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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

Mutational scenario throughout experimental evolution.Panel A. Chromosomal location of the genomic alterations observed during the in vitro passaging. The chromosomal position of each mutation (scale adjusted and given by the locus name) and the type of mutation event (inactivating events represented in red) are shown for each strain (see also S3 Table for details). Inactivating SNPs or indels refer to events leading to protein truncation (regardless the length of the resulting protein). For the strain D/CS637/11, the CT135 inactivating event involved the entire gene deletion between direct repeats (Fig 3). Panel B. Dynamics of the emergence and spread of mutations and their frequency in the evolving bacterial populations. For each time-point (passages 5–7, 10, 20, 30, 50 and 100), circular graphs show the frequency of the mutations in the bacterial population, where each color represents a different mutated locus. The number of bacterial generations was estimated taking into account the minimum and maximum values of the mean doubling time of the strains analyzed at each time-point, and assuming a conservative approach by considering 15 hours of exponential phase per bacterial life-cycle (i.e, per passage). Loci designations are based on genome annotation of the D/UW3 strain (GenBank accession number NC_000117).
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pone.0133420.g002: Mutational scenario throughout experimental evolution.Panel A. Chromosomal location of the genomic alterations observed during the in vitro passaging. The chromosomal position of each mutation (scale adjusted and given by the locus name) and the type of mutation event (inactivating events represented in red) are shown for each strain (see also S3 Table for details). Inactivating SNPs or indels refer to events leading to protein truncation (regardless the length of the resulting protein). For the strain D/CS637/11, the CT135 inactivating event involved the entire gene deletion between direct repeats (Fig 3). Panel B. Dynamics of the emergence and spread of mutations and their frequency in the evolving bacterial populations. For each time-point (passages 5–7, 10, 20, 30, 50 and 100), circular graphs show the frequency of the mutations in the bacterial population, where each color represents a different mutated locus. The number of bacterial generations was estimated taking into account the minimum and maximum values of the mean doubling time of the strains analyzed at each time-point, and assuming a conservative approach by considering 15 hours of exponential phase per bacterial life-cycle (i.e, per passage). Loci designations are based on genome annotation of the D/UW3 strain (GenBank accession number NC_000117).

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)

Mutational scenario throughout experimental evolution.Panel A. Chromosomal location of the genomic alterations observed during the in vitro passaging. The chromosomal position of each mutation (scale adjusted and given by the locus name) and the type of mutation event (inactivating events represented in red) are shown for each strain (see also S3 Table for details). Inactivating SNPs or indels refer to events leading to protein truncation (regardless the length of the resulting protein). For the strain D/CS637/11, the CT135 inactivating event involved the entire gene deletion between direct repeats (Fig 3). Panel B. Dynamics of the emergence and spread of mutations and their frequency in the evolving bacterial populations. For each time-point (passages 5–7, 10, 20, 30, 50 and 100), circular graphs show the frequency of the mutations in the bacterial population, where each color represents a different mutated locus. The number of bacterial generations was estimated taking into account the minimum and maximum values of the mean doubling time of the strains analyzed at each time-point, and assuming a conservative approach by considering 15 hours of exponential phase per bacterial life-cycle (i.e, per passage). Loci designations are based on genome annotation of the D/UW3 strain (GenBank accession number NC_000117).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133420.g002: Mutational scenario throughout experimental evolution.Panel A. Chromosomal location of the genomic alterations observed during the in vitro passaging. The chromosomal position of each mutation (scale adjusted and given by the locus name) and the type of mutation event (inactivating events represented in red) are shown for each strain (see also S3 Table for details). Inactivating SNPs or indels refer to events leading to protein truncation (regardless the length of the resulting protein). For the strain D/CS637/11, the CT135 inactivating event involved the entire gene deletion between direct repeats (Fig 3). Panel B. Dynamics of the emergence and spread of mutations and their frequency in the evolving bacterial populations. For each time-point (passages 5–7, 10, 20, 30, 50 and 100), circular graphs show the frequency of the mutations in the bacterial population, where each color represents a different mutated locus. The number of bacterial generations was estimated taking into account the minimum and maximum values of the mean doubling time of the strains analyzed at each time-point, and assuming a conservative approach by considering 15 hours of exponential phase per bacterial life-cycle (i.e, per passage). Loci designations are based on genome annotation of the D/UW3 strain (GenBank accession number NC_000117).
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