Fosmidomycin, an inhibitor of isoprenoid synthesis, induces persistence in Chlamydia by inhibiting peptidoglycan assembly.

The antibiotic, fosmidomycin (FSM) targets the methylerythritol phosphate (MEP) pathway of isoprenoid synthesis by inhibiting the essential enzyme, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) and is lethal to intracellular parasites and bacteria. The obligate intracellular bacterial pathogen, Chlamydia trachomatis, alternates between two developmental forms: the extracellular, infectious elementary body (EB), and the intracellular, replicative form called the reticulate body (RB). Several stressful growth conditions including iron deprivation halt chlamydial cell division and cause development of a morphologically enlarged, but viable form termed an aberrant body (AB). This phenotype constitutes the chlamydial developmental state known as persistence. This state is reversible as removal of the stressor allows the chlamydiae to re-enter and complete the normal developmental cycle. Bioinformatic analysis indicates that C. trachomatis encodes a homolog of Dxr, but its function and the requirement for isoprenoid synthesis in chlamydial development is not fully understood. We hypothesized that chlamydial Dxr (Dxr(CT)) is functional and that the methylerythritol phosphate (MEP) pathway is required for normal chlamydial development. Thus, FSM exposure should be lethal to C. trachomatis. Overexpression of chlamydial Dxr (Dxr(CT)) in Escherichia coli under FSM exposure and in a conditionally lethal dxr mutant demonstrated that Dxr(CT) functions similarly to E. coli Dxr. When Chlamydia-infected cultures were exposed to FSM, EB production was significantly reduced. However, titer recovery assays, electron microscopy, and peptidoglycan labeling revealed that FSM inhibition of isoprenoid synthesis is not lethal to C. trachomatis, but instead induces persistence. Bactoprenol is a critical isoprenoid required for peptidoglycan precursor assembly. We therefore conclude that FSM induces persistence in Chlamydia by preventing bactoprenol production necessary for peptidoglycan precursor assembly and subsequent cell division. Author summary Dxr is the second enzyme in the methylerythritol phosphate (MEP) pathway of isoprenoid synthesis and is inhibited by the antibiotic fosmidomycin (FSM). We used FSM as a tool to demonstrate that chlamydial Dxr functions similarly to E. coli Dxr. Furthermore, we demonstrate that FSM is not lethal to Chlamydia as in other bacteria and parasites, but instead induces persistence. Several conditions are known to cause Chlamydia to leave normal development and enter the persistent state including beta-lactam antibiotic exposure, interferon-y exposure, iron limitation and viral co-infection. Our study identifies FSM as a novel persistence inducer and isoprenoid synthesis inhibition as the underlying mechanism of FSM-induced persistence induction. By inhibiting isoprenoid synthesis, we identify isoprenoids, most likely bactoprenol, as critical molecules required for cell division in Chlamydia.