Argyrins are a family of naturally produced octapeptides that display promising antimicrobial activity against Pseudomonas aeruginosa. Argyrin B (ArgB) has been shown to interact with an elongated form of the translation elongation factor G (EF-G), leading to the suggestion that argyrins inhibit protein synthesis by interfering with EF-G binding to the ribosome. Here, using a combination of cryo-electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET), we demonstrate that rather than interfering with ribosome binding, ArgB rapidly and specifically binds EF-G on the ribosome to inhibit intermediate steps of the translocation mechanism. Our data support that ArgB inhibits conformational changes within EF-G after GTP hydrolysis required for translocation and factor dissociation, analogous to the mechanism of fusidic acid, a chemically distinct antibiotic that binds a different region of EF-G. These findings shed light on the mechanism of action of the argyrin-class antibiotics on protein synthesis as well as the nature and importance of rate-limiting, intramolecular conformational events within the EF-G-bound ribosome during late-steps of translocation.

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity.

Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of Ōmura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer activities and diverse mode-of-action at the molecular level. By a combination of structure elucidation experiments, the first total synthesis of zelkovamycin and bioassays, the zelkovamycin configuration was determined and its previously proposed molecular structure was revised. The full structure assignment proves zelkovamycin as an additional member of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system, but also guide customized argyrin NP isolation and biosynthesis studies.

The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their structure-activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising approach where simpler and fully synthetic intermediates of the natural product\'s biosynthesis can be biotechnologically incorporated. Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroalanine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence ᴅ-Ala-Dha-Sar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was formed before coupling to the Dha-containing dipeptide.

Argyrins represent a family of cyclic octapeptides exhibiting promising antimicrobial, antitumorigenic and immunosuppressant activities. They derive from a nonribosomal peptide synthetase pathway, which was identified and characterized in this study from the myxobacterial producer strain Cystobacter sp. SBCb004. Using the native biosynthetic gene cluster (BGC) sequence as template synthetic BGC versions were designed and assembled from gene synthesis fragments. A heterologous expression system was established after chromosomal deletion of a well-expressed lipopeptide pathway from the host strain Myxococcus xanthus DK1622. Different approaches were applied to engineer and improve heterologous argyrin production, which was finally increased to 160 mg/L, around 20-fold higher yields compared to the native producer. Heterologous production platform also led to identification of several novel argyrin derivatives (A2, F3, G3, I, J, K, and L). The optimized production system provides a versatile platform for future supply of argyrins and novel derivatives thereof.

The argyrins are a family of non-ribosomal peptides that exhibits different biological activities through only small structural changes. Ideally, a biologically active molecule can be tracked and observed in a variety of biological and clinical settings in a non-invasive manner. As a step towards this goal, we report here a chemical synthesis of unnatural deep blue amino acid β-(1-azulenyl)-l alanine with different fluorescence and photophysical properties, which allows a spectral separation from the native tryptophan signal. This might be especially useful for cell localization studies and visualizing the targeted proteins. In particular, the synthesis of β-(1-azulenyl)-l alanine was achieved through a Negishi coupling which proved to be a powerful tool for the synthesis of unnatural tryptophan analogs. Upon β-(1-azulenyl)-l alanine incorporation into argyrin C, deep blue octapeptide variant was spectrally and structurally characterized.

Argyrins are natural products with antibacterial activity against Gram-negative pathogens, such as Pseudomonas aeruginosa, Burkholderia multivorans, and Stenotrophomonas maltophilia We previously showed that argyrin B targets elongation factor G (FusA). Here, we show that argyrin B activity against P. aeruginosa PAO1 (MIC = 8 μg/ml) was not affected by deletion of the MexAB-OprM, MexXY-OprM, MexCD-OprJ, or MexEF-OprN efflux pump. However, argyrin B induced expression of MexXY, causing slight but reproducible antagonism with the MexXY substrate antibiotic ciprofloxacin. Argyrin B activity against Escherichia coli increased in a strain with nine tolC efflux pump partner genes deleted. Complementation experiments showed that argyrin was effluxed by AcrAB, AcrEF, and MdtFX. Argyrin B was inactive against Acinetobacter baumannii Differences between A. baumannii and P. aeruginosa FusA proteins at key residues for argyrin B interaction implied that natural target sequence variation impacted antibacterial activity. Consistent with this, expression of the sensitive P. aeruginosa FusA1 protein in A. baumannii conferred argyrin susceptibility, whereas resistant variants did not. Argyrin B was active against S. maltophilia (MIC = 4 μg/ml). Spontaneous resistance occurred at high frequency in the bacterium (circa 10-7), mediated by mutational inactivation of fusA1 rather than by amino acid substitutions in the target binding region. This strongly suggested that resistance occurred at high frequency through loss of the sensitive FusA1, leaving an alternate argyrin-insensitive elongation factor. Supporting this, an additional fusA-like gene (fusA2) is present in S. maltophilia that was strongly upregulated in response to mutational loss of fusA1.