Metabolic features of Protochlamydia amoebophila elementary bodies--a link between activity and infectivity in Chlamydiae

The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from (13)C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide evidence that metabolic activity in the extracellular stage of chlamydiae is of major biological relevance as it is a critical factor affecting maintenance of infectivity

Effect of substrate availability on maintenance of infectivity.

<p><i>P. amoebophila</i> and <i>C. trachomatis</i> cells were harvested from infected amoeba and HeLa 229 cell cultures, respectively, and incubated for indicated periods of time in different host-free media. Subsequently, incubated bacteria were used to infect amoebae (<i>P. amoebophila</i>) or HeLa 229 cells (<i>C. trachomatis</i>), which were then fixed at 48 h or 24 h p.i., respectively. Bacteria were detected by FISH (<i>P. amoebophila</i>) or immunostaining (<i>C. trachomatis</i>). The observed infectivity, relative to that observed for 2 h incubation in DGM-D (<i>P. amoebophila</i>) or 30 min incubation in DGM-D6P (<i>C. trachomatis</i>) is depicted in (A) and (C), respectively. Data represent means and standard deviations from at least three replicate host-free incubations. For each sample a minimum of 600 amoebae (A) or 300 HeLa 229 cells (C) was counted. Statistically significant differences compared to the values obtained for DGM-D (A) or DGM-D6P (C) are indicated (ANOVA; ***, p≤0.001; **, p≤0.01; *, p≤0.05). In (B) representative fluorescence and DIC images of amoebae infected with <i>P. amoebophila</i> after 48 h host-free incubation in the indicated media are shown (FISH, red). The bar indicates 10 µm. In (D) representative confocal fluorescence images of HeLa 229 cells infected with <i>C. trachomatis</i> after 2 h host-free incubation in the indicated media are shown. Bacteria were detected by immunostaining (red), host cells and DNA were stained using HCS cytoplasmic stain (grey) and DAPI (blue), respectively. The bar indicates 25 µm.</p

Respiratory activity of <i>P. amoebophila</i> developmental stages and effect of D-glucose deprivation.

<p>Fractions of <i>P. amoebophila</i> developmental forms were subjected to host-free incubation in DGM-D (or DGM-L, if indicated) containing 5 mM CTC, either immediately after purification (“0 h pre-incub”) or after a 40 h pre-incubation in the respective medium (“40 h pre-incub”), followed by the detection of bacteria with the DNA dye DAPI. Heat-inactivated EBs were included as control. The percentage of CTC-positive DAPI-stained bacteria was determined and subsequently corrected based on observed differences in the detectability of bacteria among fractions. Displayed data represent means and standard deviations of three independent experiments. For each experiment and condition, in total 1500 bacteria were considered for the quantification of CTC-positive bacteria, and 450 bacteria for the subsequent correction. Statistically significant differences compared to the EB fraction (ANOVA) are indicated by stars located directly above the bars, significant differences between immediate activity and activity after pre-incubation (t-test) are indicated by the stars at the upper edge of the diagram (***, p≤0.001; **, p≤0.01; *, p≤0.05).</p

Central carbon metabolism in <i>P. amoebophila</i> EBs deduced from mass spectrometry-based metabolite analysis.

<p>A schematic representation of the central carbon metabolism in <i>P. amoebophila</i> is shown in (A). ¹³C-labeled metabolites detected by ICR/FT-MS or UPLC-MS in extracts of DGM-D-13C15N-incubated living bacteria are indicated. The isotopologs that were observed are additionally specified by bars, consisting of a number of units equal to the number of C atoms in the molecules, whereby each unit of the bar indicates either a ¹²C (white) or a ¹³C (gray) atom. Percentage values next to the bars denote the relative abundance of the isotopologs calculated from the mass signal intensity or peak area of the respective peak (for ICR/FT-MS or UPLC-MS data, respectively) compared to peaks of the unlabeled metabolite detected in DGM-D-incubated bacteria. For citrate, for which all possible isotopologs were detected by UPLC-MS, instead of bars the complete isotopolog profile observed in DGM-D-13C15N-incubated living EBs is shown. In (B), mass signal intensities and peak areas (for ICR/FT-MS or UPLC-MS data, respectively) of selected fully ¹³C-labeled (“13C”) and corresponding unlabeled (“12C”) metabolites are displayed for DGM-D-13C15N-incubated living bacteria and the controls. Note the absence of fully ¹³C-labeled isotopologs in extracts from inactivated bacteria and the appearance of labeled intermediates and the concomitant reduction in the amount of the corresponding unlabeled metabolite in samples of DGM-D-1315N-incubated living bacteria compared to DGM-D-incubated bacteria. Exceptions were the detection of labeled glucose and phenylalanine in extracts of inactivated bacteria, presumably due to substrate adsorption to the surface of the bacterial cells.</p

Overview of ICR/FT-MS-detected annotated compounds and of metabolites discriminative for living compared to inactivated EBs.

<p>An EB-enriched fraction of <i>P. amoebophila</i> was pre-incubated for 40 h in DGM-D, followed by 48 h incubation in DGM-D or DGM-D-13C15N and subsequent mass spectrometric analysis of metabolite extracts. Heat-inactivated EBs incubated in DGM-D-13C15N were included as control. M/z features detected by ICR/FT-MS in samples of DGM-D-incubated EBs were annotated by MassTRIX <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003553#ppat.1003553-Suhre1" target="_blank">[61]</a>, followed by data analysis using PLS-DA (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003553#ppat.1003553.s006" target="_blank">Fig. S6</a>) to extract the most discriminative compounds characterizing living compared to inactivated EBs. The PLS-DA model included data from DGM-D-incubated living and DGM-D-13C15N-incubated inactivated EBs. Bars indicate the total number of annotated metabolites that were assigned to specific KEGG pathways. The number of metabolites that were found to be discriminative (black) or non-discriminative (white) for living EBs are additionally indicated. Note that metabolites from carbohydrate, nucleotide, cofactor, vitamin, and amino acid metabolism were more abundant in living EBs, whereas the pattern of lipid species was more similar between living and inactivated bacteria.</p

Annotated ¹³C-labeled metabolites detected in DGM-D-13C15N-incubated EBs by a combination of ICR/FT-MS and UPLC-MS.

<p><b>^a)</b>The detected m/z is given for each ion species with the instrument given accuracy.</p><p><b>^b)</b>Labeled metabolites were considered to be present in extracts of DGM-D-13C15N-incubated EBs when peaks corresponding to the exact mass of the unlabeled metabolites were detected in the DGM-D-incubated controls and observed mass shifts were consistent with the exchange of ¹²C by ¹³C atoms. Metabolites containing single ¹³C atoms were also observed in extracts from DGM-D-incubated EBs, due to the natural isotopic distribution of carbon, and were thus excluded.</p><p><b>^c)</b>The detection method (A: ICR/FT-MS; B: UPLC-MS) is indicated, as well as the ion species detected (H: [M-H⁺]⁻; Cl: [M+Cl⁻]⁻).</p><p><b>^d)</b>These metabolites were detected by ICR/FT-MS analysis in purchased D-[U-13C6]-glucose and were thus not considered as host-free synthesized metabolites.</p

D-glucose catabolism by host-free <i>P. amoebophila</i> EBs revealed by IRMS.

<p>Purified <i>P. amoebophila</i> EBs (EB-enriched fraction (A); highly pure EB fraction (B)) were pre-incubated for 40 h in DGM-D, followed by 48 h incubation in different media (DGM-D or DGM-D-13C (A); DGM-D, DGM-D-1-13C, DGM-D-6-13C, DGM-D-13C, or DGM-L (B)) and subsequent CO₂ measurement in the headspace of incubations by IRMS. As control, heat-inactivated bacteria (“EB-hi”) (A) or host cell lysates (B) incubated in DGM-D-13C were handled in parallel. CO₂ production (in ppm CO₂/ml; white circles) and the APE¹³C in the CO₂ (black diamonds) are displayed. Diamonds and circles indicate results from individual replicates, bars display mean values. The black solid line highlights the base line for CO₂ release and APE¹³C, which corresponds to values observed in the blanks, <i>i.e.</i> bacteria-free incubations of the respective media. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003553#s2" target="_blank">Results</a> from three independent experiments each consisting of two replicate incubations per condition are shown. An exception was the incubation in DGM-L, for which only two experiments were conducted. Statistically significant differences are indicated (for CO₂ release and APE¹³C at the upper or lower edge of the diagram, respectively) in respect to living bacteria incubated in DGM-D (ANOVA) (***, p≤0.001; **, p≤0.01; *, p≤0.05). Note that bacterial numbers that were applied per incubation were similar between replicate experiments (between 3.9×10⁹ and 5.9×10⁹ bacteria (A); between 6.3×10⁸ and 1.0×10⁹ bacteria (B)), but were significantly different between (A) and (B), explaining observed differences in the extents of CO₂ production.</p

Visualization of D-glucose uptake by host-free <i>P. amoebophila</i> EBs.

<p>Living or heat-inactivated <i>P. amoebophila</i> EBs were subjected to host-free incubation in DGM-D/2 containing 100 µM of the green-fluorescent D-glucose analog 2-NBDG. The incubation was conducted either immediately after purification or after a 40 h pre-incubation in DGM-D. Representative fluorescence and DIC images are shown. The bar indicates 10 µm.</p