Alkyl quinolones mediate heterogeneous colony biofilm architecture that improves community-level survival.

Bacterial communities exhibit complex self-organization that contributes to their survival. To better understand the molecules that contribute to transforming a small number of cells into a heterogeneous surface biofilm community, we studied acellular aggregates, structures seen by light microscopy in Pseudomonas aeruginosa colony biofilms using light microscopy and chemical imaging. These structures differ from cellular aggregates, cohesive clusters of cells important for biofilm formation, in that they are visually distinct from cells using light microscopy and are reliant on metabolites for assembly. To investigate how these structures benefit a biofilm community we characterized three recurrent types of acellular aggregates with distinct geometries that were each abundant in specific areas of these biofilms. Alkyl quinolones (AQs) were essential for the formation of all aggregate types with AQ signatures outside the aggregates below the limit of detection. These acellular aggregates spatially sequester AQs and differentiate the biofilm space. However, the three types of aggregates showed differing properties in their size, associated cell death, and lipid content. The largest aggregate type co-localized with spatially confined cell death that was not mediated by Pf4 bacteriophage. Biofilms lacking AQs were absent of localized cell death but exhibited increased, homogeneously distributed cell death. Thus, these AQ-rich aggregates regulate metabolite accessibility, differentiate regions of the biofilm, and promote survival in biofilms.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen with the ability to cause infection in the immune-compromised. It is well established that P. aeruginosa biofilms exhibit resilience that includes decreased susceptibility to antimicrobial treatment. This work examines the self-assembled heterogeneity in biofilm communities studying acellular aggregates, regions of condensed matter requiring alkyl quinolones (AQs). AQs are important to both virulence and biofilm formation. Aggregate structures described here spatially regulate the accessibility of these AQs, differentiate regions of the biofilm community, and despite their association with autolysis, correlate with improved P. aeruginosa colony biofilm survival.

Development of Inhibitory Compounds for Metallo-beta-lactamase through Computational Design and Crystallographic Analysis.

Metallo-ß-lactamases (MBL) deactivate ß-lactam antibiotics through a catalytic reaction caused by two zinc ions at the active center. Since MBLs deteriorate a wide range of antibiotics, they are dangerous factors for bacterial multidrug resistance. In this work, organic synthesis, computational design, and crystal structure analysis were performed to obtain potent MBL inhibitors based on a previously identified hit compound. The hit compound comprised 3,4-dihydro-2(1H)-quinolinone linked with a phenyl-ether-methyl group via a thiazole ring. In the first step, the thiazole ring was replaced with a tertiary amine to avoid the planar structure. In the second step, we virtually modified the compound by keeping the quinolinone backbone. Every modified compound was bound to a kind of MBL, imipenemase-1 (IMP-1), and the binding pose was optimized by a molecular mechanics calculation. The binding scores were evaluated for the respective optimized binding poses. Given the predicted binding poses and calculated binding scores, candidate compounds were determined for organic syntheses. The inhibitory activities of the synthesized compounds were measured by an in vitro assay for two kinds of MBLs, IMP-1 and New Delhi metallo-ß-lactamase (NDM-1). A quinolinone connected with an amine bound with methyl-phenyl-ether-propyl and cyclohexyl-ethyl showed a 50% inhibitory concentration of 4.8 µM. An X-ray crystal analysis clarified the binding structure of a synthesized compound to IMP-1. The δ-lactam ring of quinolinone was hydrolyzed, and the generated carboxyl group was coordinated with zinc ions. The findings on the chemical structure and binding pose are expected to be a base for developing MBL inhibitors.

Structural and mechanistic insights into Quinolone Synthase to address its functional promiscuity.

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS's steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.

Biosynthesis of Ester-Bond Containing Quinolone Alkaloids with (3R,4S) Stereoconfiguration.

Asperalins represent a novel class of viridicatin natural products with potent inhibitory activities against fish pathogens. In this study, we elucidated the biosynthesis of asperalins in the Aspergillus oryzae NSAR1 heterologous host and identified the FAD-dependent monooxygenase AplB stereoselectively hydroxylates viridicatin to yield a unique 3R,4S configuration. The monomodular NRPS AplJ catalyzes a rare intramolecular ester bond formation reaction using dihydroquinoline as a nucleophile. Subsequent modifications by cytochrome P450 AplF, chlorinase AplN, and prenyltransferase AplE tailor the anthranilic acid portion, leading to the formation of asperalins. Additionally, we explored the potential of AplB for the hydroxylation of viridicatin analogs, demonstrating its relaxed substrate specificity. This finding suggests that AplB could be developed as a biocatalyst for the synthesis of viridicatin derivatives.

Influencia do inóculo bacteriano no teste de susceptibilidade as quilonas pelo método de diluiçäo em agar

A influência do inóculo na atividade de suas quinolonas (Lomefloxacina e Pefloxacina) contra P. aeruginosa, E. coli, S. aureus e S. fecalis foi investigada pela técnica de diluiçäo em ágar. A açäo deste grupo de antimicrobianos foi reduzida quanto da utilizaçäo de um inóculo muito rico (10**5 e 10**6 ufc), principalmente em relaçäo aos gram negativos