Making and breaking antibiotics – structural studies of Bottromycin biosynthesis and a mechanism of Albicidin resistance

The emergence of antibacterial resistance is a major public health threat that warrants urgent attention. The discovery of new antibacterial natural products and unravelling of their biosynthesis, coupled with the study of resistance mechanisms employed by bacteria to evade antibiotics will be vital in ensuring that we maintain a viable antibiotic development pipeline. This thesis covers two aspects of antibiotic research: deciphering bottromycin biosynthesis and AlbA mediated antibiotic resistance. Bottromycins are potent antibacterial peptides that bind a novel bacterial target: The A-site of the ribosome. Bottromycin biosynthesis is still not fully understood, especially the role of two hydrolases, BotAH and BotH. The in-depth functional and structural characterization studies presented here demonstrate that BotAH is responsible for a key proteolytic step, whereas BotH is an atypical hydrolase-like enzyme responsible for the post-translational epimerization of L-Asp to D-Asp in bottromycin biosynthesis. AlbA is a resistance protein that had previously been reported to neutralize a potent antibacterial compound, albicidin. The structural and functional studies of AlbA presented here not only reveal the mode of albicidin binding, but also the underlying mechanism of its modification by AlbA, resulting in loss of potency. The data also demonstrate that AlbA constitutes an autoregulated antibiotic resistance system, present in a wide variety of pathogenic bacteria.Die Entstehung von Antibiotikaresistenzen ist eine ernste Gefahr für die Gesundheit der Weltbevölkerung und bedarf dringender Aufmerksamkeit. Die Entdeckung neuer Naturstoffe mit antibiotischer Wirkung und ein Entschlüsseln ihrer Biosynthese zusammen mit einem besseren Verständnis der Resistenzmechanismen, die Bakterien entwickeln um sich zur Wehr zu setzen, ist essentiell um sicherzustellen, dass wir eine Tragfähige Strategie zur Entwicklung neuer Antibiotika haben. Diese Doktorarbeit beschäftigt sich mit zwei Aspekten der Antibiotikaforschung: Einem besseren Verständnis der Bottromycin Biosynthese und dem Resistenzprotein AlbA. Bottromycine sind potente antibakterielle Peptide und ihre Biosynthese ist noch nicht vollständig erforscht, besonders die Rolle der beiden Hydrolasen BotAH und BotH. Die detaillierte funktionelle und strukturelle Charakterisierung der beiden Proteine ist Teil dieser Arbeit. BotAH ist für einen wichtigen proteolytischen Schritt in der Biosynthese verantwortlich, wohingegen BotH eine sehr atypische Hydrolase ist und die post-translationale Umwandlung von l-Asp in d-Asp katalysiert. AlbA neutralisiert den potenten antibiotischen Naturstoff Albicidin. Die strukturelle und funktionelle Untersuchung von AlbA zeigte nicht nur auf wie AlbA Albicidin bindet und es dabei modifiziert, sondern auch das AlbA ein autoinduzierbares antibiotisches Resistenzprotein ist, das in vielen pathogenen Bakterien zu finden ist

Thiazoline-specific amidohydrolase PurAH is the gatekeeper of bottromycin biosynthesis

The ribosomally synthesized and post-translationally modified peptide (RiPP) bottromycin A2 possesses potent antimicrobial activity. Its biosynthesis involves the enzymatic formation of a macroamidine, a process previously suggested to require the concerted efforts of a YcaO enzyme (PurCD) and an amidohydrolase (PurAH) in vivo. In vitro, PurCD alone is sufficient to catalyze formation of the macroamidine, but the process is reversible. We set out to probe the role of PurAH in macroamidine formation in vitro. We demonstrate that PurAH is highly selective for macroamidine-containing precursor peptides and cleaves C-terminal of a thiazoline, thus removing the follower peptide. After follower cleavage, macroamidine formation is irreversible, indicating PurAH as the gatekeeper of bottromycin biosynthesis. The structure of PurAH suggests residues involved in catalysis, which were probed through mutagenesis

Photorhabdus luminescens lectin A (PllA) : A new probe for detecting α-galactoside-terminating glycoconjugates

Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for -galactoside–terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa.We also investigated the utility of PllA as a probe for detecting -galactosides. The -Gal epitope is present on wildtype pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllA’s high specificity for -galactoside–containing ligands, and we show that PllA can be used to visualize the -Gal epitope on porcine tissues

Substrate-Inspired Fragment Merging and Growing Affords Efficacious LasB Inhibitors

Extracellular virulence factors have emerged as attractive targets in the current antimicrobial resistance crisis. The Gram-negative pathogen Pseudomonas aeruginosa secretes the virulence factor elastase B (LasB), which plays an important role in the infection process. Here, we report a submicromolar, non-peptidic, fragment-like inhibitor of LasB discovered by careful visual inspection of structural data. Inspired by the natural LasB substrate, the original fragment was successfully merged and grown. The optimized inhibitor is accessible via simple chemistry and retained selectivity with a substantial improvement in activity, which can be rationalized by the crystal structure of LasB in complex with the inhibitor. We also demonstrate an improved in vivo efficacy of the optimized hit in Galleria mellonella larvae, highlighting the significance of this class of compounds as promising drug candidates

Tackling pseudomonas aeruginosa virulence by a hydroxamic acid-absed LasB inhibitor

In search of novel antibiotics to combat the challenging spread of resistant pathogens, bacterial proteases represent promising targets for pathoblocker development. A common motif for protease inhibitors is the hydroxamic acid function, yet this group has often been related to unspecific inhibition of various metalloproteases. In this work, the inhibition of LasB, a harmful zinc metalloprotease secreted by Pseudomonas aeruginosa, through a hydroxamate derivative is described. The present inhibitor was developed based on a recently reported, highly selective thiol scaffold. Using X-ray crystallography, the lack of inhibition of a range of human matrix metalloproteases could be attributed to a distinct binding mode sparing the S1′ pocket. The inhibitor was shown to restore the effect of the antimicrobial peptide LL-37, decrease the formation of P. aeruginosa biofilm and, for the first time for a LasB inhibitor, reduce the release of extracellular DNA. Hence, it is capable of disrupting several important bacterial resistance mechanisms. These results highlight the potential of protease inhibitors to fight bacterial infections and point out the possibility to achieve selective inhibition even with a strong zinc anchor

Tutuilamides A–C: vinyl-chloride-containing cyclodepsipeptides from marine cyanobacteria with potent elastase inhibitory properties

Marine cyanobacteria (blue-green algae) have been shown to possess an enormous capacity to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Using mass-spectrometry-guided fractionation together with molecular networking, cyanobacterial field collections from American Samoa and Palmyra Atoll yielded three new cyclic peptides, tutuilamides A–C. Their structures were established by spectroscopic techniques including 1D and 2D NMR, HR-MS, and chemical derivatization. Structure elucidation was facilitated by employing advanced NMR techniques including nonuniform sampling in combination with the 1,1-ADEQUATE experiment. These cyclic peptides are characterized by the presence of several unusual residues including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid, together with a novel vinyl chloride-containing residue. Tutuilamides A–C show potent elastase inhibitory activity together with moderate potency in H-460 lung cancer cell cytotoxicity assays. The binding mode to elastase was analyzed by X-ray crystallography revealing a reversible binding mode similar to the natural product lyngbyastatin 7. The presence of an additional hydrogen bond with the amino acid backbone of the flexible side chain of tutuilamide A, compared to lyngbyastatin 7, facilitates its stabilization in the elastase binding pocket and possibly explains its enhanced inhibitory potency

Revision of the absolute configurations of chelocardin and amidochelocardin

Even with the aid of the available methods, the configurational assignment of natural products can be a challenging task that is prone to errors, and it sometimes needs to be corrected after total synthesis or single-crystal X-ray diffraction (XRD) analysis. Herein, the absolute configuration of amidochelocardin is revised using a combination of XRD, NMR spectroscopy, experimental ECD spectra, and time-dependent density-functional theory (TDDFT)-ECD calculations. As amidochelocardin was obtained via biosynthetic engineering of chelocardin, we propose the same absolute configuration for chelocardin based on the similar biosynthetic origins of the two compounds and result of TDDFT-ECD calculations. The evaluation of spectral data of two closely related analogues, 6-desmethyl-chelocardin and its semisynthetic derivative 1, also supports this conclusion

Tutuilamides A–C: vinyl-chloride-containing cyclodepsipeptides from marine cyanobacteria with potent elastase inhibitory properties

Marine cyanobacteria (blue-green algae) have been shown to possess an enormous capacity to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Using mass-spectrometry-guided fractionation together with molecular networking, cyanobacterial field collections from American Samoa and Palmyra Atoll yielded three new cyclic peptides, tutuilamides A–C. Their structures were established by spectroscopic techniques including 1D and 2D NMR, HR-MS, and chemical derivatization. Structure elucidation was facilitated by employing advanced NMR techniques including nonuniform sampling in combination with the 1,1-ADEQUATE experiment. These cyclic peptides are characterized by the presence of several unusual residues including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid, together with a novel vinyl chloride-containing residue. Tutuilamides A–C show potent elastase inhibitory activity together with moderate potency in H-460 lung cancer cell cytotoxicity assays. The binding mode to elastase was analyzed by X-ray crystallography revealing a reversible binding mode similar to the natural product lyngbyastatin 7. The presence of an additional hydrogen bond with the amino acid backbone of the flexible side chain of tutuilamide A, compared to lyngbyastatin 7, facilitates its stabilization in the elastase binding pocket and possibly explains its enhanced inhibitory potency

Structural Studies of Human Norovirus Protease Complexes with RNA and Peptides

Noroviruses are single-stranded RNA viruses. They encode a protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In addition, the protease also binds to viral RNA, and thus is thought to regulate viral replication. However, to date no structural information is available for protease-substrate complexes that might explain the interactions made by peptide residues P’-side of cleavage junctions or RNA. Here I report the work carried out to characterize these interactions in human norovirus protease using X-ray crystallography. The protease was successfully expressed, purified and the crystallization conditions were optimized to grow crystals for structure determination. Unfortunately, RNA and peptide electron density were not observed in co-crystal structures. The packing of protease molecules in one of the crystal forms shows the interaction of protease C-terminal residues with the peptide-binding groove of a neighboring molecule in the crystal, thereby providing the view of a protease-product complex

The role of protein–protein interactions in the biosynthesis of ribosomally synthesized and post-translationally modified peptides

This review covers the role of protein–protein complexes in the biosynthesis of selected ribosomally synthesized and post-translationally modified peptide (RiPP) classes. The genomic organization of RiPP systems usually allows the expression of each biosynthetic enzyme as an individual unit, which is in stark contrast to the giant assembly lines found in non-ribosomal peptide and polyketide synthesis systems. Evidence is mounting however that the formation of multi-enzyme complexes is critical for efficient RiPPs biosynthesis and that these complexes may be involved in substrate channeling or conformational sampling. In some pathways, polyfunctional enzymes have evolved, which can be viewed as perpetual protein complexes. We summarize what is currently known on enzyme complexes in RiPP systems for lasso peptides, cyanobactins, linear azolic peptides, thiopeptides, and lanthipeptides