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.
Highlights
Experimental evolution studies have exploited several organisms’ traits to obtain a real-time window on evolutionary processes, to reveal new genetic targets of selection and, in general, to get insight on the genome-scale dynamics of natural selection within microbial populations, constituting a rare opportunity to deeply uncover the correlation between genetic and phenotypic signatures [1,2,3]
Serovars A-C are normally associated with ocular infections leading to blinding trachoma, strains from serovars D-K are the major cause of bacterial sexually transmitted infections, and strains from serovars L1-L3 are the causative agents of typical bubonic lymphogranuloma venereum (LGV) or the LGV-associated proctitis [39,40,41]
We evaluated the dynamics by which mutations appear and their frequency in the evolving bacterial populations through whole-population sequencing at various time-points after C. trachomatis introduction in vitro
Summary
Experimental evolution studies have exploited several organisms’ traits to obtain a real-time window on evolutionary processes, to reveal new genetic targets of selection and, in general, to get insight on the genome-scale dynamics of natural selection within microbial populations, constituting a rare opportunity to deeply uncover the correlation (or lack of such) between genetic and phenotypic signatures [1,2,3]. Common findings include the detection of genes or mutations underlying phenotypic alterations typically observed in laboratory evolving bacterial populations, such as: evolution of new (or loss of) metabolic capabilities, development of antibiotic resistance or sensitivity, or loss of virulence (namely, reduction of the infective capacity) [1, 3,4,5,6,7,8,9] For the latter alteration, it has been observed that: i) in vitro-cultured microorganisms are in general less virulent than the corresponding wild-types; ii) virulence decreases gradually during in vitro culture; and iii) virulence can eventually be restored by in vivo passage [10,11,12,13,14]. Serovars A-C are normally associated with ocular infections leading to blinding trachoma, strains from serovars D-K are the major cause of bacterial sexually transmitted infections (where serovar E is the most clinically prevalent followed by F), and strains from serovars L1-L3 are the causative agents of typical bubonic lymphogranuloma venereum (LGV) or the LGV-associated proctitis [39,40,41] (the latter is primarily caused by L2b strains [42])
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