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Editor's Pick Research Article | Host-Microbe Biology

Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont

Lena König, Alexander Siegl, Thomas Penz, Susanne Haider, Cecilia Wentrup, Julia Polzin, Evelyne Mann, Stephan Schmitz-Esser, Daryl Domman, Matthias Horn
Angela D. Kent, Editor
Lena König
aDepartment of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria
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Alexander Siegl
aDepartment of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria
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Thomas Penz
aDepartment of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria
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Susanne Haider
aDepartment of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria
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Cecilia Wentrup
aDepartment of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria
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Julia Polzin
aDepartment of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria
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Evelyne Mann
bDepartment for Farm Animal and Public Health in Veterinary Medicine, Institute of Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, Austria
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Stephan Schmitz-Esser
bDepartment for Farm Animal and Public Health in Veterinary Medicine, Institute of Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, Austria
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Daryl Domman
aDepartment of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria
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Matthias Horn
aDepartment of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria
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Angela D. Kent
University of Illinois at Urbana-Champaign
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DOI: 10.1128/mSystems.00202-16
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  • FIG 1
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    FIG 1

    Developmental cycle of Protochlamydia amoebophila. (A) Fluorescence in situ hybridization in combination with DAPI staining was used to differentiate between RBs (pink) and EBs (blue). At 2 h postinfection EBs clearly dominate, but a few cells have already started to convert to RBs (white arrowheads). Exclusively RBs were detected at 48 hpi. The first EBs (white arrows) were seen at 72 hpi. The inset for the 24 hpi image is an enlargement of dividing RBs (to enhance clarity, the DAPI signal is shown in white). Dotted white lines indicate the outlines of amoeba host cells. If not indicated otherwise, all bars represent 10 µm. (B) The course of EB production and release was quantified by collecting intra- and extracellular bacteria, respectively, at indicated time points, and subsequent reinfection of fresh amoebae. The first intracellular EBs were present at 72 hpi, the first release of EBs was observed at 96 hpi. Values that are significantly different (P < 0.05) at the various time points by one-way analysis of variance (ANOVA) and Tukey’s posttest are indicated by an asterisk (n = 3). Prop., proportion. (C) Transmission electron micrographs visualizing developmental events at the ultrastructural level. Black arrows point to bacterial cells in overview images. Black arrowheads indicate vesicles that were observed in the inclusion lumen from 24 hpi on. Bars = 1 µm.

  • FIG 2
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    FIG 2

    Temporal classes of gene expression during the Protochlamydia developmental cycle. A total of 797 genes were detected as differentially expressed; tRNA genes (20 genes), rRNA genes (2 genes), and genes detected only in a single replicate (38 genes) were excluded from further analysis. Clustering identified three main temporal classes of gene sets (colored bars to the left of the heatmap) that could be further divided into five large subclasses (colored bars to the right of the heatmap). The largest group of genes was most highly expressed early (n = 304), whereas the expression of the smallest group of genes peaked at midcycle when only RBs were present (n = 161). The third main gene cluster generally showed highest expression at the end of the cycle and the extracellular stage (n = 273; see Data Set S1 in the supplemental material). Gene products detected in the EB proteome in a previous study (n = 231) (39) are indicated in the bar plot next to the heatmap. To illustrate the course of gene expression for each subcluster, the expression values (log2 RPKM plotted on the y axis) were averaged per time point (x axis) and visualized as line plots (error bars indicate standard deviations). Selected gene names are shown for each of the temporal clusters. RPKM, reads per kilobase per million; hpi, h postinfection; extracell., extracellular; PG, peptidoglycan synthesis; T3SS, type III secretion system.

  • FIG 3
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    FIG 3

    Enrichment of functional categories by temporal class. The overrepresentation of functional categories among genes assigned to temporal classes provides evidence for stage-specific activities during the Protochlamydia developmental cycle. Only functional categories that were significantly enriched with a false-discovery rate (FDR) of ≤0.05 are shown here; the color code indicates the degree of significance (dark red indicates highly significant). The significant enrichment of putatively type III secreted gene products was tested using Fisher’s exact test, and only P values of ≤0.05 are shown. cat, category.

  • FIG 4
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    FIG 4

    Course of Protochlamydia type III secretion system activity during the developmental cycle. This model is based on the observation that structural components of the type III secretion system and its (putative) effectors are expressed at different time points during the developmental cycle (Fig. S4). This suggests a scenario in which novel, fully assembled, and thus functional secretion systems occur only late in the developmental cycle, and type III secretion reaches its full capacity and highest activity during early stages of the infection. The indicated polarity of the active type III secretion system has been shown for C. trachomatis (54), but it is unclear whether this is also true for P. amoebophila, as the symbionts reside within single-cell inclusions. The color code for type III secretion components and effectors (nomenclature according to Hueck [105]) refers to the respective temporal gene expression classes (Fig. 2 and Fig. S4). Circles inside the cells represent chaperones. Differentially expressed components/effectors are labeled with asterisks; PEX1 and PEX2 refer to members of the expanded effector gene families in Protochlamydia (33); pc0309 is an ortholog of the putative chaperone encoded by CT274 (106). inc, inclusion membrane; hcm, host cell membrane; im, inner membrane; om, outer membrane.

  • FIG 5
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    FIG 5

    Expression maps of selected metabolic pathways of Protochlamydia. A pronounced expression of the ATP/ADP translocase (ntt1) at midcycle and an early-to-mid activity of genes involved in amino acid breakdown to pyruvate is observed, whereas pathways involved in central carbon metabolism and energy generation were generally only upregulated at later stages (with the ATPases indicated by purple boxes being notable exceptions). This suggests that a major metabolic shift occurs during the developmental cycle and provides evidence for a stage-specific metabolism. All genes marked with an asterisk were detected to be significantly differentially expressed. RPKM, reads per kilobase per million; hpi, h postinfection; extracell., extracellular.

  • FIG 6
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    FIG 6

    Biphasic metabolism of Protochlamydia during development in Acanthamoeba castellanii. This model is based on observed transcriptional patterns, enriched functional categories at different developmental stages (Fig. 2, 3, and 5 and Fig. S2A), and independent experimental evidence reported previously (see text for references). Activity of metabolic pathways as inferred from gene expression levels followed similar trends early and at midcycle, and at the two later stages, respectively. This suggests that ATP import and an amino acid-based anabolism prevails during the EB-to-RB transition and RB replication. Later stages are characterized by a glucose-based metabolism and a pronounced increase in the activity of the tricarboxylic acid (TCA) cycle and oxidative (ox.) phosphorylation pathway. Nucleotide transporters (Ntt’s) are shown in blue, and amino acid (AA) and oligopeptide transporters are shown in green. “Multi” indicates that multiple amino acid/peptide transporters with different substrate specificities are expressed. Question marks refer to hypothetical transporters not yet identified. Asterisks indicate an increased expression at the RB stage compared to the early time point. “RNA” denotes transcription, whereas “DNA” indicates DNA replication. Glc-6-P, glucose 6-phosphate; PPP, pentose phosphate pathway; glyconeog., gluconeogenesis.

Supplemental Material

  • Figures
  • TEXT S1

    Supplemental methods, results, and discussion. Download TEXT S1, PDF file, 0.1 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S1

    Similarity of Protochlamydia gene expression values between replicates and time points. The Euclidean distances between all normalized expression values (reads per kilobase per million [RPKM]) of each replicate and time point are shown, with darker colors meaning high similarity. The order of the samples is based on hierarchical cluster analysis of the distances using the complete linkage method—the clustering being illustrated by the dendrograms. The samples cluster by time point and therefore correctly reflect the experimental design. For subsequent analyses, genes were considered to be expressed at a particular time point when we obtained expression values in two or three replicates. The contribution of genes for which expression was detected in only two replicates is indicated in the right panel. Relatively low sequencing coverage at 2 h postinfection (hpi) explains the higher numbers of these genes at this early time point, and consequently also the larger distance between the 2 hpi replicates. Excl., excluding. Download FIG S1, PDF file, 0.2 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S2

    (A) Gene expression heatmaps of constitutively expressed genes and (B) genes involved in peptidoglycan synthesis and cell division. (A) All genes that were detected to be expressed (in at least two replicates) but were found not to be significantly differentially expressed (n = 712) were termed constitutively expressed. Average gene-wise expression values (log2 reads per kilobase per million [RPKM]) were hierarchically clustered based on Euclidean distances using the complete linkage method. Five clusters representing five different expression levels were defined on the basis of the dendrogram’s branching pattern. Accordingly, we found 9 very highly expressed genes, 60 highly expressed genes, 131 genes with medium expression strength, 328 genes with medium-low expression strength, and 184 genes with low expression. Each cluster was tested for the presence of overrepresented COG classes and clusters, KEGG pathways, and GO terms for which only categories with a false discovery rate (FDR) below 0.05 are listed. In addition, a few selected genes are shown. Corresponding proteins that were detected in P. amoebophila EBs in a previous study (B. S. Sixt, C. Heinz, P. Pichler, E. Heinz, J. Montanaro, et al., Proteomics 11:1868–1892, 2011, https://doi.org/10.1002/pmic.201000510 ) are indicated by black lines (n = 234). (B) Average expression values (log2 RPKM) for genes involved in peptidoglycan synthesis and cell division (N. Jacquier, P. H. Viollier, and G. Greub, FEMS Microbiol Rev 39:262–275, 2015, https://doi.org/10.1093/femsre/fuv001 ) were hierarchically clustered (average linkage) based on Pearson correlation distances. Genes found to be differentially expressed between any two consecutive time points are marked with an asterisk. hpi, h postinfection; extracell., extracellular. Download FIG S2, PDF file, 0.7 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S3

    Gene expression dynamics during the Protochlamydia developmental cycle. The intersecting gene sets of statistically significantly up- or downregulated genes between successive time point pairs are shown. Each color represents a different set of differentially expressed (DE) genes, with the height of the bar indicating the number of genes. For example, at 2 h postinfection (hpi), there are a total of 250 significantly upregulated genes of which 153 genes are downregulated at the next time point (orange bars). Note that there are overlaps between gene sets of two consecutive time points. For instance, from the orange gene set (upregulated at 2 hpi and downregulated at 48 hpi), 22 genes were subsequently also downregulated at 96 hpi (pink boxes). White areas represent DE genes that were detected as significantly regulated only once during the cycle. Download FIG S3, PDF file, 0.2 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • TABLE S1

    Sequencing and read mapping statistics. Download TABLE S1, PDF file, 0.1 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • DATA SET S1

    Gene expression values and annotation of Protochlamydia and A. castellanii. Download DATA SET S1, XLSX file, 1.8 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S4

    Expression patterns of genes encoding components of the type III secretion system and its (putative) effectors. All genes that met the following criteria were included: (i) genes that have been previously reported to encode components involved in Protochlamydia type III secretion (A. Collingro, P. Tischler, T. Weinmaier, T. Penz, E. Heinz, et al., Mol Biol Evol 28:3253–3270, 2011, https://doi.org/10.1093/molbev/msr161 ); (ii) genes that are orthologues of putative C. trachomatis effector proteins or structural components and chaperones of the chlamydial type III secretion system (H. J. Betts-Hampikian and K. A. Fields, Front Microbiol 1:114, 2010, https://doi.org/10.3389/fmicb.2010.00114 ); (iii) genes that were annotated to contain eukaryotic-like domains such as ankyrin repeats (ANK), Sel1-like repeats (Sel1), F-boxes (F-box), leucine-rich repeats (LRR, PEX1), tetratricopeptide repeats (TPR, PEX2), BTB/POZ domains (PEX1), and GDP-GTP exchange domains (RasGEF, RhoGEF); (iv) genes that were predicted to encode inclusion membrane proteins (put. inc) (E. Heinz, D. D. Rockey, J. Montanaro, K. Aistleitner, M. Wagner, et al., J Bacteriol 192:5093–5102, 2010, https://doi.org/10.1128/JB.00605-10 ); and (v) genes that encode putative effectors based on expression pattern and genomic location (data not shown). Differentially expressed genes are marked with an asterisk. Temporal classes are indicated by colored bars. The sct unified nomenclature for the naming of type III secretion system genes was used (C. J. Hueck, Microbiol Mol Biol Rev 62:379-433, 1998). RPKM, reads per kilobase per million; hpi, h postinfection; extracell., extracellular; PEX1, Protochlamydia expanded gene family 1; PEX2, Protochlamydia expanded gene family 2 (D. Domman, A. Collingro, I. Lagkouvardos, L. Gehre, T. Weinmaier, et al., Mol Biol Evol 31:2890–2904, 2014, https://doi.org/10.1093/molbev/msu227 ); put, putative; Ctr, C. trachomatis. Download FIG S4, PDF file, 1.3 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S5

    Expression of species-specific and chlamydial core genes. (A) Heatmap showing the distribution of temporarily regulated Protochlamydia genes at different levels of taxonomic conservation. The color indicates the percentage of differentially expressed (DE) genes per temporal class and taxonomic group. The absolute numbers of DE genes per temporal class and taxonomic group are indicated; the total numbers of DE genes per taxonomic group are shown in brackets. Note that the early gene set is dominated by species-specific genes (32%), whereas chlamydial core genes are most abundant at midcycle (44%). (B) Nearly all chlamydial core genes are expressed in Protochlamydia, with constitutively expressed genes contributing 62%. Download FIG S5, PDF file, 0.2 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S6

    Gene expression dynamics of the Acanthamoeba host during infection with Protochlamydia. Ten different temporal subclasses of genes can be distinguished (indicated by black lines) within three main classes (colored bars). Major gene expression shifts occurred early during infection (yellow) and at the peak of Protochlamydia RB activity (red). Only significantly differentially expressed genes are shown. The number of genes per temporal class is indicated. RPKM, reads per kilobase per million; hpi, h postinfection. Download FIG S6, PDF file, 1.2 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • TABLE S2

    Overrepresented functions among A. castellanii genes. Download TABLE S2, PDF file, 0.1 MB.

    Copyright © 2017 König et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

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Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont
Lena König, Alexander Siegl, Thomas Penz, Susanne Haider, Cecilia Wentrup, Julia Polzin, Evelyne Mann, Stephan Schmitz-Esser, Daryl Domman, Matthias Horn
mSystems May 2017, 2 (3) e00202-16; DOI: 10.1128/mSystems.00202-16

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Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont
Lena König, Alexander Siegl, Thomas Penz, Susanne Haider, Cecilia Wentrup, Julia Polzin, Evelyne Mann, Stephan Schmitz-Esser, Daryl Domman, Matthias Horn
mSystems May 2017, 2 (3) e00202-16; DOI: 10.1128/mSystems.00202-16
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KEYWORDS

Protochlamydia
RNA-seq
chlamydia
developmental cycle
gene expression
host-microbe interaction
metabolism
symbiont
type III secretion system

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