Abstract
The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC50 of 0.9 μM at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.
Highlights
Chlamydia trachomatis has a phospholipid composition that resembles its eukaryotic host, but it contains branched-chain fatty acids of chlamydial origin
Our results show that FASII is essential for the replication of C. trachomatis within its eukaryotic host
Previous work showed that the selective inhibition of LOS synthesis at the LpxC step permitted the proliferation of C. trachomatis within its cytoplasmic inclusion but blocked the terminal differentiation of reticulate body (RB) into elementary body (EB) [10]
Summary
Chlamydia trachomatis has a phospholipid composition that resembles its eukaryotic host, but it contains branched-chain fatty acids of chlamydial origin. Results: The inhibition of the enoyl-acyl carrier protein reductase (FabI) in chlamydial fatty acid synthesis blocks C. trachomatis replication. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, the cell morphology suggested differentiation into a metabolically active reticulate body These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis. AFN-1252 treatment arrests the replication of C. trachomatis when added at any time during the infectious cycle, and when administered at the beginning of the infection, it blocks development at a single cell RB-like state These data show that FASII is required for C. trachomatis replication and validate CtFabI as a suitable target for antichlamydial therapy
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