Multinucleation during C. trachomatis Infections Is Caused by the Contribution of Two Effector Pathways

Abstract

Chlamydia trachomatis is an obligate intracellular bacterial pathogen and the second leading cause of sexually transmitted infections in the US. Infections cause significant morbidity and can lead to serious reproductive sequelae, including an epidemiological link to increased rates of reproductive cancers. One of the overt changes that infected cells exhibit is the development of genomic instability leading to multinucleation. Here we demonstrate that the induction of multinucleation is not conserved equally across chlamydial species; C. trachomatis L2 caused high levels of multinucleation, C. muridarum intermediate levels, and C. caviae had very modest effects on multinucleation. Our data show that at least two effector pathways together cause genomic instability during infection leading to multinucleation. We find that the highly conserved chlamydial protease CPAF is a key effector for one of these pathways. CPAF secretion is required for the loss of centrosome duplication regulation as well as inducing early mitotic exit. The second effector pathway involves the induction of centrosome position errors. This function is not conserved in three chlamydial species tested. Together these two pathways contribute to the induction of high levels of genomic instability and multinucleation seen in C. trachomatis infections.

Integrating Chemical Mutagenesis and Whole-Genome Sequencing as a Platform for Forward and Reverse Genetic Analysis of Chlamydia

“…Indeed, mutants from this collection have been recently characterized (Chen et al, 2014; Snavely and Kokes et al, 2014; Brown et al, 2014), underscoring its utility in reverse genetic applications. Mutant strains were sequenced in pools of 20 to lower the overall DNA sequencing costs (~$15/genome).…”

Chlamydia cell biology and pathogenesis

“…Chlamydia spp. induce early mitotic exit by bypassing the spindle assembly checkpoint 111 . C. pneumoniae may mediate the arrest of the cell cycle through the T3SS effector, CopN, which has been reported to bind to microtubules and perturb their assembly in vitro ; however, whether this occurs during infection is unclear 112,113 .…”

“…CPAF is required for the induction of centrosome amplification, potentially through the degradation of one or more proteins that control centrosome duplication 111 . C. trachomatis also prevents centrosome clustering during mitosis in a CPAF-independent manner, presumably through the Inc-mediated tethering of centrosomes to the inclusion 51,111,115,116 . The cumulative effect of centro some amplification, early mitotic exit and errors in centrosome-positioning, halts cytokinesis, which results in multinucleated cells 111,114 .…”

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…5). This collection of mutants also serves as a platform for reverse genetics and has facilitated the identification of several strains carrying recently characterized mutations of interest (160)(161)(162). Furthermore, the remaining point mutations offer a comprehensive source of point mutations that can be screened for suppressors, adaptive mutations, conditional alleles, and partial loss-and gain-of-function mutations.…”