Penicillin Induced Persistence in Chlamydia trachomatis: High Quality Time Lapse Video Analysis of the Developmental Cycle

Abstract

Background Chlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non - infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non - infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence.Principal FindingsAddition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome.ConclusionsWe have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome replication this provides a defined framework to analyse the developmental cycle and to investigate and provide new insights into the effects of antibiotic treatments. Removal of penicillin allows recovery of the normal developmental cycle by 10–20 hrs and the process occurs by budding from aberrant RBs.

Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis

“…Inhibition of LOS biosynthesis did not induce the formation of enlarged abRBs that are observed during treatment with peptidoglycan assembly inhibitors or during immunological and nutritional stresses (iron and amino acid starvation) (26,32,33). The only apparent morphological consequence of LOS inhibition was the marked accumulation of RBs and the concomitant absence of EBs (Figs.…”

“…In response to cellular stresses, chlamydiae will stall the developmental cycle to generate enlarged, morphologically aberrant RBs (abRBs) that do not transition to EBs (26). This is unlikely to be the case for Chlamydia forms generated in the presence of LpxC inhibitors, because they were clearly distinct from abRB formed during treatment with ampicillin (10 μg/mL; Movie S1), indicating that loss of LOS did not induce the formation of chlamydial-persistent bodies.…”

Evolution to a Chronic Disease Niche Correlates with Increased Sensitivity to Tryptophan Availability for the Obligate Intracellular Bacterium Chlamydia pneumoniae

“…This state is reversible. Several different stimuli have been shown to induce persistence, including (i) gamma interferon treatment, (ii) penicillin treatment, (iii) amino acid (e.g., tryptophan), glucose, and iron deprivation, (iv) chlamydiophage infection, and several others (14,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Persistence therefore appears to be a mechanism that allows the organisms to ride out hostile conditions while maintaining a long-term (chronic) infection within a host cell.…”

A Coming of Age Story: Chlamydia in the Post-Genetic Era

“…While the bla marker is prohibited for use in certain serovars, it has proven robust and is the marker of choice for most researchers. The robustness of bla-mediated resistance may be due, in part, to the fact that ␤-lactams appear to be bacteriostatic for Chlamydia inducing a persistent growth state rather than death (64). This may allow for increased time for phenotypic conversion, resulting in an increased rate of transformant recovery.…”