title: Chlamydia trachomatis In Vivo to In Vitro Transition Reveals Mechanisms of Phase Variation and Down-Regulation of Virulence Factors.
abstract: Research on the obligate intracellular bacterium Chlamydia trachomatis demands culture in cell-lines, but the adaptive process behind the in vivo to in vitro transition is not understood. We assessed the genomic and transcriptomic dynamics underlying C. trachomatis in vitro adaptation of strains representing the three disease groups (ocular, epithelial-genital and lymphogranuloma venereum) propagated in epithelial cells over multiple passages. We found genetic features potentially underlying phase variation mechanisms mediating the regulation of a lipid A biosynthesis enzyme (CT533/LpxC), and the functionality of the cytotoxin (CT166) through an ON/OFF mechanism. We detected inactivating mutations in CT713/porB, a scenario suggesting metabolic adaptation to the available carbon source. CT135 was inactivated in a tropism-specific manner, with CT135-negative clones emerging for all epithelial-genital populations (but not for LGV and ocular populations) and rapidly increasing in frequency (~23% mutants per 10 passages). RNA-sequencing analyses revealed that a deletion event involving CT135 impacted the expression of multiple virulence factors, namely effectors known to play a role in the C. trachomatis host-cell invasion or subversion (e.g., CT456/Tarp, CT694, CT875/TepP and CT868/ChlaDub1). This reflects a scenario of attenuation of C. trachomatis virulence in vitro, which may take place independently or in a cumulative fashion with the also observed down-regulation of plasmid-related virulence factors. This issue may be relevant on behalf of the recent advances in Chlamydia mutagenesis and transformation where culture propagation for selecting mutants/transformants is mandatory. Finally, there was an increase in the growth rate for all strains, reflecting gradual fitness enhancement over time. In general, these data shed light on the adaptive process underlying the C. trachomatis in vivo to in vitro transition, and indicates that it would be prudent to restrict culture propagation to minimal passages and check the status of the CT135 genotype in order to avoid the selection of CT135-negative mutants, likely originating less virulent strains.
authors.affiliation: Reference Laboratory of Bacterial Sexually Transmitted Infections, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal; Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal.
authors.forename: Vítor
authors.initials: V
authors.lastname: Borges
authors.affiliation: School of Medicine, University of St Andrews, St Andrews, Scotland, United Kingdom.
authors.forename: Miguel
authors.initials: M
authors.lastname: Pinheiro
authors.affiliation: Reference Laboratory of Bacterial Sexually Transmitted Infections, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal.
authors.forename: Minia
authors.initials: M
authors.lastname: Antelo
authors.affiliation: Innovation and Technology Unit, Human Genetics Department, National Institute of Health, Lisbon, Portugal.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Sampaio
authors.affiliation: Innovation and Technology Unit, Human Genetics Department, National Institute of Health, Lisbon, Portugal.
authors.forename: Luís
authors.initials: L
authors.lastname: Vieira
authors.affiliation: Reference Laboratory of Bacterial Sexually Transmitted Infections, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal.
authors.forename: Rita
authors.initials: R
authors.lastname: Ferreira
authors.affiliation: Reference Laboratory of Bacterial Sexually Transmitted Infections, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal; Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Nunes
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: Filipe
authors.initials: F
authors.lastname: Almeida
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: Luís J
authors.initials: LJ
authors.lastname: Mota
authors.affiliation: Reference Laboratory of Bacterial Sexually Transmitted Infections, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Borrego
authors.affiliation: Reference Laboratory of Bacterial Sexually Transmitted Infections, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal; Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal.
authors.forename: João P
authors.initials: JP
authors.lastname: Gomes
mesh_terms: D002692:Chlamydia trachomatis / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D016680:Genome, Bacterial; D005838:Genotype; D011110:Polymorphism, Genetic; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D037521:Virulence Factors