, however it was structurally and functionally uncharacterised. Determination of the solution structure of MxiG-N in this study revealed it to be a forkhead associated (FHA) domain, although subsequent analyses of conserved residues suggested it does not have the canonical role in cell-signalling via phospho-threonine recognition. Subsequent positioning of the structure in the electron microscopy (EM) density for the S. flexneri NC supported models with 24-fold symmetry in the IMR. Both MxiG and MxiJ also have significant periplasmic domains, which were purified to homogeneity in this study, facilitating preliminary characterisation of their structures and intermolecular interactions. In addition, the entire IMR within the context of intact basal bodies was isolated and visualised in vitro by EM. The essential function of MxiG-N could be to localise the putative cytoplasmic ring (Cering) at the base of the T3SS. Although absolutely required for secretion, the Csring component, Spa33, was structurally uncharacterised. The crystal structure of the Cvterminal domain of Spa33 (Spa33-C) was determined in this study, showing an intertwined dimer that aligned with homologous structures and exhibited a novel interaction with the N-terminus of the ATPase regulator, MxiN. Subsequently, Spa33-C was identified as an altemative translation product of spa33 that formed a 2: 1 complex with Spa33 in vitro. This complex oligomerised further, demonstrating for the first time that Spa33 has the propensity to form the ordered, high molecular weight assemblies that would be required for C-ring formation in S. flexneri.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that has evolved to take advantage of the eukaryotic host cells it inhabits during infection. It uses bacterial effectors translocated into the host cell cytosol to manipulate host cell machinery and establish a replicative niche. In this thesis I study the function of two of these effectors, SopD and SopB, which have been shown to act cooperatively to induce phenotypes associated with gastroenteritis (fluid secretion and neutrophil influx into the intestinal lumen). In addition to promoting gastroenteritis, SopD has also been implicated in systemic and persistent infection of mice. Although recently implicated in invasion, the precise function of SopD has remained elusive. Here I show that SopD affects membrane dynamics during S. Typhimurium invasion of epithelial cells. SopD promotes membrane sealing and macropinosome formation, events that may have important consequences for efficiency of bacterial cell entry in vivo. Furthermore, we demonstrate that SopD is recruited to the invasion site membranes through the phosphatase activity of SopB, suggesting a mechanism for their cooperative action during induction of gastroenteritis. Unlike SopD, SopB has been a focus of intense research efforts and its role in invasion as a phosphoinositide phosphatase is well documented. However, we have observed that SopB also inhibits fusion of lysosomes with Salmonella-containing vacuoles (SCVs) following invasion. This ability depends on SopB-mediated reduction of negative membrane charge of the SCV during invasion by hydrolysis of the phosphoinositide PI(4,5)P2. Membrane charge alterations driven by SopB result in removal of Rab GTPases from the SCV that depend on electrostatic interactions for their targeting. Two of these Rabs, Rab23 and Rab35 were previously shown to promote phagosome-lysosome fusion. Therefore their removal from the SCV may promote SCV trafficking away from the degradative endocytic pathway of host cells. This represents a new mechanism by which an invasion associated effector controls SCV maturation. Together, this work advances our knowledge of the interaction between S. Typhimurium and its host. This research also suggests a new mechanism by which pathogens other than S. Typhimurium could promote their intracellular survival.

Despite the large arsenal of antibiotics available on the market, treatment of bacterial infections becomes more challenging in view of the fact that microbes develop resistance against existing drugs. There is an obvious need for novel drugs acting on both old and new targets in bacteria. In this thesis we have employed a whole cell bacterial assay for screening and identification of type III secretion system (T3SS) inhibitors in Yersinia pseudotuberculosis. The T3SS is a common virulence mechanism utilized by several clinically relevant Gram-negative bacteria including Salmonella, Shigella, Pseudomonas aeruginosa, Chlamydiae and Escherichia coli. Several components in the T3SS have proved to be conserved and hence data generated with Y. pseudotuberculosis as model might also be valid for other bacterial species. We have screened a 9,400 commercial compound library for T3S inhibitors in Y. pseudotuberculosis using a yopE reporter gene assay. The initial ~ 30 hits were followed up in a growth inhibition assay resulting in 26 interesting compounds that were examined in more detail. Three of the most interesting compounds, salicylanilides, 2-hydroxybenzylidene-hydrazides and 2-arylsulfonamino-benzanilides, were selected for continued investigations. The inhibitor classes show different profiles regarding the effects on T3SS in Yersinia and their use as research tools and identification of the target proteins using a chemical biology approach will increase our understanding of bacterial virulence. The 2-hydroxybenzylidene-hydrazides have been extensively studied in vitro and show potential as selective T3S inhibitors in several Gram-negative pathogens besides Y. pseudotuberculosis. The data obtained suggest that this inhibitor class targets a conserved protein in the secretion apparatus. In cell-based ex vivo infection models T3SS was inhibited to the advantage of the infected eukaryotic cells. The salicylanilides and 2-arylsulfonamino-benzanilides have been further investigated by statistical molecular design (SMD) followed by synthesis and biological evaluation in the T3SS linked reporter gene assay. Multivariate QSAR models were established despite the challenges with data obtained from assays using viable bacteria. Our results indicate that this SMD QSAR strategy is powerful in development of virulence inhibitors targeting the T3SS.

Cette thèse est composée de deux parties : Le première partie rend compte de l'étude structurale de la protéine RrgA. RrgA est associée au pilus du pathogène Streptococcus pneumoniae et participe aux premières étapes de colonisation chez l'hôte en se liant à plusieurs composés de la Matrice Extra Cellulaire. Nous avons résolu la structure de cette protéine à 1.9 Å par cristallographie aux rayons-X. RrgA possède une structure allongée formée de quatre domaines alignés d'origine eucaryote et procaryote. En effet, trois domaines ayant des similarités structurales avec les IgG et le domaine Cna-B semblent servir de piédestal pour orienter et présenter le domaine fonctionnel de type Intégrine. Nous avons confirmé la formation de deux ponts isopeptidiques stabilisateurs par spectrométrie de masse. De plus, le domaine intégrine possède deux insertions particulières dont la présence pourrait être impliquée dans la reconnaissance des divers substrats par RrgA. La deuxième partie de cette thèse est axée sur l'étude structurale du complexe ATPase et de ExsB, la pilotine présumée du système de sécrétion de type III chez Pseudomonas aeruginosa, bactérie opportuniste et jouant un rôle majeur dans l'infection des patients atteints de mucoviscidose. Pour la première fois, nous avons mis au point un protocole d'expression et de purification sous forme soluble de l'ATPase PscN en complexe avec une protéine partenaire, PscL. Des cristaux de ce complexe ont été obtenus au robot du PSB. Par ailleurs, nous avons confirmé l'expression de la lipoprotéine ExsB chez P. aeruginosa que nous avons localisée au sein de la membrane externe. De plus, nous avons résolu la structure de cette protéine qui présente un nouveau repliement et qui établie les bases structurales pour l'étude des pilotines pour tous les systèmes de sécrétion de type III de la famille Ysc. / This manuscript is made up of two parts The first part describes the structural study of RrgA from Streptococcus pneumoniae. This protein is a pilus-associated adhesin that is able to bind to several components of the Extra Cellular Matrix and thus, participates in the first steps of host colonization. We solved the structure of RrgA to 1.9 Å by X-Ray crystallography. We showed that RrgA folds into an elongated 4-domain structure, and these domains display both eukaryotic and prokaryotic origins. Actually, three out of the four domains are reminiscent of IgG and Cna-B structures and act like stalks to orient and display the large Integrin-like domain. We confirmed the presence of two isopeptide bonds by mass spectrometry and hypothesised that the two inserted arms in the integrin domain could explain the wide variety of substrates RrgA can bind. The second part of this manuscript focuses on the structural studies of the ATPase complex as well as ExsB, the putative pilotin of the type III secretion system from Pseudomonas aeruginosa. This bacterium is a major threat in hospital-acquired infections and the main pathogen found in cystic-fibrosis suffering patients. For the first time we were able to express and purify the ATPase PscN in complex with its partner PscL. Crystallization trials led to a very promising condition that is being refined. Moreover, we confirmed expression of the lipoprotein ExsB in P. aeruginosa that we localised in the outer membrane. To have a better understanding of this protein, we also solved its high-resolution structure that displays a novel fold and our study paves the way for coming studies concerning pilotins.