Penicillin Kills Chlamydia following the Fusion of Bacteria with Lysosomes and Prevents Genital Inflammatory Lesions in C. muridarum-Infected Mice

Maud Dumoux,Richard D Hayward,Colette Kanellopoulos-Langevin,Christiane Delarbre,Philippe Verbeke,Sylvain M Le Gall,Mohamed Habbeddine

doi:10.1371/journal.pone.0083511

Abstract

The obligate intracellular bacterium Chlamydia exists as two distinct forms. Elementary bodies (EBs) are infectious and extra-cellular, whereas reticulate bodies (RBs) replicate within a specialized intracellular compartment termed an ‘inclusion’. Alternative persistent intra-cellular forms can be induced in culture by diverse stimuli such as IFNγ or adenosine/EHNA. They do not grow or divide but revive upon withdrawal of the stimulus and are implicated in several widespread human diseases through ill-defined in vivo mechanisms. β-lactam antibiotics have also been claimed to induce persistence in vitro. The present report shows that upon penicillin G (pG) treatment, inclusions grow as fast as those in infected control cells. After removal of pG, Chlamydia do not revert to RBs. These effects are independent of host cell type, serovar, biovar and species of Chlamydia. Time-course experiments demonstrated that only RBs were susceptible to pG. pG-treated bacteria lost their control over host cell apoptotic pathways and no longer expressed pre-16S rRNA, in contrast to persistent bacteria induced with adenosine/EHNA. Confocal and live-video microscopy showed that bacteria within the inclusion fused with lysosomal compartments in pG-treated cells. That leads to recruitment of cathepsin D as early as 3 h post pG treatment, an event preceding bacterial death by several hours. These data demonstrate that pG treatment of cultured cells infected with Chlamydia results in the degradation of the bacteria. In addition we show that pG is significantly more efficient than doxycycline at preventing genital inflammatory lesions in C. muridarum-C57Bl/6 infected mice. These in vivo results support the physiological relevance of our findings and their potential therapeutic applications.

Highlights

Obligate intracellular bacteria of the genus Chlamydiaceae are the causal agents of a spectrum of human diseases of public health importance
In contrast to the bacterial inclusions produced in this persistence model (Ad/EHNA), inclusions produced in infected cells treated by penicillin G (pG) are growing at the same rate as the inclusions developing in untreated cells, both lysing at 100 hpi
We conclude that unlike Ad/EHNA, pG treatment leads to the formation of very enlarged bacterial bodies, which we call ‘‘pGforms’’, contained within a growing inclusion, a phenotype inconsistent with classical persistence

Summary

Introduction

Obligate intracellular bacteria of the genus Chlamydiaceae are the causal agents of a spectrum of human diseases of public health importance. Host cell infection is caused by a small, infectious yet metabolically inert form, the elementary body (EB, 0.3 mm diameter). EBs differentiate into a larger, metabolically active form, the ‘reticulate body’ (RB, 1 mm). RBs forge a replicative niche within a membrane-bound compartment, termed an ‘inclusion’, by hijacking key eukaryotic processes. This includes blocking cellular apoptosis and the fusion of lysosomes with the inclusion [3,4,5,6,7]. RBs subsequently replicate by binary fission, prior to re-differentiating into EBs, which are released to re-infect neighboring cells [8]

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Conclusion