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

Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects monocytes and macrophages, and causes human monocytic ehrlichiosis, an emerging life-threatening infectious disease. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system effector, is essential for Ehrlichia 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 E. chaffeensis-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 K-D values of 1-10 mu M) that efficiently enter the cytosol of mammalian cells. Peptides B7, C8, B7-131-5, B7-133-3, and B7-133-8 significantly inhibited Ehrlichia 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 E. chaffeensis-inclusion membranes, but not Etf-1 localization to the mitochondria. Our results not only affirm the critical role of Etf-1 functions in E. chaffeensis infection, but also demonstrate the feasibility of developing macrocyclic peptides as powerful chemical probes and potential treatment of diseases caused by Ehrlichia and other intracellular pathogens.

Automated summary

This study identified peptides that can bind to Etf-1 and demonstrated their ability to inhibit Ehrlichia infection. Mechanistic studies revealed that these peptides inhibit the binding of Etf-1 to Beclin 1 and its localization to inclusion membranes. These findings not only confirm the critical role of Etf-1 in E.chaffeensis infection but also demonstrate the potential of developing macrocyclic peptides as chemical probes and treatments for diseases caused by Ehrlichia and other intracellular pathogens.