Inhibition of infection by cell-permeable macrocyclic peptides that bind type IV secretion effector Etf-1.

is an obligatory intracellular bacterium that infects monocytes and macrophages, and causes human monocytic ehrlichiosis, an emerging life-threatening infectious disease. translocated factor-1 (Etf-1), a type IV secretion system effector, is essential for infection of host cells. Etf-1 translocates to mitochondria to block host apoptosis; furthermore, it can bind Beclin 1 (ATG6) to induce cellular autophagy and localize to -inclusion membrane to obtain host-cell cytoplasmic nutrients. In this study, we screened a synthetic library of over 320,000 cell-permeable macrocyclic peptides, which consist of an ensemble of random peptide sequences in the first ring and a small family of cell-penetrating peptides in the second ring, for Etf-1 binding. Library screening followed by hit optimization identified multiple Etf-1-binding peptides (with values of 1-10 μM) that efficiently enter the cytosol of mammalian cells. Peptides B7, C8, B7-131-5, B7-133-3, and B7-133-8 significantly inhibited infection of THP-1 cells. Mechanistic studies revealed that peptide B7 and its derivatives inhibited the binding of Etf-1 to Beclin 1, and Etf-1 localization to -inclusion membranes, but not Etf-1 localization to the mitochondria. Our results not only affirm the critical role of Etf-1 functions in infection, but also demonstrate the feasibility of developing macrocyclic peptides as powerful chemical probes and potential treatment of diseases caused by and other intracellular pathogens.