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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

Comparative analysis of global gene expression (RNA-seq) between D/CT135-positive and D/CT135-negative populations.Panels A-B. Comparison of gene expression between biological replicates for the D/CT135-positive (A) and D/CT135-negative (B) populations. Pearson correlation coefficients are shown. Panel C. Comparison of gene expression between the D/CT135-negative and D/CT135-positive populations. The red points mark genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected P-values were below 0.05. For panels A to C, axes are log10-transformed normalized expression levels (FPKM). Panel D. Volcano plot of –log2 fold change (D/CT135-positive versus D/CT135-negative) versus –log10 adjusted P-values. In order to better fit the scale to data, corrected P-values ≤10−3 were set as 10−3. Points in red indicate genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected P-values were below 0.05.
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pone.0133420.g006: Comparative analysis of global gene expression (RNA-seq) between D/CT135-positive and D/CT135-negative populations.Panels A-B. Comparison of gene expression between biological replicates for the D/CT135-positive (A) and D/CT135-negative (B) populations. Pearson correlation coefficients are shown. Panel C. Comparison of gene expression between the D/CT135-negative and D/CT135-positive populations. The red points mark genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected P-values were below 0.05. For panels A to C, axes are log10-transformed normalized expression levels (FPKM). Panel D. Volcano plot of –log2 fold change (D/CT135-positive versus D/CT135-negative) versus –log10 adjusted P-values. In order to better fit the scale to data, corrected P-values ≤10−3 were set as 10−3. Points in red indicate genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected P-values were below 0.05.

Mentions: The mechanism by which CT135 promotes C. trachomatis virulence is unknown. Taking into account the rapid and parallel loss of the CT135 in all epithelial-genital strains, we wonder if the in vitro inactivation of CT135 is beneficial likely because it reduces the expression of several genes, and hence the associated energetic costs of unused functions. We performed RNA-seq analyses to compare the global gene expression of the serovar D CT135-positive and CT135-negative strains. Here, we are assuming that potential changes in gene expression are essentially due to the CT135 loss, but the deletion of 1452-bp also partially involved the flanking genes CT134 (encodes a putative Inc protein) and CT136 (encodes a Lysophospholipase esterase) (Fig 3), so a synergistic effect cannot be ruled out. This particularly stands for CT134, as it is predicted to belong to the same operon as CT135 [46]. By using a false discovery rate cutoff of 0.05 and a fold-change cutoff of >2.0, we identified 48 significantly differentially expressed genes and one non-coding RNA, all being down-regulated in the CT135-negative strain (Table 1 and Fig 6). To confirm these results, we used RT-qPCR to evaluate the expression of a set of the highlighted genes and found a good level of correlation (slope 1.01, Pearson correlation 0.958, N = 7) (S2 Fig). Remarkably, the pool of genes that are down-regulated after the occurrence of the deletion event involves multiple virulence-associated genes (some of them belonging to the same operon) (Table 1). We highlight genes coding for: i) the most prominent adhesins (e.g., CT443/OmcB) [107, 108] and T3S effectors (e.g., CT456/Tarp, CT694 and CT875/TepP) known to play a role in the C. trachomatis host-cell invasion process [109–112]; ii) multiple T3S-related proteins, such as substrates (e.g., CT082, CT619-20 and CT847-9) [38, 99, 113–115], a chaperone (CT576/Scc2) [116] and components of the translocation pore (e.g., CT578/CopB and CT579/CopD) [117]; iii) genes encoding proteins putatively related to the chlamydial protease/proteasome-like activity factor (CPAF), either potential substrates (e.g., CT005, CT288, CT443/OmcB, CT456/Tarp and CT694-5) [118–120] or other virulence proteases acting in the same pathways in the subversion of host-cellular functions (e.g., CT441/Tsp and CT868/ChlaDub1) [121–124]; and iv) virulence-associated proteins regulated by the plasmid-encoded Pgp4 (e.g., CT049-CT051, CT142-CT144 and CT798/GlgA) [51]. Regarding the latter set of genes, we looked at the differential expression of their regulator-encoded gene (ORF6/pgp4) and observed a slight decrease of expression (~1.4-fold) in the CT135-negative strain. Thus, we hypothesize that the down-regulation of those genes might have been mediated by pgp4 underexpression. Of note, the down-regulated genes include all members (CT619, CT620, CT621, CT711, and CT712) except CT621 of a family of chlamydial T3S substrates characterized by a domain of unknown function (DUF582 proteins) that are believed to target nuclear cell functions [115, 125]. The highly down-regulated non-coding small RNA (sRNA) (located between CT080 and CT082, and previously designated ctrR0332 in the L2b/UCH-1 strain) (Table 1) was previously found to be overrepresented in EBs, which is consistent with the profile found for the majority of the affected transcripts [126]. Noteworthy, we observed only one differentially expressed gene (CT377/ltuA) in the control experiment with the Ia/CS190/96 strain. This result suggests that the short-term laboratory propagation of C. trachomatis does not substantially change gene expression in absence of genomic alterations.


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)

Comparative analysis of global gene expression (RNA-seq) between D/CT135-positive and D/CT135-negative populations.Panels A-B. Comparison of gene expression between biological replicates for the D/CT135-positive (A) and D/CT135-negative (B) populations. Pearson correlation coefficients are shown. Panel C. Comparison of gene expression between the D/CT135-negative and D/CT135-positive populations. The red points mark genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected P-values were below 0.05. For panels A to C, axes are log10-transformed normalized expression levels (FPKM). Panel D. Volcano plot of –log2 fold change (D/CT135-positive versus D/CT135-negative) versus –log10 adjusted P-values. In order to better fit the scale to data, corrected P-values ≤10−3 were set as 10−3. Points in red indicate genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected P-values were below 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133420.g006: Comparative analysis of global gene expression (RNA-seq) between D/CT135-positive and D/CT135-negative populations.Panels A-B. Comparison of gene expression between biological replicates for the D/CT135-positive (A) and D/CT135-negative (B) populations. Pearson correlation coefficients are shown. Panel C. Comparison of gene expression between the D/CT135-negative and D/CT135-positive populations. The red points mark genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected P-values were below 0.05. For panels A to C, axes are log10-transformed normalized expression levels (FPKM). Panel D. Volcano plot of –log2 fold change (D/CT135-positive versus D/CT135-negative) versus –log10 adjusted P-values. In order to better fit the scale to data, corrected P-values ≤10−3 were set as 10−3. Points in red indicate genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected P-values were below 0.05.
Mentions: The mechanism by which CT135 promotes C. trachomatis virulence is unknown. Taking into account the rapid and parallel loss of the CT135 in all epithelial-genital strains, we wonder if the in vitro inactivation of CT135 is beneficial likely because it reduces the expression of several genes, and hence the associated energetic costs of unused functions. We performed RNA-seq analyses to compare the global gene expression of the serovar D CT135-positive and CT135-negative strains. Here, we are assuming that potential changes in gene expression are essentially due to the CT135 loss, but the deletion of 1452-bp also partially involved the flanking genes CT134 (encodes a putative Inc protein) and CT136 (encodes a Lysophospholipase esterase) (Fig 3), so a synergistic effect cannot be ruled out. This particularly stands for CT134, as it is predicted to belong to the same operon as CT135 [46]. By using a false discovery rate cutoff of 0.05 and a fold-change cutoff of >2.0, we identified 48 significantly differentially expressed genes and one non-coding RNA, all being down-regulated in the CT135-negative strain (Table 1 and Fig 6). To confirm these results, we used RT-qPCR to evaluate the expression of a set of the highlighted genes and found a good level of correlation (slope 1.01, Pearson correlation 0.958, N = 7) (S2 Fig). Remarkably, the pool of genes that are down-regulated after the occurrence of the deletion event involves multiple virulence-associated genes (some of them belonging to the same operon) (Table 1). We highlight genes coding for: i) the most prominent adhesins (e.g., CT443/OmcB) [107, 108] and T3S effectors (e.g., CT456/Tarp, CT694 and CT875/TepP) known to play a role in the C. trachomatis host-cell invasion process [109–112]; ii) multiple T3S-related proteins, such as substrates (e.g., CT082, CT619-20 and CT847-9) [38, 99, 113–115], a chaperone (CT576/Scc2) [116] and components of the translocation pore (e.g., CT578/CopB and CT579/CopD) [117]; iii) genes encoding proteins putatively related to the chlamydial protease/proteasome-like activity factor (CPAF), either potential substrates (e.g., CT005, CT288, CT443/OmcB, CT456/Tarp and CT694-5) [118–120] or other virulence proteases acting in the same pathways in the subversion of host-cellular functions (e.g., CT441/Tsp and CT868/ChlaDub1) [121–124]; and iv) virulence-associated proteins regulated by the plasmid-encoded Pgp4 (e.g., CT049-CT051, CT142-CT144 and CT798/GlgA) [51]. Regarding the latter set of genes, we looked at the differential expression of their regulator-encoded gene (ORF6/pgp4) and observed a slight decrease of expression (~1.4-fold) in the CT135-negative strain. Thus, we hypothesize that the down-regulation of those genes might have been mediated by pgp4 underexpression. Of note, the down-regulated genes include all members (CT619, CT620, CT621, CT711, and CT712) except CT621 of a family of chlamydial T3S substrates characterized by a domain of unknown function (DUF582 proteins) that are believed to target nuclear cell functions [115, 125]. The highly down-regulated non-coding small RNA (sRNA) (located between CT080 and CT082, and previously designated ctrR0332 in the L2b/UCH-1 strain) (Table 1) was previously found to be overrepresented in EBs, which is consistent with the profile found for the majority of the affected transcripts [126]. Noteworthy, we observed only one differentially expressed gene (CT377/ltuA) in the control experiment with the Ia/CS190/96 strain. This result suggests that the short-term laboratory propagation of C. trachomatis does not substantially change gene expression in absence of genomic alterations.

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