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Chlamydia trachomatis In Vivo to In Vitro Transition Reveals Mechanisms of Phase Variation and Down-Regulation of Virulence Factors.

Borges V, Pinheiro M, Antelo M, Sampaio DA, Vieira L, Ferreira R, Nunes A, Almeida F, Mota LJ, Borrego MJ, Gomes JP - PLoS ONE (2015)

Bottom Line: 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.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).Finally, there was an increase in the growth rate for all strains, reflecting gradual fitness enhancement over time.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

CT135 mRNA decay analysis.Panel A shows the comparison of the relative amount of transcripts at 4 h post-infection (pi) and after 10 min of transcriptional blockage with rifampicin (10 μg/ml) between the ancestral (grey) and the evolved populations (black). The assay was performed for all strains with emergent CT135-negative clones (i.e., all epithelial-genital isolates) and for the strain L2b/CS19/08 (control). The number of transcripts was quantified by independent RT-qPCR targeting the two genes of the operon CT134-CT135 (see methods for details), except for serovar D strain as the evolved population lacks CT135. Data was normalized against the number of C. trachomatis genomes quantified on the corresponding DNA samples. In order to facilitate the comparative analysis, the normalized value before rifampicin treatment (4h pi) was arbitrarily set to 1. Panel B shows the mRNA half-life times calculated based on the fit of an exponential decay between the quantified values at 4h pi and the values calculated 10 minutes after the transcriptional arrest.
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pone.0133420.g005: CT135 mRNA decay analysis.Panel A shows the comparison of the relative amount of transcripts at 4 h post-infection (pi) and after 10 min of transcriptional blockage with rifampicin (10 μg/ml) between the ancestral (grey) and the evolved populations (black). The assay was performed for all strains with emergent CT135-negative clones (i.e., all epithelial-genital isolates) and for the strain L2b/CS19/08 (control). The number of transcripts was quantified by independent RT-qPCR targeting the two genes of the operon CT134-CT135 (see methods for details), except for serovar D strain as the evolved population lacks CT135. Data was normalized against the number of C. trachomatis genomes quantified on the corresponding DNA samples. In order to facilitate the comparative analysis, the normalized value before rifampicin treatment (4h pi) was arbitrarily set to 1. Panel B shows the mRNA half-life times calculated based on the fit of an exponential decay between the quantified values at 4h pi and the values calculated 10 minutes after the transcriptional arrest.

Mentions: The mechanisms for controlling the mRNA processing and decay play an important role in the continuous adjustment of the levels of gene expression according to the protein needs, with bacterial mRNA half-life times ranging from seconds to hours [62, 106]. We evaluated whether the molecular stability of CT135 transcripts was affected after protein truncation. We observed that the mRNA half-life time decreased for the epithelial-genital isolates (Fig 5), whereas it remained unaltered for the LGV clinical isolate. These results are concordant with an adaptive scenario where both the genomic inactivation and mRNA processing mechanisms concomitantly act to increment the competitive fitness of the CT135-negative clones. Curiously, when looking at the mRNA half-life time of the ancestral strains, we observed that the CT135-transcript is more labile for the LGV strain.


Chlamydia trachomatis In Vivo to In Vitro Transition Reveals Mechanisms of Phase Variation and Down-Regulation of Virulence Factors.

Borges V, Pinheiro M, Antelo M, Sampaio DA, Vieira L, Ferreira R, Nunes A, Almeida F, Mota LJ, Borrego MJ, Gomes JP - PLoS ONE (2015)

CT135 mRNA decay analysis.Panel A shows the comparison of the relative amount of transcripts at 4 h post-infection (pi) and after 10 min of transcriptional blockage with rifampicin (10 μg/ml) between the ancestral (grey) and the evolved populations (black). The assay was performed for all strains with emergent CT135-negative clones (i.e., all epithelial-genital isolates) and for the strain L2b/CS19/08 (control). The number of transcripts was quantified by independent RT-qPCR targeting the two genes of the operon CT134-CT135 (see methods for details), except for serovar D strain as the evolved population lacks CT135. Data was normalized against the number of C. trachomatis genomes quantified on the corresponding DNA samples. In order to facilitate the comparative analysis, the normalized value before rifampicin treatment (4h pi) was arbitrarily set to 1. Panel B shows the mRNA half-life times calculated based on the fit of an exponential decay between the quantified values at 4h pi and the values calculated 10 minutes after the transcriptional arrest.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514472&req=5

pone.0133420.g005: CT135 mRNA decay analysis.Panel A shows the comparison of the relative amount of transcripts at 4 h post-infection (pi) and after 10 min of transcriptional blockage with rifampicin (10 μg/ml) between the ancestral (grey) and the evolved populations (black). The assay was performed for all strains with emergent CT135-negative clones (i.e., all epithelial-genital isolates) and for the strain L2b/CS19/08 (control). The number of transcripts was quantified by independent RT-qPCR targeting the two genes of the operon CT134-CT135 (see methods for details), except for serovar D strain as the evolved population lacks CT135. Data was normalized against the number of C. trachomatis genomes quantified on the corresponding DNA samples. In order to facilitate the comparative analysis, the normalized value before rifampicin treatment (4h pi) was arbitrarily set to 1. Panel B shows the mRNA half-life times calculated based on the fit of an exponential decay between the quantified values at 4h pi and the values calculated 10 minutes after the transcriptional arrest.
Mentions: The mechanisms for controlling the mRNA processing and decay play an important role in the continuous adjustment of the levels of gene expression according to the protein needs, with bacterial mRNA half-life times ranging from seconds to hours [62, 106]. We evaluated whether the molecular stability of CT135 transcripts was affected after protein truncation. We observed that the mRNA half-life time decreased for the epithelial-genital isolates (Fig 5), whereas it remained unaltered for the LGV clinical isolate. These results are concordant with an adaptive scenario where both the genomic inactivation and mRNA processing mechanisms concomitantly act to increment the competitive fitness of the CT135-negative clones. Curiously, when looking at the mRNA half-life time of the ancestral strains, we observed that the CT135-transcript is more labile for the LGV strain.

Bottom Line: 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.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).Finally, there was an increase in the growth rate for all strains, reflecting gradual fitness enhancement over time.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus