Insertional mutagenesis in the zoonotic pathogen Chlamydia caviae.

Kimberly Filcek,Katarina Vielfort,Samada Muraleedharan,Barbara S Sixt,Johan Henriksson,Raphael H Valdivia,Patrik M Bavoil

doi:10.1371/journal.pone.0224324

Kimberly Filcek, Katarina Vielfort + Show 5 more

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https://doi.org/10.1371/journal.pone.0224324

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Abstract

The ability to introduce targeted genetic modifications in microbial genomes has revolutionized our ability to study the role and mode of action of individual bacterial virulence factors. Although the fastidious lifestyle of obligate intracellular bacterial pathogens poses a technical challenge to such manipulations, the last decade has produced significant advances in our ability to conduct molecular genetic analysis in Chlamydia trachomatis, a major bacterial agent of infertility and blindness. Similar approaches have not been established for the closely related veterinary Chlamydia spp., which cause significant economic damage, as well as rare but potentially life-threatening infections in humans. Here we demonstrate the feasibility of conducting site-specific mutagenesis for disrupting virulence genes in C. caviae, an agent of guinea pig inclusion conjunctivitis that was recently identified as a zoonotic agent in cases of severe community-acquired pneumonia. Using this approach, we generated C. caviae mutants deficient for the secreted effector proteins IncA and SinC. We demonstrate that C. caviae IncA plays a role in mediating fusion of the bacteria-containing vacuoles inhabited by C. caviae. Moreover, using a chicken embryo infection model, we provide first evidence for a role of SinC in C. caviae virulence in vivo.

Highlights

The bacterial genus Chlamydia is comprised of multiple human and animal pathogenic species that are capable of causing significant morbidity and mortality [1]
To enable molecular genetic characterization of virulence factors in C. caviae GPIC, we tested the applicability of the TargeTron system for site-specific insertional mutagenesis in this species
The successful application of the TargeTron system for the generation of IncA- and SinC-deficient C. caviae GPIC strains (Fig 1) marks the first time that stable, site-specific mutations have been introduced in a member of the C. caviae-C. abortus-C. felis-C. psittaci lineage

Summary

Introduction

The bacterial genus Chlamydia is comprised of multiple human and animal pathogenic species that are capable of causing significant morbidity and mortality [1]. The infective stage, the elementary body (EB), invades the host cell in a process that leads to the formation of a pathogen-containing vacuole, named inclusion. Within this inclusion, the EB differentiates into the replicative stage, the reticulate body (RB). The main human pathogenic Chlamydia spp. are Chlamydia trachomatis, responsible for both urogenital and ocular infections [4, 5], and Chlamydia pneumoniae, an agent of respiratory tract infections [6].

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