Cell-cell communication via LuxR solos in Photorhabdus species

Bacteria constantly need to monitor their environment and adapt the bacterial group-coordinated behaviour to changing habitats like nutrition alterations or host variations. Commonly cell-cell communication via acyl homoserine lactones (AHLs)is used to synchronise the behaviour of a bacterial population dependent on cell size. This process is referred to as quorum sensing (QS) and predominantly occurs in Gram-negative bacteria. The typical QS system consists of a LuxI-synthase that synthesises AHLs, and a LuxR-type receptor, which then responds to these AHLs. Upon AHL-binding, the LuxR-type receptor regulates the expression of different target genes and thus influences several processes, like biofilm formation, virulence, antibiotic production or cell-cell interaction. Interestingly, many proteobacteria possess additional LuxR homologs, but lack a cognate LuxI-type synthase. Those LuxR-type receptors are referred to as LuxR orphans or LuxR solos and can expand the regulatory QS network. Photorhabdus species are insect pathogenic bacteria, living in symbiosis with entomopathogenic nematodes. They all possess an exceptionally high number of LuxR solos, but lack LuxI homologs and therefore do not produce AHLs. The function of these LuxR solos, their role in cell-cell communication and the identification of their cognate signalling molecules in Photorhabdus species is the main focus of this work. In this thesis a novel signalling molecule used for QS could be identified for the first time in P. luminescens. This novel QS molecule is an α-pyrone named photopyrone (PPY) and produced endogenously by the photopyrone synthase (PpyS). The PPYs are specifically recognized by the LuxR solo regulator PluR, which then activates expression of the pcf (Photorhabdus clumping factor) operon leading to cell clumping of P. luminescens cells. Moreover, the PpyS/PluR quorum sensing system and its induced cell clumping contribute to the overall toxicity of P. luminescens. Furthermore, a second novel signalling molecule sensed by a LuxR solo of Photorhabdus species could be identified besides PPYs. The insect and human pathogenic bacteria P. asymbiotica lacks a PpyS homolog as well as a LuxI homolog, but harbours a pcf operon and a homologue to PluR, which is named PauR. The signalling molecule sensed by the LuxR-type receptor PauR could be identified, which is neither an AHL nor a PPY. PauR recognises a 2,5-dialkylresorcinol (DAR) produced by the DarABC pathway. Upon binding of the cognate signalling molecule, Summary XII PauR activates expression of the pcf operon. This also leads to cell clumping in P. asymbiotica. Furthermore, the DarABC/PauR QS system also contributes to the overall pathogenicity of P. asymbiotica against Galleria mellonella insect larvae. A bioinformatics approach revealed a high number of LuxR solos present in P. temperata and P. asymbiotica like in P. luminescens. Thereby, several conserved motives of amino acids could be identified, which are potentially important for signalbinding and -specificity. Variations in these amino acid motifs are assumed to reflect the overall variety of signals that can be sensed by LuxR solos. Furthermore, the specificity of the two LuxR solos PluR and PauR towards their cognate signalling molecules, PPYs and DARs, respectively, was analysed. Thereby, it could be shown that the previously identified conserved amino acid motives in the signal-binding domain (SBD), the TYDQCS-motif of PluR and the TYDQYI-motif of PauR, are essential but not sufficient for ligand-binding. Similar as for AHLs, it was unclear how the signalling molecules PPYs and DARs can cross the bacterial cell membrane. In the last part of this thesis the import mechanism for the Photorhabdus-specific signalling compounds PPYs and DARs were identified. Initial evidence could be provided that the membrane-integrated transporter FadL is mainly involved in the import of these hydrophobic compounds, and that they are not transported via simple diffusion across the cell membrane, which is assumed for AHLs. In conclusion, the data that is compiled presents two LuxR solos of Photorhabdus species adapted to sense and respond to novel non-AHL signalling molecules used for QS. Therefore, this thesis reveals that cell-cell communication via LuxR-type receptors goes far beyond AHL-signalling in nature

Cell-cell communication via LuxR solos in Photorhabdus species

Bacteria constantly need to monitor their environment and adapt the bacterial group-coordinated behaviour to changing habitats like nutrition alterations or host variations. Commonly cell-cell communication via acyl homoserine lactones (AHLs)is used to synchronise the behaviour of a bacterial population dependent on cell size. This process is referred to as quorum sensing (QS) and predominantly occurs in Gram-negative bacteria. The typical QS system consists of a LuxI-synthase that synthesises AHLs, and a LuxR-type receptor, which then responds to these AHLs. Upon AHL-binding, the LuxR-type receptor regulates the expression of different target genes and thus influences several processes, like biofilm formation, virulence, antibiotic production or cell-cell interaction. Interestingly, many proteobacteria possess additional LuxR homologs, but lack a cognate LuxI-type synthase. Those LuxR-type receptors are referred to as LuxR orphans or LuxR solos and can expand the regulatory QS network. Photorhabdus species are insect pathogenic bacteria, living in symbiosis with entomopathogenic nematodes. They all possess an exceptionally high number of LuxR solos, but lack LuxI homologs and therefore do not produce AHLs. The function of these LuxR solos, their role in cell-cell communication and the identification of their cognate signalling molecules in Photorhabdus species is the main focus of this work. In this thesis a novel signalling molecule used for QS could be identified for the first time in P. luminescens. This novel QS molecule is an α-pyrone named photopyrone (PPY) and produced endogenously by the photopyrone synthase (PpyS). The PPYs are specifically recognized by the LuxR solo regulator PluR, which then activates expression of the pcf (Photorhabdus clumping factor) operon leading to cell clumping of P. luminescens cells. Moreover, the PpyS/PluR quorum sensing system and its induced cell clumping contribute to the overall toxicity of P. luminescens. Furthermore, a second novel signalling molecule sensed by a LuxR solo of Photorhabdus species could be identified besides PPYs. The insect and human pathogenic bacteria P. asymbiotica lacks a PpyS homolog as well as a LuxI homolog, but harbours a pcf operon and a homologue to PluR, which is named PauR. The signalling molecule sensed by the LuxR-type receptor PauR could be identified, which is neither an AHL nor a PPY. PauR recognises a 2,5-dialkylresorcinol (DAR) produced by the DarABC pathway. Upon binding of the cognate signalling molecule, Summary XII PauR activates expression of the pcf operon. This also leads to cell clumping in P. asymbiotica. Furthermore, the DarABC/PauR QS system also contributes to the overall pathogenicity of P. asymbiotica against Galleria mellonella insect larvae. A bioinformatics approach revealed a high number of LuxR solos present in P. temperata and P. asymbiotica like in P. luminescens. Thereby, several conserved motives of amino acids could be identified, which are potentially important for signalbinding and -specificity. Variations in these amino acid motifs are assumed to reflect the overall variety of signals that can be sensed by LuxR solos. Furthermore, the specificity of the two LuxR solos PluR and PauR towards their cognate signalling molecules, PPYs and DARs, respectively, was analysed. Thereby, it could be shown that the previously identified conserved amino acid motives in the signal-binding domain (SBD), the TYDQCS-motif of PluR and the TYDQYI-motif of PauR, are essential but not sufficient for ligand-binding. Similar as for AHLs, it was unclear how the signalling molecules PPYs and DARs can cross the bacterial cell membrane. In the last part of this thesis the import mechanism for the Photorhabdus-specific signalling compounds PPYs and DARs were identified. Initial evidence could be provided that the membrane-integrated transporter FadL is mainly involved in the import of these hydrophobic compounds, and that they are not transported via simple diffusion across the cell membrane, which is assumed for AHLs. In conclusion, the data that is compiled presents two LuxR solos of Photorhabdus species adapted to sense and respond to novel non-AHL signalling molecules used for QS. Therefore, this thesis reveals that cell-cell communication via LuxR-type receptors goes far beyond AHL-signalling in nature

Cell-cell communication via LuxR solos in Photorhabdus species

Bacteria constantly need to monitor their environment and adapt the bacterial group-coordinated behaviour to changing habitats like nutrition alterations or host variations. Commonly cell-cell communication via acyl homoserine lactones (AHLs)is used to synchronise the behaviour of a bacterial population dependent on cell size. This process is referred to as quorum sensing (QS) and predominantly occurs in Gram-negative bacteria. The typical QS system consists of a LuxI-synthase that synthesises AHLs, and a LuxR-type receptor, which then responds to these AHLs. Upon AHL-binding, the LuxR-type receptor regulates the expression of different target genes and thus influences several processes, like biofilm formation, virulence, antibiotic production or cell-cell interaction. Interestingly, many proteobacteria possess additional LuxR homologs, but lack a cognate LuxI-type synthase. Those LuxR-type receptors are referred to as LuxR orphans or LuxR solos and can expand the regulatory QS network. Photorhabdus species are insect pathogenic bacteria, living in symbiosis with entomopathogenic nematodes. They all possess an exceptionally high number of LuxR solos, but lack LuxI homologs and therefore do not produce AHLs. The function of these LuxR solos, their role in cell-cell communication and the identification of their cognate signalling molecules in Photorhabdus species is the main focus of this work. In this thesis a novel signalling molecule used for QS could be identified for the first time in P. luminescens. This novel QS molecule is an α-pyrone named photopyrone (PPY) and produced endogenously by the photopyrone synthase (PpyS). The PPYs are specifically recognized by the LuxR solo regulator PluR, which then activates expression of the pcf (Photorhabdus clumping factor) operon leading to cell clumping of P. luminescens cells. Moreover, the PpyS/PluR quorum sensing system and its induced cell clumping contribute to the overall toxicity of P. luminescens. Furthermore, a second novel signalling molecule sensed by a LuxR solo of Photorhabdus species could be identified besides PPYs. The insect and human pathogenic bacteria P. asymbiotica lacks a PpyS homolog as well as a LuxI homolog, but harbours a pcf operon and a homologue to PluR, which is named PauR. The signalling molecule sensed by the LuxR-type receptor PauR could be identified, which is neither an AHL nor a PPY. PauR recognises a 2,5-dialkylresorcinol (DAR) produced by the DarABC pathway. Upon binding of the cognate signalling molecule, Summary XII PauR activates expression of the pcf operon. This also leads to cell clumping in P. asymbiotica. Furthermore, the DarABC/PauR QS system also contributes to the overall pathogenicity of P. asymbiotica against Galleria mellonella insect larvae. A bioinformatics approach revealed a high number of LuxR solos present in P. temperata and P. asymbiotica like in P. luminescens. Thereby, several conserved motives of amino acids could be identified, which are potentially important for signalbinding and -specificity. Variations in these amino acid motifs are assumed to reflect the overall variety of signals that can be sensed by LuxR solos. Furthermore, the specificity of the two LuxR solos PluR and PauR towards their cognate signalling molecules, PPYs and DARs, respectively, was analysed. Thereby, it could be shown that the previously identified conserved amino acid motives in the signal-binding domain (SBD), the TYDQCS-motif of PluR and the TYDQYI-motif of PauR, are essential but not sufficient for ligand-binding. Similar as for AHLs, it was unclear how the signalling molecules PPYs and DARs can cross the bacterial cell membrane. In the last part of this thesis the import mechanism for the Photorhabdus-specific signalling compounds PPYs and DARs were identified. Initial evidence could be provided that the membrane-integrated transporter FadL is mainly involved in the import of these hydrophobic compounds, and that they are not transported via simple diffusion across the cell membrane, which is assumed for AHLs. In conclusion, the data that is compiled presents two LuxR solos of Photorhabdus species adapted to sense and respond to novel non-AHL signalling molecules used for QS. Therefore, this thesis reveals that cell-cell communication via LuxR-type receptors goes far beyond AHL-signalling in nature

LuxR solos in Photorhabdus species

Bacteria communicate via small diffusible molecules to mediate group-coordinated behavior, a process designated as quorum sensing. The basic molecular quorum sensing system of Gram-negative bacteria consists of a Luxl-type autoinducer synthase producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor detecting the AHLs to control expression of specific genes. However, many proteobacteria possess one or more unpaired LuxR-type receptors that lack a cognate Luxl-like synthase, referred to as LuxR solos. The enteric and insect pathogenic bacteria of the genus Photorhabdus harbor an extraordinarily high number of LuxR solos, more than any other known bacteria, and all lack a Luxl-like synthase. Here, we focus on the presence and the different types of LuxR solos in the three known Photorhabdus species using bioinformatics analyses. Generally, the N-terminal signal-binding domain (SBD) of LuxR-type receptors sensing AHLs have a motif of six conserved amino acids that is important for binding and specificity of the signaling molecule. However, this motif is altered in the majority of the Photorhabdus-specific LuxR solos, suggesting the use of other signaling molecules than AHLs. Furthermore, all Photorhabdus species contain at least one LuxR solo with an intact AHL-binding motif, which might allow the ability to sense AHLs of other bacteria. Moreover, all three species have high AHL-degrading activity caused by the presence of different AHL-lactonases and AHL-acylases, revealing a high quorum quenching activity against other bacteria. However, the majority of the other LuxR solos in Photorhabdus have a N-terminal so-called PAS4-domain instead of an AHL-binding domain, containing different amino acid motifs than the AHL-sensors, which potentially allows the recognition of a highly variable range of signaling molecules that can be sensed apart from AHLs. These PAS4-LuxR solos are proposed to be involved in host sensing, and therefore in inter-kingdom signaling. Overall, Photorhabdus species are perfect model organisms to study bacterial communication via LuxR solos and their role for a symbiotic and pathogenic life style

Transferrin-modified chitosan nanoparticles for targeted nose-to-brain delivery of proteins

Nose-to-brain delivery presents a promising alternative route compared to classical blood-brain barrier passage, especially for the delivery of high molecular weight drugs. In general, macromolecules are rapidly degraded in physiological environment. Therefore, nanoparticulate systems can be used to protect biomolecules from premature degradation. Furthermore, targeting ligands on the surface of nanoparticles are able to improve bioavailability by enhancing cellular uptake due to specific binding and longer residence time. In this work, transferrin-decorated chitosan nanoparticles are used to evaluate the passage of a model protein through the nasal epithelial barrier in vitro. It was demonstrated that strain-promoted azide-alkyne cycloaddition reaction can be utilized to attach a functional group to both transferrin and chitosan enabling a rapid covalent surface-conjugation under mild reaction conditions after chitosan nanoparticle preparation. The intactness of transferrin and its binding efficiency were confirmed via SDS-PAGE and SPR measurements. Resulting transferrin-decorated nanoparticles exhibited a size of about 110-150 nm with a positive surface potential. Nanoparticles with the highest amount of surface bound targeting ligand also displayed the highest cellular uptake into a human nasal epithelial cell line (RPMI 2650). In an air-liquid interface co-culture model with glioblastoma cells (U87), transferrin-decorated nanoparticles showed a faster passage through the epithelial cell layer as well as increased cellular uptake into glioblastoma cells. These findings demonstrate the beneficial characteristics of a specific targeting ligand. With this chemical and technological formulation concept, a variety of targeting ligands can be attached to the surface after nanoparticle formation while maintaining cargo integrity

Cell and molecular transitions during efficient dedifferentiation

Dedifferentiation is a critical response to tissue damage, yet is not well understood, even at a basic phenomenological level. Developing Dictyostelium cells undergo highly efficient dedifferentiation, completed by most cells within 24 hr. We use this rapid response to investigate the control features of dedifferentiation, combining single cell imaging with high temporal resolution transcriptomics. Gene expression during dedifferentiation was predominantly a simple reversal of developmental changes, with expression changes not following this pattern primarily associated with ribosome biogenesis. Mutation of genes induced early in dedifferentiation did not strongly perturb the reversal of development. This apparent robustness may arise from adaptability of cells: the relative temporal ordering of cell and molecular events was not absolute, suggesting cell programmes reach the same end using different mechanisms. In addition, although cells start from different fates, they rapidly converged on a single expression trajectory. These regulatory features may contribute to dedifferentiation responses during regeneration

Display Selection of a Hybrid Foldamer–Peptide Macrocycle

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer–peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition

Specificity of Signal-Binding via Non-AHL LuxR-Type Receptors.

Quorum sensing is a typical communication system among Gram-negative bacteria used to control group-coordinated behavior via small diffusible molecules dependent on cell number. The key components of a quorum sensing system are a LuxI-type synthase, producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor that detects AHLs to control expression of specific target genes. Six conserved amino acids are present in the signal-binding domain of AHL-sensing LuxR-type proteins, which are important for ligand-binding and -specificity as well as shaping the ligand-binding pocket. However, many proteobacteria possess LuxR-type regulators without a cognate LuxI synthase, referred to as LuxR solos. The two LuxR solos PluR and PauR from Photorhabdus luminescens and Photorhabdus asymbiotica, respectively, do not sense AHLs. Instead PluR and PauR sense alpha-pyrones and dialkylresorcinols, respectively, and are part of cell-cell communication systems contributing to the overall virulence of these Photorhabdus species. However, PluR and PauR both harbor substitutions in the conserved amino acid motif compared to that in AHL sensors, which appeared to be important for binding the corresponding signaling molecules. Here we analyze the role of the conserved amino acids in the signal-binding domain of these two non-AHL LuxR-type receptors for their role in signal perception. Our studies reveal that the conserved amino acid motif alone is essential but not solely responsible for ligand-binding