RESUMO
Bacteria produce a plethora of specialized metabolites (SM), with the ecological function of most of them not known. A major group of SM are peptides derived from nonribosomal peptide synthetases (NRPS). In entomopathogenic bacteria of the genus Xenorhabdus, PAX (peptide-antimicrobial-Xenorhabdus) were described as NRPS-derived lipopeptides, which show antimicrobial activities against bacteria and fungi. We analyzed the production of PAX in Xenorhabdus doucetiae and found the majority bound to the cells. We derivatized PAX with fluorophores and show binding to cells when added externally using super-resolution microscopy. Externally added PAX in X. doucetiae and E. coli as well as inducible PAX production in X. doucetiae showed a protective effect against various antimicrobial peptides (AMPs) from insects, where they are used as a defense mechanism against pathogens. Because AMPs are often positively charged, our results suggest a PAX-induced repulsive force due to positive charge at the bacterial cell wall.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Produtos Biológicos/química , Insetos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Mecanismos de Defesa , Escherichia coli/química , Corantes Fluorescentes/química , Lipopeptídeos/química , Peptídeo Sintases/metabolismo , Xenorhabdus/químicaRESUMO
A new cyclic lipopeptide, phototemtideâ A (1), was isolated from Escherichia coli expressing the biosynthetic gene cluster pttABC from Photorhabdus temperata Meg1. The structure of 1 was elucidated by HR-ESI-MS and NMR experiments. The absolute configurations of amino acids and 3-hydroxyoctanoic acid in 1 were determined by using the advanced Marfey's method and comparison after total synthesis of 1, respectively. Additionally, three new minor derivatives, phototemtides B-D (2-4), were identified by detailed HPLC-MS analysis. Phototemtideâ A (1) showed weak antiprotozoal activity against Plasmodium falciparum, with an IC50 value of 9.8â µm. The biosynthesis of phototemtides A-D (1-4) was also proposed.
Assuntos
Antiprotozoários/farmacologia , Escherichia coli/metabolismo , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Photorhabdus/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Trypanosoma/efeitos dos fármacos , Escherichia coli/genética , Família Multigênica , Photorhabdus/genética , Trypanosoma/classificaçãoRESUMO
Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.
Assuntos
Produtos Biológicos/química , Vias Biossintéticas/genética , Metabolômica/métodos , HumanosRESUMO
Xenorhabdus doucetiae, the bacterial symbiont of the entomopathogenic nematode Steinernema diaprepesi produces several different fatty acid amides. Their biosynthesis has been studied using a combination of analysis of gene deletions and promoter exchanges in X. doucetiae and heterologous expression of candidate genes in E. coli. While a decarboxylase is required for the formation of all observed phenylethylamides and tryptamides, the acyltransferase XrdE encoded in the xenorhabdin biosynthesis gene cluster is responsible for the formation of short chain acyl amides. Additionally, new, long-chain and cytotoxic acyl amides were identified in X. doucetiae infected insects and when X. doucetiae was grown in Galleria Instant Broth (GIB). When the bioactivity of selected amides was tested, a quorum sensing modulating activity was observed for the short chain acyl amides against the two different quorum sensing systems from Chromobacterium and Janthinobacterium.