Streptothricins are components of the natural product, nourseothricin; each containing identical streptolidine and gulosamine aminosugar moieties attached to varying numbers of linked b-lysines. Nourseothricin was discovered by Waksman and colleagues in the early 1940's, generating intense interest because of excellent Gram-negative activity. However, the natural product mixture was associated with toxicity, and subsequent exploration was limited. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin-F (S-F, one lysine) and streptothricin D (S-D, three lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE). The MIC50 and MIC90 for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. There was >10-fold in vitro selectivity of S-F compared with S-D on LLC-PK1 and J774 cell lines. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain at dosing levels without observable or minimal toxicity. Resistance mutations obtained in single ribosomal operon E. coli identify novel interactions with 16S rRNA helix 34, i.e., C1504A and A1196G/C conferred high level resistance to nourseothricin. Based on promising, unique activity, we suggest that the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, Gram-negative pathogens
Robust kinetochore-microtubule (kMT) attachment is critical for accurate chromosome segregation. G2/M-specific depletion of human Cdt1 that localizes to kinetochores in an Ndc80 complex-dependent manner, leads to abnormal kMT attachments and mitotic arrest. This indicates an independent mitotic role for Cdt1 in addition to its prototypic function in DNA replication origin licensing. Here, we show that Cdt1 directly binds to microtubules (MTs). Endogenous or transiently expressed Cdt1 localizes to both mitotic spindle MTs and kinetochores. Deletion mapping of Cdt1 revealed that the regions comprising the middle and C-terminal winged-helix domains but lacking the N-terminal unstructured region was required for efficient MT-binding. Mitotic kinase Aurora B interacts with and phosphorylates Cdt1. Aurora B-phosphomimetic Cdt1 exhibited attenuated MT-binding and its cellular expression induced defective kMT attachments with a concomitant delay in mitotic progression. Thus we provide mechanistic insight into how Cdt1 affects overall kMT stability in an Aurora B kinase phosphorylation-dependent manner; which is envisioned to augment the MT-binding of the Ndc80 complex.eTOC summary• Cdt1 binds to microtubules• The middle and the C-terminal winged-helix domains of Cdt1 are involved in MT-binding• Aurora B Kinase phosphorylates Cdt1 and influences its MT-binding• Aurora B-mediated Cdt1 phosphorylation is necessary for kMT stability and mitotic progression
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