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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

Schematic representation of the CT135 deletion in the serovar D strain.The inactivating event of CT135 involved the complete gene deletion between direct repeats (in blue) and the putative formation of a fusion gene enrolling the two flanking genes (CT134 and CT136). The underlying mechanism likely relied in one of three major pathways: A—intermolecular crossing over between direct repeats followed by recombination (yielding both a tandem duplication and a deletion); B—looping out in between direct repeats followed by recombination; and C—DNA polymerase slippage during DNA replication [89–91]. The figure also shows the position of all CT135 frameshift mutations (labeled by Ψ) reported here and elsewhere [46, 48, 131, 149, 150, 164], demonstrating that the strains evolved towards CT135 inactivation regardless the “genetic pathway” that drove that inactivation The bilobal hydrophobic domains that putatively enable the insertion of the CT134 and CT135 proteins into the inclusion membrane [33] are highlighted in grey.
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pone.0133420.g003: Schematic representation of the CT135 deletion in the serovar D strain.The inactivating event of CT135 involved the complete gene deletion between direct repeats (in blue) and the putative formation of a fusion gene enrolling the two flanking genes (CT134 and CT136). The underlying mechanism likely relied in one of three major pathways: A—intermolecular crossing over between direct repeats followed by recombination (yielding both a tandem duplication and a deletion); B—looping out in between direct repeats followed by recombination; and C—DNA polymerase slippage during DNA replication [89–91]. The figure also shows the position of all CT135 frameshift mutations (labeled by Ψ) reported here and elsewhere [46, 48, 131, 149, 150, 164], demonstrating that the strains evolved towards CT135 inactivation regardless the “genetic pathway” that drove that inactivation The bilobal hydrophobic domains that putatively enable the insertion of the CT134 and CT135 proteins into the inclusion membrane [33] are highlighted in grey.

Mentions: We evaluated the emergence and spread of adaptive mutations throughout C. trachomatis experimental evolution (Fig 2). No emergent mutations were detected for the LGV-proctitis strain (L2b/CS19/08) at any time-point. In contrast, we observed inactivating mutations in the virulence gene CT135 for all the epithelial-genital strains, which rapidly rose in frequency. We estimated that, in each 10 in vitro passages of epithelial-genital strains, a mean of 23.1% (SD ±11.9) of the emergent clones will carry inactivating mutations in this virulence gene. An extreme example stands for D/CS637/11, where CT135-negative clones reached a frequency of 100% at passage 20. For this strain, the inactivating mutation consisted of a 1452-bp deletion between direct repeats leading to the putative formation of a fusion gene involving the flanking genes CT134 and CT136 (Fig 3). Three major alternative mechanisms may have mediated this event: i) intermolecular crossing over between DNA direct repeats; ii) intramolecular pairing of repeats by looping out followed by homologous recombination; and iii) DNA polymerase slippage during DNA replication [89–91]. Of note, deletions between direct repeats have been proposed to have played a role in the genome reductive evolution of Chlamydia bacteria [85, 92, 93], namely in the cytotoxin loss in the ocular C. trachomatis serovars A, Ba and C [85]. In support of the expression of the fusion protein CT134-CT136, we observed the existence of transcripts (in RNA-seq) compatible with this novel genomic structure. However, immunoblotting attempts to detect the protein (polyclonal antibody targeting CT136) in culture lysates obtained at mid-cycle were unsuccessful (data not shown).


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)

Schematic representation of the CT135 deletion in the serovar D strain.The inactivating event of CT135 involved the complete gene deletion between direct repeats (in blue) and the putative formation of a fusion gene enrolling the two flanking genes (CT134 and CT136). The underlying mechanism likely relied in one of three major pathways: A—intermolecular crossing over between direct repeats followed by recombination (yielding both a tandem duplication and a deletion); B—looping out in between direct repeats followed by recombination; and C—DNA polymerase slippage during DNA replication [89–91]. The figure also shows the position of all CT135 frameshift mutations (labeled by Ψ) reported here and elsewhere [46, 48, 131, 149, 150, 164], demonstrating that the strains evolved towards CT135 inactivation regardless the “genetic pathway” that drove that inactivation The bilobal hydrophobic domains that putatively enable the insertion of the CT134 and CT135 proteins into the inclusion membrane [33] are highlighted in grey.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133420.g003: Schematic representation of the CT135 deletion in the serovar D strain.The inactivating event of CT135 involved the complete gene deletion between direct repeats (in blue) and the putative formation of a fusion gene enrolling the two flanking genes (CT134 and CT136). The underlying mechanism likely relied in one of three major pathways: A—intermolecular crossing over between direct repeats followed by recombination (yielding both a tandem duplication and a deletion); B—looping out in between direct repeats followed by recombination; and C—DNA polymerase slippage during DNA replication [89–91]. The figure also shows the position of all CT135 frameshift mutations (labeled by Ψ) reported here and elsewhere [46, 48, 131, 149, 150, 164], demonstrating that the strains evolved towards CT135 inactivation regardless the “genetic pathway” that drove that inactivation The bilobal hydrophobic domains that putatively enable the insertion of the CT134 and CT135 proteins into the inclusion membrane [33] are highlighted in grey.
Mentions: We evaluated the emergence and spread of adaptive mutations throughout C. trachomatis experimental evolution (Fig 2). No emergent mutations were detected for the LGV-proctitis strain (L2b/CS19/08) at any time-point. In contrast, we observed inactivating mutations in the virulence gene CT135 for all the epithelial-genital strains, which rapidly rose in frequency. We estimated that, in each 10 in vitro passages of epithelial-genital strains, a mean of 23.1% (SD ±11.9) of the emergent clones will carry inactivating mutations in this virulence gene. An extreme example stands for D/CS637/11, where CT135-negative clones reached a frequency of 100% at passage 20. For this strain, the inactivating mutation consisted of a 1452-bp deletion between direct repeats leading to the putative formation of a fusion gene involving the flanking genes CT134 and CT136 (Fig 3). Three major alternative mechanisms may have mediated this event: i) intermolecular crossing over between DNA direct repeats; ii) intramolecular pairing of repeats by looping out followed by homologous recombination; and iii) DNA polymerase slippage during DNA replication [89–91]. Of note, deletions between direct repeats have been proposed to have played a role in the genome reductive evolution of Chlamydia bacteria [85, 92, 93], namely in the cytotoxin loss in the ocular C. trachomatis serovars A, Ba and C [85]. In support of the expression of the fusion protein CT134-CT136, we observed the existence of transcripts (in RNA-seq) compatible with this novel genomic structure. However, immunoblotting attempts to detect the protein (polyclonal antibody targeting CT136) in culture lysates obtained at mid-cycle were unsuccessful (data not shown).

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