Search

Chlamydia are obligate intracellular bacteria that reside within a membrane-bound compartment called the chlamydial inclusion inside a eukaryotic host cell. These pathogens have a complex biphasic developmental cycle, which involves conversion between a replicating, but noninfectious, reticulate body (RB) and an infectious elementary body (EB). Small molecule inhibitors have been reported to have deleterious effects on the intracellular Chlamydia infection, but these studies have typically been limited in terms of assays and time points of analysis. We compared published and novel inhibitors and showed that they can differentially alter inclusion size, chlamydial number and infectious EB production, and that these effects can vary over the course of the intracellular infection. Our results provide the justification for analysis with multiple assays performed either at the end of the infection or over a time course. We also show that this approach has the potential to identify the particular step in the developmental cycle that is impacted by the inhibitor. We furthermore propose that the magnitude of inhibitor-induced progeny defects are best quantified and compared by using a new value called maximal progeny production (Progenymax). As a demonstration of the validity of this systematic approach, we applied it to inhibitors of Akt and AMPK, which are host kinases involved in lipid synthesis and cholesterol trafficking pathways. Both inhibitors reduced EB production, but Akt disruption primarily decreased RB-to-EB conversion while AMPK inhibition paradoxically enhanced RB replication. IMPORTANCE Chlamydia is the most reported cause of bacterial, sexually transmitted infection in the United States. This bacterium infects human cells and reproduces within a cytoplasmic inclusion via an unusual developmental cycle involving two specialized chlamydial forms. Small molecule compounds have been reported to negatively affect the inclusion as well as chlamydial replication and infectious progeny production, but we showed that these effects can be discordant and vary over the course of the 48- to 72-hour long intracellular infection. We propose approaches to analyze these nonuniform effects, including measurements at the end of the intracellular infection, and more detailed analysis with multiple assays performed over the course of the developmental cycle. We then applied this approach to investigate and compare the anti-chlamydial effects of two inhibitors that alter host lipid synthesis and cholesterol trafficking.

Chlamydia is an obligate intracellular bacterium and the most common reportable cause of human infection in the United States. This pathogen proliferates inside a eukaryotic host cell, where it resides within a membrane-bound compartment called the chlamydial inclusion. It has an unusual developmental cycle, marked by conversion between a replicating form, the reticulate body (RB), and an infectious form, the elementary body (EB). We found that the small molecule H89 slowed inclusion growth and decreased overall RB replication by 2-fold but caused a 25-fold reduction in infectious EBs. This disproportionate effect on EB production was mainly due to a defect in RB-to-EB conversion and not to the induction of chlamydial persistence, which is an altered growth state. Although H89 is a known inhibitor of specific protein kinases and vesicular transport to and from the Golgi apparatus, it did not cause these anti-chlamydial effects by blocking protein kinase A or C or by inhibiting protein or lipid transport. Thus, H89 is a novel anti-chlamydial compound that has a unique combination of effects on an intracellular Chlamydia infection.

ABSTRACT Chlamydia are obligate intracellular bacteria that reside within a membrane-bound compartment called the chlamydial inclusion inside a eukaryotic host cell. These pathogens have a complex biphasic developmental cycle, which involves conversion between a replicating, but noninfectious, reticulate body (RB) and an infectious elementary body (EB). Small molecule inhibitors have been reported to have deleterious effects on the intracellular Chlamydia infection, but these studies have typically been limited in terms of assays and time points of analysis. We compared published and novel inhibitors and showed that they can differentially alter inclusion size, chlamydial number and infectious EB production, and that these effects can vary over the course of the intracellular infection. Our results provide the justification for analysis with multiple assays performed either at the end of the infection or over a time course. We also show that this approach has the potential to identify the particular step in the developmental cycle that is impacted by the inhibitor. We furthermore propose that the magnitude of inhibitor-induced progeny defects are best quantified and compared by using a new value called maximal progeny production (Progenymax). As a demonstration of the validity of this systematic approach, we applied it to inhibitors of Akt and AMPK, which are host kinases involved in lipid synthesis and cholesterol trafficking pathways. Both inhibitors reduced EB production, but Akt disruption primarily decreased RB-to-EB conversion while AMPK inhibition paradoxically enhanced RB replication. IMPORTANCE Chlamydia is the most reported cause of bacterial, sexually transmitted infection in the United States. This bacterium infects human cells and reproduces within a cytoplasmic inclusion via an unusual developmental cycle involving two specialized chlamydial forms. Small molecule compounds have been reported to negatively affect the inclusion as well as chlamydial replication and infectious progeny production, but we showed that these effects can be discordant and vary over the course of the 48- to 72-hour long intracellular infection. We propose approaches to analyze these nonuniform effects, including measurements at the end of the intracellular infection, and more detailed analysis with multiple assays performed over the course of the developmental cycle. We then applied this approach to investigate and compare the anti-chlamydial effects of two inhibitors that alter host lipid synthesis and cholesterol trafficking.