Moonlighting chaperone activity of the enzyme PqsE contributes to RhlR-controlled virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa, with the rhl circuit playing the leading role in late and chronic infections. The majority of traits controlled by rhl transcription factor RhlR depend on PqsE, a dispensable thioesterase in Pseudomonas Quinolone Signal (PQS) biosynthesis that interferes with RhlR through an enigmatic mechanism likely involving direct interaction of both proteins. Here we show that PqsE and RhlR form a 2:2 protein complex that, together with RhlR agonist N-butanoyl-L-homoserine lactone (C4-HSL), solubilizes RhlR and thereby renders the otherwise insoluble transcription factor active. We determine crystal structures of the complex and identify residues essential for the interaction. To corroborate the chaperone-like activity of PqsE, we design stability-optimized variants of RhlR that bypass the need for C4-HSL and PqsE in activating PqsE/RhlR-controlled processes of P. aeruginosa. Together, our data provide insight into the unique regulatory role of PqsE and lay groundwork for developing new P. aeruginosa-specific pharmaceuticals

Design und Charakterisierung von stabilen Varianten des Quorum Sensing Regulator RhlR aus Pseudomonas aeruginosa

During the past decades, the opportunistic pathogen Pseudomonas aeruginosa rose to a central threat in human health care. It is able to cause fatal infections. Intrinsic and acquired multidrug resistance limit possible treatments. Therefore, new approaches are strongly needed to combat the bacterium. One approach is the inhibition of bacterial quorum sensing (QS), a cell-to-cell communication system which regulates the expression of multiple virulence factors. In P. aeruginosa, three intertwined QS systems are known: the las-, the rhl- and the pqs-circuit. The las-circuit can be found at the top of a QS cascade, triggering the other two circuits. However, the interplay of two proteins from the rhl- and the pqs-circuit is crucial for pathogenicity. Recent studies suggest a direct protein-protein-interaction between the thioesterase PqsE and the transcription factor RhlR. This interaction is a key element of virulence in P. aeruginosa: when either of these proteins is missing, the bacterium stops producing several important virulence factors. Further, different classes of RhlR-regulated genes and their (in-)dependency on PqsE were found. In this study, the intrinsically instable LuxR-type transcription factor RhlR was investigated in detail. By computational stability design, five stability-optimized RhlR variants were designed and characterized. All of these showed greatly improved expression levels and significantly higher thermal stability than the wildtype protein. By X-ray crystallography, detailed structural insights on three variants could be gained. As expected, the designed RhlR variants show typical LuxR characteristics such as dimerization and the division into two functional domains: an N-terminal ligand-binding domain (LBD) and a C-terminal DNA-binding domain (DBD). Analysis of the LBD showed binding of the synthetic autoinducer analog mBTL and revealed deep insights into the distinctive ligand-binding motif of RhlR variants. In addition to structural characterization, the generated RhlR variants were also investigated at the functional level. An inability of RhlR variants to interact with PqsE in vitro was noted, which likely is a consequence of mutations in the PqsE interaction interface of RhlR introduced by the stability-optimized design. Additionally, in vitro ligand- and DNA-binding of the stability-optimized RhlR variants was investigated, providing insights into affinity determinants and specificity of RhlR. Further, pyocyanin production of different P. aeruginosa knockout mutants was monitored by an in vivo assay. Subsequently, these mutants were complemented with a plasmid-encoded RhlR variant, revealing that the stability-optimized proteins were able to restore pyocyanin production up to wildtype level independently from PqsE or the cognate autoinducer C4-HSL. Overall, this study provides detailed insights into the structure and function of stability-optimized variants of the LuxR-type transcription factor RhlR. Here, a structural, biochemical and biophysical characterization of the generated variants was possible. This is the first time that structural data of RhlR could be obtained. Further, the understanding of the interplay of RhlR and PqsE in P. aeruginosa could be expanded by the in vivo experiments of this study. Ultimately, these insights might provide the basis for the development of new anti-infectives and treatment strategies.In den vergangenen Jahrzehnten hat sich der opportunistische Erreger Pseudomonas aeruginosa zu einer zentralen Bedrohung für die menschliche Gesundheit entwickelt, da er tödliche Infektionen verursachen kann. Intrinsische und erworbene Antibiotika-Resistenzen begrenzen mögliche Behandlungen. Daher sind neue Ansätze zur Bekämpfung des Bakteriums dringend erforderlich. Ein Ansatz ist dabei die Hemmung des bakteriellen Quorum Sensing (QS), eines zellulären Kommunikationssystems, das die Expression verschiedener Virulenzfaktoren reguliert. Bei P. aeruginosa sind drei miteinander verflochtene QS-Systeme bekannt: das las-, das rhl- und das pqs-System. Das las-System befindet sich an der Spitze einer QS-Kaskade und löst die beiden anderen Systeme aus. Entscheidend für die Pathogenität ist jedoch das Zusammenspiel zweier Proteine aus dem rhl- und dem pqs-System. Neuere Studien deuten auf eine direkte Protein-Protein-Interaktion zwischen der Thioesterase PqsE und dem Transkriptionsfaktor RhlR hin. Diese Wechselwirkung ist ein Schlüsselelement der Virulenz von P. aeruginosa: wenn eines der Proteine fehlt, stoppt die Produktion verschiedener Virulenzfaktoren. Des Weiteren wurden verschiedene Klassen von RhlR-regulierten Genen und deren (Un-) Abhängigkeit von PqsE entdeckt. In dieser Studie wurde der intrinsisch instabile, LuxR-typische Transkriptionsfaktor RhlR detailliert untersucht. Durch computergestütztes Stabilitätsdesign wurden fünf stabilitätsoptimierte RhlR-Varianten entworfen und charakterisiert. Alle zeigten eine deutlich verbesserte Expression und höhere thermische Stabilität als das Wildtyp-Protein. Durch Röntgenkristallographie konnten detaillierte strukturelle Erkenntnisse zu drei Varianten gewonnen werden. Die untersuchten RhlR-Varianten weisen erwartungsgemäß typische LuxR-Merkmale wie Dimerisation und die Aufteilung in zwei funktionelle Domänen auf: eine N-terminale Liganden-Bindungsdomäne (LBD) und eine C-terminale DNA-Bindungsdomäne (DBD). Die Analyse der LBD zeigte die Bindung des synthetischen Autoinduktors mBTL und lieferte tiefe Einblicke in das charakteristische Liganden-Bindungsmotiv der RhlR-Varianten. Neben der strukturellen Charakterisierung wurden die generierten RhlR-Varianten auch auf funktioneller Ebene untersucht. Es wurde festgestellt, dass RhlR-Varianten in vitro nicht mit PqsE interagieren können, was wahrscheinlich eine Folge von RhlR-seitigen Mutationen in der PqsE-Interaktionsschnittstelle ist, die durch das stabilitätsoptimierte Design eingeführt wurden. Zusätzlich wurde die in vitro Liganden- und DNA-Bindung der stabilitätsoptimierten RhlR-Varianten untersucht, was Aufschluss über Affinitäten und die Spezifität von RhlR gab. Darüber hinaus wurde die Pyocyanin-Produktion verschiedener P. aeruginosa Knockout-Mutanten durch einen in vivo Assay untersucht. Diese Mutanten wurden anschließend mit einer Plasmid-kodierten RhlR-Variante komplementiert, was zeigte, dass die stabilitätsoptimierten Proteine in der Lage waren, die Pyocyanin-Produktion bis zum Wildtyp-Niveau unabhängig von PqsE oder dem Autoinduktor C4-HSL wiederherzustellen. Insgesamt liefert die Studie detaillierte Einblicke in die Struktur und Funktion stabilitätsoptimierter Varianten des LuxR-typischen Transkriptionsfaktors RhlR. Es war eine strukturelle, biochemische und biophysikalische Charakterisierung der generierten Varianten möglich. Erstmals konnten damit Strukturdaten von RhlR gewonnen werden. Darüber hinaus konnte das Verständnis des Zusammenspiels von RhlR und PqsE in P. aeruginosa durch die in vivo Experimente dieser Studie erweitert werden. Diese Erkenntnisse könnten letztlich die Grundlage für die Entwicklung neuer Anti-Infektiva und Behandlungsstrategien bilden

The crystal structure of the heme d biosynthesis-associated small c-type cytochrome NirC reveals mixed oligomeric states in crystallo.

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC

Biocatalytically active and stable cross-linked enzyme crystals of halohydrin dehalogenase HheG by protein engineering

A major drawback for practical application of halohydrin dehalogenase HheG in biocatalysis is its rather low thermal stability and low organic solvent tolerance. We therefore pursued a stabilization of HheG via immobilization as cross-linked enzyme crystals. Since glutaraldehyde inactivates HheG, we introduced a cysteine residue in the crystal interface, which enabled thiol-specific cross-linking at predefined cross-linking sites. Variant HheG D114C displayed improved crystallizability and yielded stable and catalytically active CLECs using bis-maleimidoethane as cross-linker. Effective cross-linking at the predefined site could be confirmed via the CLEC crystal structure. Compared to soluble enzyme, the CLECs displayed significantly improved stability and activity at higher temperatures, lower pH values and in the presence of water-miscible organic solvents, which enabled their reuse over 21 days in the azidolysis of cyclohexene oxide