Avväpning av bakterier : ett strukturellt tillvägagångssätt för utvecklingen av ett anti-virulent läkemedel mot Listeria monocytogenes

Antibiotic resistances are one of the biggest threats to global health and if we don’t change our behavior and way of using antibiotics we will end up in a ‘post-antibiotic era’, in which common infections and minor injuries can once kill again and up to 10 million deaths per year may occur by 2050. Therefore, there is a high need for new anti-bacterial drugs, especially of alternatives to existing antibiotics with already described resistances. Classical antibiotics target the essential processes of survival and growth in bacteria and therefore put a high selective pressure on them to develop resistances. In contrast, the ability to infect or damage a host, the virulence, is less essential for bacteria. Thus, targeting the virulence is supposed to cause a lower selective pressure and this alternative mode-of-action could help to decelerate the development of antibiotic resistances. The aims in this work were to proceed with the structure-based design of an anti-virulence drug against the food-borne pathogen Listeria monocytogenes, but also to deepen our understanding of the complex regulation system for the virulence of this bacterium. PrfA, the master regulator of virulence in Listeria monocytogenes, is a member of a large family of bacterial transcription factors, which are regulated by a conformational change and allosteric modulation by different regulator molecules. Furthermore, its critical role in virulence regulations makes is a suitable target for an anti-virulence drug. In this work new lead compounds based on the previously identified ring-fused 2-pyridone scaffold were designed, synthesized and analyzed by different biological, biophysical, computational and structural biology methods. Three new binding sites and binding modes of these compounds in PrfA were evaluated for their potential use in future designs and a compound with improved activity was identified. In a second study another structurally different lead compound was discovered to inhibit PrfA. Furthermore, the studies on proposed natural regulators of PrfA uncovered the underlying mechanism for the virulence regulation by the peptide signature of the environment and in a follow-up study the structural basis of the binding of inhibitory peptides to PrfA was further investigated. Finally, a structural review on all available structure of PrfA provided more insights into the allosteric regulation mechanism of PrfA activity. This work will hopefully support in the successful development of an anti-virulence drug against Listeria monocytogenes and thus contribute to the reduction of the problem of antibiotic resistances

Structural basis for transthyretin amyloid formation in vitreous body of the eye

Amyloid transthyretin (ATTR) amyloidosis is characterized by the abnormal accumulation of ATTR fibrils in multiple organs. However, the structure of ATTR fibrils from the eye is poorly understood. Here, we used cryo-EM to structurally characterize vitreous body ATTR fibrils. These structures were distinct from previously characterized heart fibrils, even though both have the same mutation and type A pathology. Differences were observed at several structural levels: in both the number and arrangement of protofilaments, and the conformation of the protein fibril in each layer of protofilaments. Thus, our results show that ATTR protein structure and its assembly into protofilaments in the type A fibrils can vary between patients carrying the same mutation. By analyzing and matching the interfaces between the amino acids in the ATTR fibril with those in the natively folded TTR, we are able to propose a mechanism for the structural conversion of TTR into a fibrillar form

Insights into Enzymatic Catalysis from Binding and Hydrolysis of Diadenosine Tetraphosphate by E. coli Adenylate Kinase

Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from Escherichia coli interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics

Nonclassical Pathways of Protein Crystallization in the Presence of Multivalent Metal Ions

Using bovine β-lactoglobulin as a model system, we have studied the crystallization pathways in the presence of the di- and trivalent salts ZnCl₂ and YCl₃. Previous work has shown that protein solutions undergo a reentrant condensation (RC) phase behavior in the presence of YCl₃, i.e., a condensed phase occurs in between two boundary salt concentrations, <i>c</i>*<i>< c</i>**. In the presence of ZnCl₂, <i>c</i>* also exists, but protein solutions with high salt concentrations do not become completely clear at higher protein concentrations (<i>></i>20 mg/mL). We thus denote this diffuse transition as <i>pseudo</i>-<i>c</i>**. Small angle X-ray scattering measurements show that the effective interactions change from repulsion to attraction near <i>c</i>*, and attractive interactions dominate around <i>pseudo</i>–<i>c</i>**. Solutions near <i>c</i>* and <i>pseudo</i><i>-c</i>** provide the optimized conditions for growth of high quality protein single crystals but with different pathways. While crystal growth near <i>c</i>* follows the classical one-step nucleation pathway, crystallization around <i>pseudo</i>-<i>c</i>** (for zinc) or <i>c</i>** (for yttrium) follows a nonclassical process with an intermediate phase appearing before crystallization. Furthermore, the intermediate phases strongly depend on the crystallization temperature. Samples with high salt concentrations exhibit a typical transition temperature, <i>T</i>_tr, below which the solutions become turbid. When crystallizing near <i>T</i>_tr, the intermediate phase consists of protein clusters; below <i>T</i>_tr, the intermediate phase corresponds to macroscopic protein aggregates which can further relax into a dense liquid phase before crystallization. However, the experimental data cannot distinguish whether nucleation occurs within or outside of the intermediate phase. Possible scenarios are discussed based on the equilibrium phase behavior of colloidal systems with a short-range attraction. The crystallographic analysis of the resulting crystals shows that metal ions are an integral part of the crystal lattice. Both types of metal ions can create new protein contacts in the crystal lattice via bridging; however, yttrium creates more bridging contacts compared to zinc

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.Originally included in thesis in manuscript form. </p