Genome Copy Number Regulates Inclusion Expansion, Septation, and Infectious Developmental Form Conversion in Chlamydia trachomatis

Abstract

DNA replication is essential for the growth and development of Chlamydia trachomatis, however it is unclear how this process contributes to and is controlled by the pathogen’s biphasic lifecycle. While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature sensitive dnaE mutant (dnaEts) that exhibits a ∼100-fold reduction in genome copy number at the non-permissive temperature (40°C), but replicates similarly to the parent at the permissive temperature of 37°C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in dnaEts at 40°C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. dnaEts formed fewer and smaller pathogenic vacuoles (inclusions) at 40°C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernable peptidoglycan and polymerized MreB, the major cell division organizing protein in Chlamydia responsible for nascent peptidoglycan biosynthesis. We also found that absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion reversed when dnaEts was shifted from 40°C to 37°C early in infection, and intragenic suppressor mutations in dnaE also restored dnaEts genome replication and inclusion expansion at 40°C. Overall, our results show that genome replication in C. trachomatis is required for inclusion expansion, septum formation, and the transition between the microbe’s replicative and infectious forms. SIGNIFICANCE Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and non-infectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen's infectious cycle has not been characterized. We show that genome replication is dispensable for EB to RB conversion, but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in Chlamydia that facilitate the ordering of events in the microbe's biphasic life cycle. Our findings suggest that Chlamydia utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment.

Differential Effects of Small Molecule Inhibitors on the Intracellular Chlamydia Infection

“…Detailed quantitative and volumetric analyses have shown a tight correlation between chlamydial number and inclusion size over the entire course of a wild-type infection ( 13 ). Similarly, a correlation between chlamydial genome copy number and inclusion growth has been reported ( 14 ). As inclusion size can be readily monitored by immunofluorescence microscopy, it has often been used as a surrogate measure for the overall infection.…”

Chlamydia trachomatis: quest for an eye-opening vaccine breakthrough

“…It is in these inclusions that C. trachomatis transform into reticulate bodies and replicate by binary fission -the second phase of C. trachomatis life cycle. In this stage, C. trachomatis utilizes nutrients and adenosine triphosphate from the host cytoplasm, and when these resources run out, the reticulate bodies transform back into elementary bodies and are secreted into the extracellular environment to infect neighboring cells [12].…”

Localized Peptidoglycan Biosynthesis in Chlamydia trachomatis Conforms to the Polarized Division and Cell Size Reduction Developmental Models

“…Another study has reported that C. trachomatis RBs undergo cell size reduction prior to differentiating into their non-replicative, infectious forms ( Lee et al, 2018 ), effectively linking the processes of cell size regulation, division, and differentiation. The processes of DNA replication and septation are decoupled in C. trachomatis ( Lambden et al, 2006 ); however, the inhibition of replication has been shown to directly affect PG septal formation ( Brothwell et al, 2021 ). The exact mechanism by which RBs regulate their division process and by proxy their cell size, remains unknown.…”