Biosynthesis and Mechanism of Action of the Cell Wall Targeting Antibiotic Hypeptin.

Hypeptin is a cyclodepsipeptide antibiotic produced by Lysobacter sp. K5869, isolated from an environmental sample by the iChip technology, dedicated to the cultivation of previously uncultured microorganisms. Hypeptin shares structural features with teixobactin and exhibits potent activity against a broad spectrum of gram-positive pathogens. Using comprehensive in vivo and in vitro analyses, we show that hypeptin blocks bacterial cell wall biosynthesis by binding to multiple undecaprenyl pyrophosphate-containing biosynthesis intermediates, forming a stoichiometric 2:1 complex. Resistance to hypeptin did not readily develop in vitro. Analysis of the hypeptin biosynthetic gene cluster (BGC) supported a model for the synthesis of the octapeptide. Within the BGC, two hydroxylases were identified and characterized, responsible for the stereoselective ß-hydroxylation of four building blocks when bound to peptidyl carrier proteins. In vitro hydroxylation assays corroborate the biosynthetic hypothesis and lead to the proposal of a refined structure for hypeptin.

The MraY Inhibitor Muraymycin D2 and Its Derivatives Induce Enlarged Cells in Obligate Intracellular Chlamydia and Wolbachia and Break the Persistence Phenotype in Chlamydia.

Chlamydial infections and diseases caused by filarial nematodes are global health concerns. However, treatment presents challenges due to treatment failures potentially caused by persisting Chlamydia and long regimens against filarial infections accompanied by low compliance. A new treatment strategy could be the targeting of the reduced peptidoglycan structures involved in cell division in the obligate intracellular bacteria Chlamydia and Wolbachia, the latter being obligate endosymbionts supporting filarial development, growth, and survival. Here, cell culture experiments with C. trachomatis and Wolbachia showed that the nucleoside antibiotics muraymycin and carbacaprazamycin interfere with bacterial cell division and induce enlarged, aberrant cells resembling the penicillin-induced persistence phenotype in Chlamydia. Enzymatic inhibition experiments with purified C. pneumoniae MraY revealed that muraymycin derivatives abolish the synthesis of the peptidoglycan precursor lipid I. Comparative in silico analyses of chlamydial and wolbachial MraY with the corresponding well-characterized enzyme in Aquifex aeolicus revealed a high degree of conservation, providing evidence for a similar mode of inhibition. Muraymycin D2 treatment eradicated persisting non-dividing C. trachomatis cells from an established penicillin-induced persistent infection. This finding indicates that nucleoside antibiotics may have additional properties that can break bacterial persistence.