title: Effect of long-term laboratory propagation on Chlamydia trachomatis genome dynamics.
abstract: It is assumed that bacterial strains maintained in the laboratory for long time shape their genome in a different fashion from the nature-circulating strains. Here, we analyzed the impact of long-term in vitro propagation on the genome of the obligate intracellular pathogen Chlamydia trachomatis. We fully-sequenced the genome of a historical prototype strain (L2/434/Bu) and a clinical isolate (E/CS88), before and after one-year of serial in vitro passaging (up to 3500 bacterial generations). We observed a slow adaptation of C. trachomatis to the in vitro environment, which was essentially governed by four mutations for L2/434/Bu and solely one mutation for E/CS88, corresponding to estimated mutation rates from 3.84 × 10(-10) to 1.10 × 10(-9) mutations per base pair per generation. In a speculative basis, the mutations likely conferred selective advantage as: (i) mathematical modeling showed that selective advantage is mandatory for frequency increase of a mutated clone; (ii) transversions and non-synonymous mutations were overrepresented; (iii) two non-synonymous mutations affected the genes CTL0084 and CTL0610, encoding a putative transferase and a protein likely implicated in transcription regulation respectively, which are families known to be highly prone to undergone laboratory-derived advantageous mutations in other bacteria; and (iv) the mutation for E/CS88 is located likely in the regulatory region of a virulence gene (CT115/incD) believed to play a role in subverting the host cell machinery. Nevertheless, we found no significant differences in the growth rate, plasmid load, and attachment/entry rate, between strains before and after their long-term laboratory propagation. Of note, from the mixture of clones in E/CS88 initial population, an inactivating mutation in the virulence gene CT135 evolved to 100% prevalence, unequivocally indicating that this gene is superfluous for C. trachomatis survival in vitro. Globally, C. trachomatis revealed a slow in vitro adaptation that only modestly modifies the in vivo-derived genomic evolutionary landscape.
authors.affiliation: Department of Infectious Diseases, National Institute of Health, Av Padre Cruz, 1649-016 Lisbon, Portugal.
authors.forename: Vítor
authors.initials: V
authors.lastname: Borges
authors.forename: Rita
authors.initials: R
authors.lastname: Ferreira
authors.forename: Alexandra
authors.initials: A
authors.lastname: Nunes
authors.forename: Mafalda
authors.initials: M
authors.lastname: Sousa-Uva
authors.forename: Miguel
authors.initials: M
authors.lastname: Abreu
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Borrego
authors.forename: João P
authors.initials: JP
authors.lastname: Gomes
mesh_terms: D002460:Cell Line; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000254:growth & development; D056084:Genetic Fitness; D016680:Genome, Bacterial; D042822:Genomic Instability; D006801:Humans; D015233:Models, Statistical; D009154:Mutation; D059645:Mutation Rate